Date Fruit (Phoenix dactylifera L.) Cultivar Extracts: Nanoparticle Synthesis, Antimicrobial and Antioxidant Activities

The pharmaceutical research sector’s inability to produce new drugs has made it difficult to keep up with the rate at which microbial resistance is developing. Recently, nanotechnology and its combinations with natural products have been the saviors against multidrug resistant bacteria. In the present investigation, different Egyptian and Saudi date cultivars were extracted and then phytochemically analyzed and tested for possible antimicrobial activities against multidrug resistant (MDR) microbes. The results revealed that extract of the flesh of fresh “Hayany” fruit (Egyptian date) showed the highest antimicrobial activity, with high levels of phenolic, flavonoid, and tannin concentrations (538.578 µg/mL, 28.481 µg/mL, and 20.888 µg/mL, respectively) and high scavenging activity, with an IC50 reaching 10.16 µg/mL. The highest synergistic activity was found between fresh “Hayany” fruit extract and amikacin. Novel nano-fresh fruit of “Hayany” date extract was synthesized using a ball-milling technique. The vesicle size was 21.6 nm, while the PDI and zeta potential were 0.32 and +38.4 mV, respectively. The inhibition zone diameters of nano-fresh fruit of “Hayany” date extract/amikacin reached 38 mm and 34 mm, with complete microbial eradication after 9 h and 6 h, against Candida albicans and Staphylococcus aureus, respectively. In conclusion, date fruit extract could be used as a candidate bioactive compound in the fight against infectious diseases.


Introduction
Multidrug resistant (MDR) bacteria pose a serious hazard to the entire world and have lessened the treatment options against infectious diseases. There is a need for more effective antimicrobial agents against MDR infections even though traditional antimicrobial drugs are fairly effective against some communicable diseases [1]. There is growing urgency for the invention of plant-based natural pharmaceutical products for the treatment and prevention of pathogenic diseases. Herbal medicines and phytochemicals have been employed for their promising antibacterial action since the prehistoric period. Moreover, the application of antimicrobial agent/phytochemical combinations in pharmaceutical industry can weaken the resistance mechanisms of MDR bacteria and enable the antibiotic to regain its effectiveness against multidrug resistant microorganisms [1,2]. These phytochemicals possess antimicrobial activity through inhibiting the efflux pump, degrading some resistant enzymes, etc. There have been several reports of herbal extracts, essential oils, and isolated pure chemicals acting in combination with some already available antibiotics, antifungals, and chemotherapeutics to increase the potency of these medications. The sensitivity patterns of numerous microorganisms towards natural/antimicrobial combinations showed considerable declines in the minimum inhibitory concentrations (MICs) [1]. Many secondary metabolites extracted from plants have antibacterial activities [10,11]. The tested fruit and epicarp extracts demonstrated encouraging antibacterial activity against S. pyogenes [12]. Abuharfeil [12] demonstrated that date fruit extract inhibited streptolysin O's hemolytic activity by neutralizing it, and 96% suppression was achieved at a relatively low dose. Date fruit, epicarp, and seed extract have been proven to be effective against Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Bacillus cereus). The most resistant Gram-negative bacteria were Shigella dysenteriae, Salmonella Typhi, and Klebsiella pneumoniae, and the most resistant Gram-positive bacteria was Staphylococcus aureus, while the Candida albicans fungus was the most resistant against all the tested extracts of the palm date extracts [13].   Figure 1). In FADE extract, pentanoic acid, α-chloro-D-alanine, 2-furancarboxylic acid 3-methyl-methyl ester, and 1,2-benzenediol, O-(4-fluorobenzoyl)-O-(1-naphthoyl) were identified with 18.37, 28.70, 36.82, and 8.88 area percentages, respectively. In FZDE extract, only nicotinic acid was identified, with 72.44 area percentage. In FHDE extract, N-(phosphonomethyl) glycine, α-chloro-D-alanine, hexadecanoic acid methyl ester, and 9-octadecenoic acid (Z)-methyl ester were identified with 56.12, 31.60, 5.24, and 1.81 area percentages, respectively (Table 4).  Various levels of polyphenols and flavanols such as nicotinic acid, p-hydroxybenzoi acid, hexadecanoic acid, protocatechuic acid, pentanoic acid, vanillic acid, gallic acid, O coumaric acid, and caffeic acid have been identified in palm date extracts [14]. Primar and secondary metabolite profiles of 21 date varieties from Egypt were analyzed by Fara et al. [15], who reported that caffeic acid conjugates (O-caffeoyl shikimic acid), flavonoid (quercetin and luteolin glycosides), sphingolipids, fatty acids, and flavonoids wer detected as the most abundant classes amongst all the examined P. dactylifera frui samples.       Various levels of polyphenols and flavanols such as nicotinic acid, p-hydroxybenzoic acid, hexadecanoic acid, protocatechuic acid, pentanoic acid, vanillic acid, gallic acid, O-coumaric acid, and caffeic acid have been identified in palm date extracts [14]. Primary and secondary metabolite profiles of 21 date varieties from Egypt were analyzed by Farag et al. [15], who reported that caffeic acid conjugates (O-caffeoyl shikimic acid), flavonoids (quercetin and luteolin glycosides), sphingolipids, fatty acids, and flavonoids were detected as the most abundant classes amongst all the examined P. dactylifera fruit samples.
The observed differences in phenolic component composition could have resulted from the differences in date cultivars, solvents, extraction techniques, and geographic location, which may have caused the observed antibacterial activity variance across the tested date extracts [14]. Authors who have studied the antibacterial properties of date extracts have also noted the presence of polyphenols, flavonoids, and tannins, which together have an antioxidant effect. Another important pharmacological activity is the inhibition of microbial growth by these secondary metabolites, which can be useful in powerful natural products and phytochemicals [16]. These compounds may act individually as a biologically active compounds or provide a synergistic effect when added to antibiotics to achieve higher antibacterial effect [17]. Several date cultivars were extracted and assessed for their total  Ibourki et al. [19] assessed eight different types of Moroccan date palms. "Boufgous", "Agondari", and "Bouskri" had the highest weight percentages for whole fruit, flesh, and seeds, respectively. Proteins (1.60-3.53%), moisture (5.31-17.31%), ash (2.08-2.50%), lipids (0.32-1.09%), carbohydrates (76.69-90.18%), energy value (338.30-385.89 kcal/100 g), and reducing capacity (100.14-1607.12 mg GAE/100 g) were the fruit flesh's greatest advantages.

Antioxidant Activity
The scavenging activity of FADE, FHDE, and FZDE extracts against 2,2-diphenyl-1picrylhydrazyl (DPPH) radicals increased in a concentration-dependent way ( Figure 2). The maximum scavenging activity was shown by FHDE extract at 71.21%, with IC50 10.16 µg/mL, followed by FZDE (61.75%; IC50 10.45 µg/mL) then FADE (47.85%; IC50 12.49 µg/mL) extracts (Table 6). Similarly, Mrabet et al. [20] showed that date extracts had strong radical scavenging activity, with IC50 values in the range of 1.65-8.25 µg/mL, by using the DPPH antioxidant method [20]. Another study revealed that the tested date extracts showed good DPPH radical scavenging (IC50 103-177 µg/mL) and hydroxyl radical scavenging (IC50 1.1-1.55 mg/mL) activity and total antioxidant capacity (IC50 87-192 µg/mL). The significant variation in the degree of antioxidant activity can be attributed to the differences in the content and composition of phenolic compounds [21]. Similar results were observed with spent coffee grounds (SCGs), which showed high antioxidant activity as assessed by DPPH (up to 92.12 ± 0.22%) and DPPH IC50 (up to 53.73 ± 1.03 µg mL −1 ) [22]. µg/mL) extracts (Table 6). Similarly, Mrabet et al. [20] showed that date extracts had strong radical scavenging activity, with IC50 values in the range of 1.65-8.25 µg/mL, by using the DPPH antioxidant method [20]. Another study revealed that the tested date extracts showed good DPPH radical scavenging (IC50 103-177 µg/mL) and hydroxyl radical scavenging (IC50 1.1-1.55 mg/mL) activity and total antioxidant capacity (IC50 87-192 µg/mL). The significant variation in the degree of antioxidant activity can be attributed to the differences in the content and composition of phenolic compounds [21]. Similar results were observed with spent coffee grounds (SCGs), which showed high antioxidant activity as assessed by DPPH (up to 92.12 ± 0.22%) and DPPH IC50 (up to 53.73 ± 1.03 µg mL −1 ) [22].    Hence, principal component analysis (PCA) was used as a multivariate statistical method to be an efficient discriminative approach to differentiate among the date palm varieties and the observed scavenging and phytochemical analyses (dependent variables). The first two principal components (PCs) explained over 74% of the total data variabilities. The first component (PC1) seemed to separate between the tested dates extracts and the observed phytochemical analyses (Figure 3). Fresh fruit of "Amhaat" and "Hayany" date extracts were on the positive side of the first component (PC1), while "Zaghloul" date extract was on the opposite side. Furthermore, total flavonoids (TF), total phenolics (TP), and total tannins (TT) were linked and related as dependent variables. Ibourki et al. [19] mentioned that "Ismmarti", "Boufgous", "Amouch", and "Jihel" interacted, with higher scores of seed weight, fruit weight, flesh weight, and flesh percentage, while "Ismmarti", "Agondari", and "Boufgous" interacted with greater values of reducing capacity and mineral elements.

Effects of Combinations of FADE, FHDE, and FZDE Extracts with Commonly Used Antibiotics
The interactions of FADE, FHDE, and FZDE with commonly known antibiotics were evaluated against C. albicans, E. coli, and S. aureus individually using disc diffusion method. Data shown in Table 7 revealed that the combined actions of FHDE with amikacin (AK), ampicillin/cloxacillin (AX), amoxicillin/clavulanate (AMC), gentamicin (GEN), and cloxacillin (COX) showed synergistic effects against C. albicans, while the combinations with AK and COX showed synergistic action against S. aureus. In addition, when polymyxin B (PB) was combined with FHDE, it showed an indifferent effect against S. aureus. Furthermore, FZDE showed a synergistic effect against E. coli when combined with COX. However, other combinations showed antagonistic effects. The most potent combination against the tested pathogens was FHDE combined with AK. Therefore, this combination was subjected to further investigations. Ibourki et al. [19] mentioned that "Ismmarti", "Boufgous", "Amouch", and "Jihel" interacted, with higher scores of seed weight, fruit weight, flesh weight, and flesh percentage, while "Ismmarti", "Agondari", and "Boufgous" interacted with greater values of reducing capacity and mineral elements.

Effects of Combinations of FADE, FHDE, and FZDE Extracts with Commonly Used Antibiotics
The interactions of FADE, FHDE, and FZDE with commonly known antibiotics were evaluated against C. albicans, E. coli, and S. aureus individually using disc diffusion method. Data shown in Table 7 revealed that the combined actions of FHDE with amikacin (AK), ampicillin/cloxacillin (AX), amoxicillin/clavulanate (AMC), gentamicin (GEN), and cloxacillin (COX) showed synergistic effects against C. albicans, while the combinations with AK and COX showed synergistic action against S. aureus. In addition, when polymyxin B (PB) was combined with FHDE, it showed an indifferent effect against S. aureus. Furthermore, FZDE showed a synergistic effect against E. coli when combined with COX. However, other combinations showed antagonistic effects. The most potent combination against the tested pathogens was FHDE combined with AK. Therefore, this combination was subjected to further investigations. Ampicillin/cloxacillin AX Alone 6.0 ± 0.2 6.0 ± 0.6 6.0 ± 0.5 +FADE 6.0 ± 0.2 14.0 ± 0.2 6.0 ± 0.2 +FZDE 6.0 ± 0.1 6.0 ± 0.9 6.0 ± 0.5 +FHDE 28.0 ± 0.1 6.0 ± 0.7 6.0 ± 0.7 Amoxicillin/clavulanate AMC Alone 6.0 ± 0.2 6.0 ± 0.5 23.0 ± 0.9 +FADE 6.0 ± 0.7 6.0 ± 0. An efficient strategy for dealing with the resistance problem is the synergistic interaction of natural substances with commonly known antibiotics. The interaction of an antimicrobial agent with its target within the pathogen may be improved or designed to facilitate a combination between natural substances, which would stop the development of resistance. Because it allows for the use of lower doses of both drugs, this technique can minimize drug toxicity [23,24]. The emergence of microbial resistance to antibiotics has made it incredibly challenging to identify new treatments. Abreu et al. [24] and Betts et al. [25] isolated some phytochemicals from herbs that were biologically active. For instance, reserpine, isopimarane, EGCG, and carnosic acid blocked bacterial efflux pumps, whereas corilagin, gallic acid, and thymol increased the permeability of bacterial outer membranes. Tellimagrandin and corilagin both inhibited the penicillin binding proteins (PBP2a) [24]. Berberine and 5 -methoxyhydnocarpin, two phytochemicals typically found in barberry plants, worked together to concentrate inside bacteria and inhibit the MDR efflux pump [24]. Chaqroune and Taleb [26] declared that Soxhlet extraction combined with polar solvents (ethanol and methanol) led to the best MIC (8.17 ± 1.04-24.20 ± 0.98 µg/mL) and IC50 values (50.02 ± 0.08-390.00 ± 1.00 µg/mL) obtained from wild rosemary.

Nano-Date Palm Fruit Extract Synthesis and Characterization
FHDE nanoparticles were physically synthesized using the ball-milling technique. Then, the synthesized nano-FHDE (NFHDE) was combined with AK (FHDE/AK, 1:1 w/w). TEM images of the nano-FHDE/AK (NFHDE/AK) showed it to comprise homogenous unilamellar vesicles. These were well identified as having nearly perfect sphere-like shapes and smooth vesicle surfaces (Figure 4). The recorded vesicle size was 21.6 nm, while the PDI and zeta potential were 0.32 and +38.4 mV, respectively, which proved the nanoparticles' homogeneity and stability. Alothman et al. [27] revealed that nano-date extract was a heterogeneous mix of spherical and irregular shapes. Evidently, the particles' sphere-like structure may provide them highly maintained flexibility for useful applications. According to TEM investigations, the size range was between 30 and 110 nm. Similarly, Saba et al. [28] reported that the nanosize of date fruit (using the ball-milling process) was up to 100 nm. FHDE nanoparticles were physically synthesized using the ball-milling technique. Then, the synthesized nano-FHDE (NFHDE) was combined with AK (FHDE/AK, 1:1 w/w). TEM images of the nano-FHDE/AK (NFHDE/AK) showed it to comprise homogenous unilamellar vesicles. These were well identified as having nearly perfect sphere-like shapes and smooth vesicle surfaces (Figure 4). The recorded vesicle size was 21.6 nm, while the PDI and zeta potential were 0.32 and +38.4 mV, respectively, which proved the nanoparticles' homogeneity and stability. Alothman et al. [27] revealed that nano-date extract was a heterogeneous mix of spherical and irregular shapes. Evidently, the particles' sphere-like structure may provide them highly maintained flexibility for useful applications. According to TEM investigations, the size range was between 30 and 110 nm. Similarly, Saba et al. [28] reported that the nanosize of date fruit (using the ball-milling process) was up to 100 nm.

Antimicrobial Activity of the Synthesized NFHDE/AK Extract
Data shown in Table 8 revealed that the inhibition zone (IZ) diameter of NFHDE/AK against C. albicans was 38 mm, with an MIC, MBC, and MIC index of 25 µg/mL, 50 µg/mL and 2, respectively. Its IZ diameter against S. aureus was 34 mm, with an MIC, MBC, and MIC index of 12.5 µg/mL, 25 µg/mL, and 2, respectively. In order to test the eradication  Table 8 revealed that the inhibition zone (IZ) diameter of NFHDE/AK against C. albicans was 38 mm, with an MIC, MBC, and MIC index of 25 µg/mL, 50 µg/mL and 2, respectively. Its IZ diameter against S. aureus was 34 mm, with an MIC, MBC, and MIC index of 12.5 µg/mL, 25 µg/mL, and 2, respectively. In order to test the eradication time for FHDE-and NFHDE/AK-treated C. albicans and S. aureus cells, microbial lethality curves were evaluated. The results, as shown in Figure 5, showed a steady increase in the growth of S. aureus and C. albicans during the log phase (first 7 h and 8 h, respectively) after which microbial growth sharply decreased until complete eradication after 6 h and 9 h for NFHDE/AK formula-treated S. aureus and C. albicans cells respectively. When the microbial cells were treated with FHDE, the cells were eradicated after 10 h. Transmission electron microscopy (TEM) was applied to NFHDE/AK-treated S. aureus and C. albicans cells. Figure 6a,b shows that the microbial cells treated with NFHDE/AK revealed excessive damage to the cell wall and huge leakage of intracellular components that led to cell death. The nanoparticles of date extract were able to reduce Gram-positive and Gram-negative bacterial growth by disrupting the cell membrane with 100-fold higher antibacterial activity than the crude palm date extract [29]. death. The nanoparticles of date extract were able to reduce Gram-positive and Gramnegative bacterial growth by disrupting the cell membrane with 100-fold higher antibacterial activity than the crude palm date extract [29].

Sample Collection
Date palm fruits were collected at the "Tamr stage" (full ripeness) from local markets in Saudi Arabia and Egypt during September 2021. The local Arabic names of the cultivars used in this study were "Suqaey", "Barhi", and "Ajwa" (red, yellow, and brown, respectively) for the Saudi Arabian dates and "Zaghloul", "Hayany", and "Amhaat" (red, yellow, and brown, respectively) for the Egyptian dates. Samples were cleaned and rinsed with sterile distilled water, air dried, and ground into powder before being conserved at −20 • C.

Extraction of Bioactive Material
The conventional Soxhlet method was used for the extraction of bioactive agents from the fresh fruit flesh, dried fruit flesh, epicarp, dried epicarp, and seeds of different Egyptian and Saudi date palm cultivars. One at a time, 15 g of each of the powdered dates was extracted by methanol/chloroform (2:1) [30].

Antibacterial Effect of Date Extracts
Antimicrobial activity was investigated using the disk diffusion method according to CLSI guidelines [31]. By measuring the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and microbial lethality curve, further antibacterial activity was evaluated [32]. Using gallic acid, quercetin, and tannic acid as standards, the total phenolic content (TPC), total flavonoid content (TFC), and total tannin content (TTC), respectively, of the date extracts were measured [21,34]. According to standard curve calculations, the findings of the TPC, TFC, and TTC contents are represented as gallic acid equivalent (mg GAE/g), quercetin equivalent (mg QE/g), and tannic acid equivalent (mg TAE/g), respectively.

DPPH Free Radical Scavenging Assay
The technique of Zihad et al. [21] was used to assess the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity of the date extracts. Aliquots (50 µL) of serially diluted extract solutions in methanol (500-0.98 µg/mL) were combined with 5 mL of DPPH solution in methanol (40 µg/mL). The reaction mixture was completely vortexed and kept at room temperature for 30 min in the dark. After then, a UV spectrophotometer (Shimadzu 2000) was used to assess the mixture's absorbance at 517 nm. Percent inhibition was obtained using ascorbic acid as the positive control using Equation (1): where Abs control is the absorbance of the DPPH solution dissolved in methanol and Abs sample is the absorbance of the tested date extract or the standard solution dissolved in methanol.

Total Antioxidant Capacity
For this experiment, a modified version of Yang et al.'s technique was applied [35]. In this test, 3 mL of the reagent mixture (4 mM ammonium molybdate, 0.6 M sulfuric acid, and 28 mM trisodium phosphate) was blended with 0.3 mL of the tested date extract or a standard solution (ascorbic acid). This reaction mixture was heated to 90 • C and then allowed to cool to room temperature for 90 min. At 695 nm, the reaction mixture's absorbance was appraised by comparing the absorbance of the blank preparation with the IC50 values.

Statistical Analyses
Results are expressed as means ± standard deviations (SD). Principal component analysis (PCA) was performed to differentiate among date extract varieties and the estimated phytochemicals using ORIGINPro ® 2022b (OriginLab, Inc., Northampton, MA, USA).

Effects of Combinations of the of the Most Promising Date Extracts with Commonly Used Antibiotics Assessed Using the Disk Diffusion Method
Different antibiotics were selected in the present experiment, namely, ceftriaxone (CTR, 30 µg), amikacin (AK, 30 µg), amoxicillin/clavulanate (AMC, 20/10 µg), gentamicin (GEN, 10 µg), ampicillin/cloxacillin (AX, 10 µg), cloxacillin (COX, 5 µg), ciprofloxacin (CIP, 5 µg), oxacillin (OX, 1 µg), polymyxin (PB, 5 µg), and cotrimoxazole (COT, 25 µg). The antibiotic discs were loaded with 20 µL of each date extract (20 µg) one at a time and placed on the surface of the inoculated Mueller-Hinton agar. When the combined effect was equal to the sum of the individual effects, the action was considered as additive. Antagonism was observed when the effect of the combined compounds was less than that of the individual compounds. Synergism was observed when the effect of the combined compounds was greater than the sum of the individual effects, while indifference was observed in the absence of interaction [36].

Physical Synthesis of Nanoparticles of the Most Promising Extract Using Ball-Milling Technique
With slight modifications, the milling procedure was carried out in accordance with the methodology of Saba et al. [28]. Using 10 mm zirconia balls in a 15 mL zirconia container, the date extract-to-ball ratio was maintained at 1:10. An entire number of 30 min cycles was maintained, with a 15 min break in between each cycle. The RPM was maintained at 300 throughout the operation.

Characterization of the Synthesized Nanoextract
The prepared nanoparticles were mixed in a constant ratio that equaled 1:1 (w/w) with the most potent antibiotic (which proved promising synergistic activity with the crude date extract). The potent formula was characterized through dynamic light scattering (DLS) techniques to determine the vesicle size, polydispersity index (PDI), and zeta potential. The ultrastructure of the prepared nanoparticles was analyzed through transmission electron microscopic (TEM) study. Moreover, the antibacterial activity of the prepared nanoparticles was evaluated using the disk diffusion method via the minimal inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and microbial lethality curve along with TEM examination of the treated microbial cells [37].

Conclusions
According to the data provided, it was concluded that Egyptian date extracts ("Zaghloul", "Hayany", and "Amhaat" (red, yellow, and brown Egyptian dates)) showed high antimicrobial activity against all of the tested multidrug resistant microbes, especially Candida albicans and Staphylococcus aureus. The extract of the fresh fruit of "Hayany" (yellow) dates showed the highest antimicrobial activities, with a promising antioxidant activity and the highest concentrations of total phenolics, flavonoids, and tannins. The combination between the extract of the fresh fruit of "Hayany" dates and amikacin had synergistic activity. The novel nanoformula (NFHDE/AK, nano-fresh fruit of "Hayany" date extract/amikacin) showed the highest antimicrobial activity. Egyptian date extracts can be used as bioactive healthy compound sources. However, extensive preclinical and clinical studies must be undertaken for the benefit of humankind.