Assessment of polyphenol composition, antioxidant and antimicrobial properties of various extracts of Date Palm Pollen (DPP) from two Tunisian cultivars

2.6.1 DPPH free radical scavenging activity

DPPH free radical scavenging activity was determined according to a slightly modified version of the method of Barros et al. (2007). In brief, 1 ml DPPH (0.1 mM) was dissolved in methanol and added to a 0.5 ml solution of DPP extracts at different concentrations. Samples were kept in the dark for 30 min at room temperature, and decrease in absorption was measured at 517 nm against a blank using a spectrophotometer (Bio-Rad SmartSpecTM plus). The blank was prepared from the reaction mixture without DPPH solution. BHT and vitamin E were used as positive controls. The experiment was performed in triplicate.

The decrease in the absorption of the DPPH solution was calculated by the following equation: $$\text{DPPH radical scavenging activity}(\%)=\left[\frac{A0-A1}{A0}\right]\times 100$$ where A0 refers to the absorbance of the control reaction containing all reagents except the tested compound, and A1 to the absorbance of the test compound.

The results were expressed as IC₅₀ (μg/ml) values, referring the amount of sample necessary to decrease the absorbance of DPPH radicals by 50%. All measurements were performed in triplicate.

2.6.2 β-carotene–linoleic acid bleaching assay

The antioxidant activity of the extracts under investigation was evaluated using a β-carotene–linoleate model system following a slightly modified version of the method described by Siddhuraju and Becker (2003). In brief, 2 mg of β-carotene was added to 10 ml of chloroform. After that, 20 μl linoleic acid and 200 mg Tween 40 were mixed with 1 ml of the chloroform solution. After evaporation of the chloroform under vacuum at 45 °C, 50 ml oxygenated water was added, and the mixture was vigorously shaken. Four milliliters of aliquot of this mixture and 0.2 ml of each extract at different concentrations or BHT (control antioxidant) were placed in test tubes and mixed thoroughly. The tubes were immediately incubated at 50 °C for 120 min. The oxidation of the β-carotene emulsion was monitored spectrophotometrically by measuring absorbance at 470 nm. The antioxidant activity (Inhibition %) of the samples was calculated using the following equation: $$\mathit{AA}(\%)=\left[1-\frac{A0-\mathit{At}}{A^{\prime }0-A^{\prime }t}\right]\times 100$$ where A0 and A′0 refer to the absorbance values measured at zero time for the test sample and control, respectively, and At and A′t refer to the corresponding absorbance values of test sample and control measured after incubation for 120 min, respectively. All tests were carried out for three sample replications.

2.7.1 Microbial strains and growth conditions

The antibacterial activities of DPP were tested against 10 strains of bacteria. These included Gram-positive bacteria: Bacillus subtilis JN 934392, Bacillus cereus JN 934390, Staphylococcus aureus ATCC 6538, Enterococcus faecalis, Micrococcus luteus, Listeria monocytogenes, and Gram-negative bacteria: Salmonella enteric serotype Enteritidis ATCC43972, Salmonella enteric serotype Typhimurium, Escherichia coli ATCC 25922, and Klebsiella pneumoniae. Antifungal activities were tested using four fungal strains: Aspergillus niger, Fusarium phyllophilum AB587006, Fusarium sp. JX391934, Fusarium oxysporum AB586994, Penicillium sp., Pythium catenulatum AY598675, and Rhizoctonia solani.

The test bacteria were cultured on Petri dishes containing Mueller Hinton Agar (MHA) and incubated for 18–24 h. From these dishes, a bacterial culture was prepared in 3 ml MH broth with agitation (200 rpm) for 24 h at 37 °C, except for Bacillus species, which were incubated at 30 °C. Optical density read at 625 nm ranged from 0.08 to 0.10, a density equivalent to 10⁷ CFU/ml (Mohammedi, 2006).

For fungal strains, growth was carried out at 30 °C for 4 days on Sabouraud agar until mycelia growth covered the entire dishes, from which, a spore suspension was obtained in 10 ml sterile water containing 0.1% Tween 80. Optical density was also adjusted to 0.08–0.10 to obtain a solution equivalent to 10⁶ spores/ml (Ben Hsouna et al., 2011).

2.7.2 Antimicrobial activity detection by agar diffusion method

Antibacterial and antifungal activities were detected by the agar well diffusion test using a slightly modified version of the method described by Ben Hsouna et al. (2011). In brief, a cavity (wells) of 6 mm was created in the MHA using a sterile Pasteur pipette. A freshly prepared bacterial suspension or spore solution (100 μl) adjusted to 10⁷ CFU/ml for bacteria and 10⁶ spores/ml for fungus was inoculated onto the surface of agar plates using a sterile swab. Each well was then filled with 80 μl of each extract (125 mg/ml DMSO). A negative control was carried out simultaneously with the DMSO. The plate was left at +4 °C for 2 h to facilitate the diffusion of the extracts in the agar (Trigui et al., 2013), and then incubated at 37 °C for 24 h for bacterial strains and at 30 °C for 4–7 h for fungal strains. Antimicrobial activity was determined by measuring the diameter of inhibition zone around the well.

2.7.3 Determination of MIC and MFC by micro-dilution well method

The MIC values, representing the lowest extract concentrations that prevented the visible growth of microorganisms, were determined by the method of Gulluce et al. (2007) in a sterile 96-well microplate, with a final volume of 200 μl per well. A stock solution of each extract (125 mg/ml) was prepared in DMSO, which is known to have no strong antimicrobial activity (Gachkar et al., 2007). Twofold serial dilutions of the extracts were prepared in the microplate wells over the range 0.98–125 mg of extract/ml DMSO. Each well was supplemented with 100 μl of each extract dilution, and 10 μl of cell suspension to a final inoculum concentration of 10⁷ CFU/ml and 10⁶ spores/ml for bacteria or 90 μl of MH broth and PDB broth for fungi. The last well, which contained only bacteria or fungi in the adequate medium without the addition of extract, was considered as a positive growth control. The one containing DMSO without extract was used as a negative control. After content homogenization, the plates were covered with sterile plate covers and incubated for 24 h at 37 °C for bacterial strains and for 3 days at 30 °C for fungal strains.

Microorganism viability assays involved the addition of 25 μl of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H-tetrazolium bromide) (0.5 mg/ml sterile distilled water), as an oxidation reduction indicator, to each well and subsequent incubation of the mixture for 30 min at 37 °C. In this assay, the wells involving microbial growth inhibition stay clear after incubation with MTT (Ben Hsouna et al., 2011). The MIC was defined as the lowest concentration of the compounds to inhibit the growth of the microorganisms. The MBC values were interpreted as the highest dilution (lowest concentration) of the sample, which showed clear fluid with no turbidity development and without visible growth of microorganisms after incubation for 48 h at 37 °C (Diao et al., 2014; Mighri et al., 2010).

Minimum fungicidal concentrations (MFC) were defined by the first wells with no visible growth and determined by serial subcultivation of 10 μl in Potatoes dextrose agar (PDA) plates and incubation for 3–4 days at 30 °C. The MFC was considered as the lowest concentration that prevented mycelium growth (Zarrin et al., 2010).

3.3.1 DPPH free radical scavenging activity

The DPPH radical scavenging assay is an easy, rapid, and sensitive method commonly used for the screening and evaluation of the antioxidants activity and free radical scavenging ability of plant extracts. This test provides information on how capable an antioxidant is in inhibiting oxidative cell damages by preventing reactive radical species from attacking key biomolecules, such as lipoproteins, and polyunsaturated fatty acids. The results presented in Table 1 are expressed in IC₅₀ values (extract concentrations required to scavenge DPPH radical by 50%); thus, a lower IC₅₀ value would reflect greater antioxidant activity of the sample. The findings revealed that all extracts exhibited dose-dependent scavenging effects, with DPP-T showing higher antioxidant activity than DPP-K. Among all extracts, the acetone extract displayed the highest free radical scavenging activity (IC₅₀ = 46.56 ± 0.28 μg/ml), followed by ethyl acetate (IC₅₀ = 67.50 ± 0.95 μg/ml). The scavenging activities of the other extracts were, however, significantly low when compared to the BHT reference standard (IC₅₀ = 32.17 ± 0.42 μg/ml) used at the same dose. These results implied that ethyl acetate and particularly acetone led to the extraction of more compounds, with possibly more antioxidant activity. The differences observed between the DPPH scavenging ability of the DPP-T and DPP-K extracts could presumably be attributed to differences in their polyphenolic contents. As mentioned earlier, the antioxidant activities of the acetone and ethyl acetate extracts could be ascribed to their high of phenolic contents. These findings confirm the earlier obtained data on the DPPH scavenging activity of Egyptian Date Palm Pollen methanolic extract with IC₅₀ value of 0.8 μg/ml (Khider et al., 2013).

The antioxidant activity of phenolic compounds is due to the presence of a hydroxyl group which donates protons to free radicals and scavenges them (Fukumoto and Mazza, 2000). It was previously reported that epicatechin, vanillic, coumarin and caffeic acid have a great potential of antioxidant activity (Konyaloglu et al., 2005). Although free phenolic acids exist in lower amounts than the bound ones, they affect antioxidant properties. Epicatechin and catechin, a polyphenol very abundant in tea plants, have been reported to represent a strong radical scavenger and metal chelator in model chemical systems, and these effects correlate well with the present study (Lambert and Elias, 2010)

The diversity observed in chemical composition could present a principal factor in negative synergism when antioxidant results are compared since phenolic compounds and flavonoid aglycones are considered to possess more potent antioxidants than their corresponding glycosides (Gao et al., 1999).

3.3.2 β-carotene–linoleic acid bleaching assay

The antioxidant activities of the DPP extracts were evaluated through the measurement of percent inhibition of peroxidation in a linoleic acid system using the β-carotene bleaching test. The presence of antioxidants is known to avoid the destruction of the β-carotene conjugate system and minimize its oxidation by hydroperoxides (must be neutralized by the presence of antioxidants in the extracts). The results presented in Table 1 show that the DPP-T and DPP-K extracts were able to inhibit lipid peroxidation in a dose-dependent pattern. The concentrations providing 50% inhibition were expressed in IC₅₀ values. The antioxidant potential of the DPP-T extracts in terms of inhibition percentages was noted to vary with the extraction solvent according to the following order: acetone > ethyl acetate > BHT > chloroform > aqueous > hexane extracts. However, the antioxidant potential of the DPP-K varied according to the following order: BHT ≈ acetone > ethyl acetate > chloroform > aqueous > hexane. The inhibitory concentrations required for each extract to provide 50% inhibition were compared to BHT and vitamin E as reference standards. The results indicated that DPP-T exhibited higher antioxidant activity than DPP-K, with the acetone extract showing the highest rate of activity, which was nearly similar to that of BHT. The variability observed for the antioxidant activities of both DPP extracts could presumably be attributed to differences in extraction procedures, sample processing or drying. During the processing of extracts, some active volatile compounds may have been destroyed or evaporated (Naczk and Shahidi, 2006).

As regards the antioxidant activity of some plants, we demonstrated that the antioxidant activity of DPP could be attributed to the differences in their chemical composition and primarily related to their hydroxylation and methylation patterns, to the presence of many phenolic compounds, such as flavonoids and polyphenols and others molecules such as proteins, ascorbate, β carotene, α-tocopherol and lycopene which enhance the antioxidant activity. Caffeic acid, catechin, and epicatechin have previously been reported for their abilities to provide stronger protective benefits against lipid oxidation, which may be helpful for oxidation-related disease prevention (Liao and Yin, 2000). Overall, these results are of significant importance, given the scarcity of data on the antioxidant activity of DPP in the literature.

3.5.1 Effect of DPP-T ethyl acetate extract on viable counts of S. aureus

Time-kill kinetic assays provide more accurate description of the antibacterial activity of antimicrobial agents than MIC assays. Accordingly, the effect of DPP-T ethyl acetate extract on the viable counts of selected bacteria such as S. aureus was investigated to further confirm its effect and clarify its mechanism of action. The results presented in Fig. 1 indicate that, compared to the control, the S. aureus strain treated with the DPP-T ethyl acetate extract at the MIC and 2MIC values (0.98 and 1.95 mg/ml, respectively) showed a decrease in the number of viable cells over the 60 min period of the test, reaching 3 and 2 log CFU/ml, respectively. At higher concentrations of 4MIC and 8MIC (3.92 and 7.84 mg/ml), no viable cells were observed after 10 min. As a consequence, the bactericidal concentration required for DPP-T to kill S. aureus should be less than 3.92 mg/ml (4MIC). The above results confirm the postulation that the antimicrobial substances in the tested species might be destroyed by the peptidoglycan layer of the cell wall, thus suggesting that the mode of action of the DPP-T extracts was cell-wall related (Okemo et al., 2001). Accordingly, DPP-T could be considered a promising candidate for application as natural preservative additive in various food and pharmaceutical products and formulations.

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Figure 1

Bactericidal effect of Date Palm pollen ethyl acetate extract from Tozeur on Staphylococcus aureus strain. Samples were taken at different incubation times and viability was determined by the plate colony count procedure (CFU: colony forming unit).

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3.5.2 Effect of DPP-K ethyl acetate extract on viable counts of L. monocytogenes

According to the sensitivity of the tested microorganisms on DPP-K, L. monocytogenes was selected as a model to confirm the antibacterial effects of the DPP-K ethyl acetate extract and investigate its mode of action. As shown in Fig. 2, the number of L. monocytogenes viable cells decreased progressively from 6.7 to 2.61 log CFU/ml within 60 min after the addition of 0.98 mg/ml (1MIC) of extract. However, no viable cells were detected after 60 min with the 2MIC (1.95 mg/ml) concentration. No viable cells were also detected after 5 min with the addition of higher concentrations of 4MIC and 8MIC (3.92 and 7.84 mg/ml). However, the control (without extract) revealed approximately the same CFU/ml value throughout the 60 min, and no L. monocytogenes growth inhibition was observed. As a consequence, the bactericidal concentration required to kill L. monocytogenes should be less than 1.95 mg/ml (2MIC).

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Figure 2

Bactericidal effect of pollen ethyl acetate extract from Kerkennah on Listeria monocytogenes strain. Samples were taken at different incubation times and viability was determined by the plate colony count procedure (CFU: colony forming unit).

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Taken together, the results showed the effect of time and DPP-K extract on bactericidal behavior. The comparison of the results presented in this work (5 min and 3.92 mg/ml) and the findings previously reported elsewhere for the T. hirsuta extract (10 min and 7.50 mg/ml) using the same strain pointed to the potent antibacterial effect of the DPP-K extract to inhibit the growth of L. monocytogenes (Trigui et al., 2013).

This suggested that the ethyl acetate DPP-K extract is a good potential inhibitor of food spoiling microbial growth and could be a highly effective therapeutic choice for human and plant infections.