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Table of Contents
Abstract
1. Introduction
2. Materials And Methods
2.1. Sample Preparation
2.2. Reagents
2.3. Solid-Liquid Extraction
2.4. Lyophilization Of Date Seeds Extracts
2.5. Determination Of The Phenolic Profile Using HPLC-DAD-ESI-MS
2.6. Determination Of The Antioxidant Activity Of Date Seeds Using Spectrophotometric Assays
2.6.1. ABTS Assay
2.6.2. DPPH Assay
2.7. Determination Of The Antibacterial Activity Of Date Seeds Using The Agar Well Diffusion Method
2.7.1. Preparation Of Date Seeds Samples
2.7.2. Preparation Of Bacterial Inocula
2.7.3. Assessment Of Antibacterial Activity
2.8. Extraction Of Date Seeds Oil Using Soxhlet Extraction
2.9. Determination Of The Fatty Acid Profile By GCMS– Analysis
2.10. Statistical Analysis
3. Results And Discussion
3.1. Phenolic Composition
3.2. Antioxidant Activity
3.3. Antibacterial Activity
3.4. Oil Yield And Fatty Acids Profile
4. Conclusion
Acknowledgments
Abstract
Date palm fruit (Phoenix dactylifera L.) is a well-known fruit that has become increasingly popular among consumers due to its pleasant flavor, nutrient-dense content, and nutraceutical characteristics. Date seeds are the main by-product of date fruit consumption and processing. Date seeds are usually incorporated in animal feed; however, their application in human contexts, such as food products, remains minimal. Nonetheless, they contain significant amounts of bioactive phenolic compounds, primarily flavonoids, which possess potential health benefits. The current study investigated and compared the phenolic profile, antioxidant, and antibacterial activities of aqueous extracts of nine different date seeds varieties. The phenolic profile, analyzed using highperformance liquid chromatography (HPLC-DAD-ESI-MS), showed that total phenolic compounds ranged from 1.05 to 1.24 mg/g in Reziz and Naghal varieties, respectively, with flavan-3-ols predominating in all varieties. Results of the ABTS and DPPH assays revealed that the antioxidant activities against free radicals were directly correlated with the overall phenolic content, particularly for caffeic acid. Indeed, all date seeds possessed antibacterial capabilities, with a stronger effect observed on Listeria monocytogenes than Escherichia coli. Additionally, date seeds demonstrated a promising source of oil rich in oleic and lauric acids, both of which are considered beneficial for health. According to our findings, date seeds are a significant source of bioactive compounds, which renders them ideal candidates for potential inclusion in human diets as food additives and nutraceuticals.
Assessment of the phenolic profile and biological activities of aqueous date seed extracts: A comparative analysis Tareq M. Osaili a,b,c,*, Aseel Swaidan d, Anas Al-Nabulsi c, Amin Olaimat e, Susanne Neugart f, Layla Engelhardt f, Tuba Esatbeyoglu g, Mutamed Ayyash h, Leila Cheikh Ismail a,b, Maher M Al-Dabbas i,j, Reyad S. Obaid a,b, Nada El Darra d,* a Department of Clinical Nutrition and Dietetics, College of Health Sciences, University of Sharjah, P. O. Box 27272 Sharjah, UAE b Research Institute for Medical and Health Sciences, University of Sharjah, P.O. Box 27272 Sharjah, UAE c Department of Nutrition and Food Technology, Faculty of Agriculture, Jordan University of Science and Technology, P.O. Box 3030, Irbid 22110, Jordan d Department of Nutrition and Dietetics, Faculty of Health Sciences, Beirut Arab University, Tarik El Jedidah, Riad El Solh, P.O. Box 115020, Beirut 1107 2809, Lebanon e Department of Clinical Nutrition and Dietetics, Faculty of Applied Medical Sciences, The Hashemite University, P.O. Box 150459, Zarqa 13115, Jordan f Department of Crop Sciences, Division Quality and Sensory of Plant Products, Georg-August-Universität Göttingen, Carl-Sprengel-Weg 1, 37075 Goettingen, Germany g Department of Molecular Food Chemistry and Food Development, Institute of Food and One Health, Gottfried Wilhelm Leibniz University Hannover, Am Kleinen Felde 30, 30167 Hannover, Germany h Department of Food Science, College of Food and Agriculture, United Arab Emirates University, UAE i Nutrition and Dietetics program, College of Pharmacy, Al Ain University, Abu Dhabi, UAE j Department of Nutrition and Food Technology, Faculty of Agriculture, The University of Jordan, Amman 11942, Jordan A R T I C L E I N F O Keywords: Phoenix dactylifera Phenolic compound Antioxidant Antibacterial Date seeds oil Nutraceuticals A B S T R A C T Date palm fruit (Phoenix dactylifera L.) is a well-known fruit that has become increasingly popular among consumers due to its pleasant flavor, nutrient-dense content, and nutraceutical characteristics. Date seeds are the main by-product of date fruit consumption and processing. Date seeds are usually incorporated in animal feed; however, their application in human contexts, such as food products, remains minimal. Nonetheless, they contain significant amounts of bioactive phenolic compounds, primarily flavonoids, which possess potential health benefits. The current study investigated and compared the phenolic profile, antioxidant, and antibacterial activities of aqueous extracts of nine different date seeds varieties. The phenolic profile, analyzed using highperformance liquid chromatography (HPLC-DAD-ESI-MS), showed that total phenolic compounds ranged from 1.05 to 1.24 mg/g in Reziz and Naghal varieties, respectively, with flavan-3-ols predominating in all varieties. Results of the ABTS and DPPH assays revealed that the antioxidant activities against free radicals were directly correlated with the overall phenolic content, particularly for caffeic acid. Indeed, all date seeds possessed antibacterial capabilities, with a stronger effect observed on Listeria monocytogenes than Escherichia coli. Additionally, date seeds demonstrated a promising source of oil rich in oleic and lauric acids, both of which are considered beneficial for health. According to our findings, date seeds are a significant source of bioactive compounds, which renders them ideal candidates for potential inclusion in human diets as food additives and nutraceuticals.
1. Introduction
In the last few decades, numerous studies have indicated the beneficial properties of polyphenols González-Vallinas et al., 2013; Tenore et al., 2020; Ali et al., 2023). Polyphenols are naturally occurring compounds that are commonly found in plant-based foods, including tea, fruits, and vegetables (Alara et al., 2021). Phenolic compounds (e.g., flavonoids, phenolic acids, hydroxycinnamic acids, tannins) in these List of Abbreviations: ABTS, 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid); DPPH, 2,2-Diphenyl-1-picrylhydrazyl; DM, Dry Matter; ESI, Electrospray Ionization; LOD, Limit of Detection; LOQ, Limit of Quantification; QQQ, Triple Quadrupole; SST, Stainless Steel Teflon; TEAC, Trolox Equivalent Antioxidant Capacity. * Corresponding author. E-mail addresses: tosaili@sharjah.ac.ae, tosaili@just.edu.jo (T.M. Osaili), n.aldarra@bau.edu.lb (N. El Darra). Contents lists available at ScienceDirect Applied Food Research journal homepage: www.elsevier.com/locate/afres https://doi.org/10.1016/j.afres.2024.100493 Received 21 June 2024; Received in revised form 9 August 2024; Accepted 30 August 2024 Applied Food Research 4 (2024) 100493 Available online 7 September 2024 2772-5022/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC license ( http://creativecommons.org/licenses/bync/4.0/ ). foods have gained researchers’ interest as they exhibit remarkable biological activities and health benefits, including anti-inflammatory, antioxidant, and antibacterial properties (Higdon & Frei, 2003; Ali et al., 2023). However, the consumption of these foods generates a large amount of by-products (Campoy-Muñoz et al., 2021). In fact, it has been estimated that 90 million tons of food wastes are generated in the European Union annually from various food sources, which represent a severe threat to the environment (Tapia-Quirós et al., 2022). On the other hand, agri-byproducts were shown to possess immense biological activities, such as anticancer, antioxidant, anti-diabetic, and gastro protective, due to their rich content of bioactive compounds, including polyphenols (Saleh et al., 2017; Cheaib et al., 2018; Raafat et al., 2020; Selim et al., 2021). Date palm fruit (Phoenix dactylifera L.) is a commonly consumed fruit owing to its potential rich source of nutrients, fibers, vitamins (thiamin, riboflavin, ascorbic acid, niacin), minerals (potassium, calcium, magnesium), flavonols, anthocyanidins, phenolic acids, procyanidins, and tannins (Arjmandi et al., 2023). Dates can promote the health of the heart, brain and digestive tract, while preventing oxidative stress and chronic diseases like diabetes, hypertension, and cardiovascular illnesses (Al-Shahib & Marshall, 2009). It is among the most widely cultivated fruits in the Middle East and a well-known consumed staple food worldwide (Habib et al., 2014). The Food and Agriculture Organization (FAO) estimated the world production of dates to be around 9 million tons annually (“FAO Publ. Cat. 2022,” 2022). Contributing to 12% of the global date production, the United Arab Emirates (UAE) ranks as the fourth-largest date producer in the world (Al-Muaini et al., 2019). While examining the primary by-products left behind this vast production and consumption of date fruit, it is obvious that the pits are generated in significant amounts (M. A. Al-Farsi & Lee, 2008). According on the variety, date pits typically account for 6.11–11.47% of the total weight of the fruit (Habib and Ibrahim, 2011b). Date seeds have long been utilized as soil fertilizers and animal feed (Rehman et al., 2017). Recently, their application in the processing of functional foods and drinks, such as muffins, cakes, bread, biscuits, gluten-free cookies, cooking oil, and non-caffeinated coffee, as well as in cosmetics, medicine and human dietary supplements, has been recommended owing to their extensive phenolic content (M. Al-Farsi et al., 2007; Ghnimi et al., 2015; Bentrad & Gaceb-Terrak, 2020). In fact, research has correlated date seeds’ high levels of polyphenols to their health benefits and biological activities (Habib, El-Fakharany, Souka, et al., 2022; Swaidan et al., 2023). Date seeds nutritional content ranges from 75–81% carbohydrates, 6–12% oil, 5–10% moisture, 4–7% protein, and 1% ash (Bouhlali et al., 2017). Macro elements, including potassium (229–400 mg/100 g), magnesium (51–282 mg/100 g), phosphorus (68–83 mg/100 g), calcium (28–62 mg/100 g), and iron (2–4 mg/100 g), are present in considerable amounts as well (Attia et al., 2021). From date seeds, edible oil can be extracted, and the fatty acid composition is notably variable depending on the cultivar and conditions of the growth season. Lauric (C12:0) (10–20%), myristic (C14:0) (9–14%), palmitic (C16:0) (9–12%), and stearic (C18:0) (3–5%) are the most commonly identified saturated fatty acids. Additionally, oleic acid (C18:1) (41–50%) and linoleic acid (C18:2) (10–19%) are the two primary unsaturated fatty acids (Mrabet et al., 2020b). Nonetheless, date seeds’ polyphenol content has been extensively researched. It varies greatly depending on the variety, with an average of 51.1 g/kg (Majid et al., 2023c). Meanwhile, only a few studies have examined the phenolic profile of date seeds (Bai et al., 2013; Habib et al., 2014; Hilary et al., 2021; Majid et al., 2023a). Flavan-3-ols, which are mainly found in date seeds as polymeric proanthocyanidins, have been shown to be the predominant class of polyphenols, especially catechins and epicatechins, with concentrations ranging from 47.91 to 50.18 g/kg flavan-3-ols. In addition to flavan-3-ols, phenolic acids such as protocatechuic acid, p-hydroxybenzoic acid, and caffeoylshikimic acid appeared to exist in significant amounts (Habib et al., 2014). With all the provided data, date seeds consequently represent a potentially valuable source of bioactive compounds. As such, they may contribute to the management and/or prevention of chronic diseases. This claim has been supported through previous in-vivo research on humans and animals. For example, date seeds ingestion in rats was able to decrease tissue oxidation levels significantly and subsequently improve the antioxidant status (Habib and Ibrahim, 2011a). Furthermore, a study revealed that Arabic pita bread supplemented with date seeds and served with breakfast for human participants over a period of two weeks was able to boost the antioxidant activity as measured by glutathione (GSH) reduction (Platat et al., 2019). The high phenolic content of date seeds likely contributes to their ability to eliminate free radicals and mitigate reactive oxygen species (ROS), hence protecting cell components like DNA (Majid et al., 2023). Furthermore, recent scientific research has been oriented towards discovering natural antibacterial substances in response to the rise of antibiotic-resistant microorganisms. Due to their excellent source of bioactive compounds, mainly polyphenols, the antibacterial potential of various date pit varieties has been reported against Gram-positive as well as Gram-negative bacterial strains, with greater effect noticed against Gram-positive bacteria (Samad et al., 2016; Aljazy et al., 2019; Selim et al., 2021). According to these findings, date pits should no longer be considered waste products. Indeed, they represent an excellent candidate for inclusion in the human diet as a new functional element. To our knowledge, so far, the comprehensive phenolic profile and phenolic content, as well as the biological activities of date seeds aqueous extracts have not been thoroughly studied for the varieties included in this research. Due to the fact that date seeds composition can vary widely according to their variety, there is a need to conduct more research aiming to compare the phenolic and fatty acid compositions of date seeds so that their potential health benefits can be clearly understood. Therefore, the aim of the current study was to assess the phenolic composition of nine aqueous date seeds extracts originating from the UAE (Fard, Shishi, Lulu, Khalas, Whenetri, Naghal, Bumaan, Barhi, Reziz), as well as to examine and compare their biological properties, mainly antioxidant and antibacterial. To our knowledge, most research that examined date seeds used organic solvents such as methanol, ethanol, and acetone, or their combination with water for extraction and analysis of phenolic compounds, as well as the assessment of their biological activities. In this study, 100% distilled water was used as an eco-friendly and cheap alternative extraction solvent aiming to address the current gap, especially for the included nine varieties which are commonly produced and consumed in the UAE. Moreover, the research aims to identify, quantify, and compare the fatty acid content of date seeds oil.
2. Materials and methods
2.1. Sample preparation
Date seeds of nine varieties (Fard, Shishi, Lulu, Khalas, Whenetri, Naghal, Bumaan, Barhi, Reziz) originating from the UAE were assessed in this study. One kilogram of each type of date was bought from a wellknown commercial store in the UAE. The dates were selected randomly, with no consideration given to their size, color, or appearance. After pitting the dates, the obtained seeds were washed with potable water to get rid of any sticky flesh, and were further dried overnight at room temperature. The next day, date seeds were initially crushed up into small pieces with a hammer, then thoroughly ground using an electric blender. After allowing the powder to pass through a 0.5 mm sieve, the samples were frozen until required.
2.2. Reagents
All the reagents used in this study were of analytical grade and the water was distilled. ABTS●+ (2,2′-azino-bis (3-ethylbenzothiazoline-6- sulfonic acid) diammonium salt) (≥ 98%), diethyl ether, and tert-butyl methyl ether were purchased from Sigma-Aldrich (Steinheim, Germany). DPPH● (2,2-diphenyl-1-picrylhydrazyl) radical (95%), Trolox® (97%), were obtained from Thermo Fisher (Darmstadt, Germany). Acetonitrile (ACN; HPLC grade), methanol (MeOH; HPLC grade), potassium persulfate (≥ 99%) and HPLC standards were purchased from Carl Roth (Karlsruhe, Germany). For the antibacterial assay, DMSO (dimethylsulfoxide, Roth Art.Nr. 4720.4, ≥ 99.8 %), Trypton broth (8.5 g/L NaCl, 1 g/L Trypton), CASO agar (Roth, Art.Nr. X937.2), Penicillin G sodium salt (MP Biomedicals, Art.Nr. 100548) were used.
2.3. Solid-liquid extraction
Distilled water was used to extract polyphenols from date seeds in a ratio equivalent to 1:100 (w/v), meaning that for every 1 g of date seeds powder, 100 mL of distilled water was used. The extraction mixture was left on continuous stirring at room temperature using a magnetic stirrer. Multiple extractions were performed, with three repetitive extractions of 20 minutes each. In each extraction, the obtained extract was filtered, and the date seeds powder was collected and mixed with another 100 mL of distilled water. After that, all supernatants were mixed together and centrifuged at 6000 rpm for 10 minutes.
2.4. Lyophilization of date seeds extracts
For the lyophilization of date seeds extracts, each sample was split into four flasks and allowed to freeze for 24 hours at -20 ◦C. Then, the frozen flasks were attached directly to the ports of a manifold benchtop lyophilizer (TELSTAR® CRYODOS—50, Madrid, Spain) for a duration of 48 hours. After that, the lyophilized sample was collected and stored at -20 ◦C for further use.
2.5. Determination of the phenolic profile using HPLC-DAD-ESI-MS
Samples were prepared using the method outlined by Neugart et al. (2015) for the analysis of phenolic compounds by HPLC-MS analysis. For this, 600 μL of 60% aqueous methanol was used to extract 20 mg of the lyophilized materials, and the extraction was left for 1 h with constant stirring at room temperature. After that, the extract was centrifuged for 10 min at 19,000 × g and the supernatant was collected. After two further repetitions with 400 μL and 200 μL for 20 min and 10 min, respectively, all supernatants were mixed together and allowed to dry using a rotary evaporator. Following that, 200 μL of distilled water was used to dissolve the dried residue, and the produced extract was then filtered using centrifuge tube filters with a 0.22 μm cellulose acetate membrane (Corning®-Costar®-Spin-X®, Sigma Aldrich Chemical Co., St. Louis, MO). The HPLC analysis was carried out in accordance to the method described by Engelhardt et al. (2022). The HPLC system was supplied with a diode array detector (DAD), a CBM-20A communication bus module, a refrigerated SIL-20AC HT auto sampler, a DGU-20A5 degasser, an LC-20 AT liquid chromatograph quaternary pump, and a CTO-10AS VP column oven. A Supelco guard column (5.0 × 3.0 mm, 5 µm) and a Supelco Ascentis®Express F5 (150 × 3.0 mm, 5 µm) were also used, in addition to a 0.2-micron SST Frits for UltraLine (Restek, Bad Homburg, Germany). A temperature of 30 ◦C was chosen for the column. Eluent A (1% acetic acid) and Eluent B (100% acetonitrile) made up the mobile phase. The separation was according to the following gradient: 0–7 min, 5% B; 7–30 min, 5–20% B; 30–49.5 min, 20–90% B; 49.5–52 min, 90% B; 52–52.7 min, 90–5% B; 52.7–59 min, 5% B. The injection volume was 30 µL, and the flow rate was 0.3 mL/min. The quantification was done using standard calibration curves for the phenolic components. The limit of detection (LOD; factor 3.3) and quantification (LOQ; factor 10) were calculated using standard error of the intercepts and slopes of the calibration curves. In order to identify phenolic compounds, samples were analyzed in a negative ion mode using an Agilent 6460 HPLC-QQQ fitted with an electrospray ionization (ESI) interface, applied to perform mass spectrometry. The gas temperature was set to 350 ◦C, the nebulizer was set to 60 psi, the capillary voltage was set to +4000/− 4000 V, and the gas flow rate was set to 13 L/min. The dissociation was triggered by applying a fragmentation voltage of 135 V.
2.6. Determination of the antioxidant activity of date seeds using spectrophotometric assays
2.6.1. ABTS assay
The ABTS-radical scavenging activity was determined as described by Engelhardt et al. (2022) and was expressed as Trolox equivalent. For the preparation of the stock solution, 9.6 mg of ABTS and 1.66 mg of potassium persulfate were filled up to 25 mL with distilled water. To allow for complete radicalization, the solution was incubated for 12 to 16 h at room temperature in the dark. 5 mL of the stock solution were diluted with 100% methanol to prepare 25 mL of the working solution. After that, 150 µL of the working solution was added to 10 µL of sample and the mixture was incubated for 5 min. Using a UV–Vis spectrophotometer, the absorbance of the samples was measured at 734 nm against a 100% methanol blank solution, after 1 min of medium-speed orbital shaking and 1 min of resting of the samples. The control was prepared following the same procedure, except by using 10 µL distilled water instead of date seeds extract. A standard curve with concentrations ranging between 0.025 and 0.7 mM Trolox scavenging activity (R2> 0.99) was prepared and the results were expressed as Trolox Equivalents [mmol TE/g sample (dry weight)] according to the following equation: TEAC = C × V × DF /m Where C is the concentration obtained from Trolox calibration curve (mmoL/mL), V is the total volume of the sample prepared (mL), DF is the dilution factor, and m is the weight of dry sample (g).
2.6.2. DPPH assay
The DPPH radical scavenging activity of the nine date seeds was assessed in-vitro in accordance with the method described by Engelhardt et al. (2022). The method can be described as follows: 7.88 mg of DPPH were dissolved in 100 mL of 100% methanol, then left for two hours in the dark at room temperature for incubation. Subsequently, 180 µL of the prepared DPPH solution was added to 20 µL of each of the nine date seeds extract, and the mixture was left for 28 min at room temperature, while wrapped with aluminum foil to ensure darkness. After 1 min of medium-speed orbital shaking, followed by another minute of delay, the absorbance was measured at 515 nm. The blank was composed of 100% methanol, while the control was prepared with the same procedure, but with 20 µL of distilled water instead of date seeds extract. A standard curve with concentrations ranging between 0.025 and 0.7 mM Trolox was prepared (R2> 0.99) and the results were reported as Trolox Equivalents [mmol TE/g sample (dry weight)] according to the same equation used for ABTS assay: TEAC = C × V × DF /m
2.7. Determination of the antibacterial activity of date seeds using the agar well diffusion method
The antibacterial activity of date seeds was assessed against two bacterial strains: Listeria monocytogenes (DSM 20,600) and Escherichia coli (DSM 498), which were obtained from the Leibniz-Institut DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen). L. monocytogenes is a Gram-positive bacterium, while E. coli is a Gramnegative bacterium. The agar well diffusion test was applied according to Behera et al. (2017) which can be described as follows:
2.7.1. Preparation of date seeds samples
Samples were prepared by dissolving lyophilized date seeds in DMSO (≥ 99.8 %) to reach a final concentration of 60 mg/mL. The extracts were then placed in the ultrasonic bath for 3–4 min for complete dissolving.
2.7.2. Preparation of bacterial inocula
For evaluating the antimicrobial activity, fresh subcultures of E. coli and L. monocytogenes bacterial strains were suspended in Trypton broth (8.5 g/L NaCl, 1 g/L Trypton) to adjust the absorbance of the bacterial suspension between 0.1and 0.2 at a wavelength of 600 nm. The suspensions were diluted with Trypton broth (1:100). The inoculum must have a germ density of 1 × 106 to 5 × 106 cfu/mL.
2.7.3. Assessment of antibacterial activity
The inoculum was spread evenly onto a Petri dish containing CASO agar (Roth, Art.Nr.X937.2) using sterile swabs and allowed to soak into the culture medium for 5 to 10 min before punching the wells (5 mm deep, diameter 8 mm). After that, each well of the inoculated plates was filled with 100 µL of the sample. The extracts were allowed to diffuse into the agar for 10 minutes at room temperature before incubation. After the incubation period (E. coli 18 ± 2 h, 36 ± 1 ◦C; L. monocytogenes 48 ± 2 h, 36 ± 1 ◦C), the plates were observed. A clear inhibition zone surrounding the well containing the date seeds extract indicated an antibacterial activity. The edges of the growth, which is clearly reduced in colony size, served as the borders. A measuring scale in millimeters (mm) was used to measure the inhibition zone. Penicillin G sodium salt and DMSO were used as the positive and negative controls, respectively.
2.8. Extraction of date seeds oil using soxhlet extraction
A cellulose thimble, about 15 mm × 85 mm, was placed into a Soxhlet extractor and filled with about 8 g of date seed powder. Diethyl ether was the extraction solvent, and 150 mL of it was transferred to the extractor connected to a condenser. After that, the entire assembly was placed on the temperature-controlled heater to attain and regulate the required temperature (60–70 ◦C). The extraction was stopped after 6 h, and date seeds oil was removed from the mixture under vacuum by rotary evaporation at 40 ◦C and 30 rpm. The oil mass was calculated by subtracting the weight of the empty flask from the weight of the flask containing the oil (de Souza et al., 2020). The following formula was used to calculate the oil yield (%): Oil Yield (%) = Mass of extracted oil Mass of solid date seeds used for extraction × 100
2.9. Determination of the fatty acid profile by GCMS– analysis
The fatty acid profile of the extracted date seeds oil was assessed using the GC–MS. In order to create fatty acid methyl esters, the sample was added to tert-butyl methyl ether in a 1:1 ratio. In split mode, the injection volume was 5 µL, with a ratio of 1:10. A GC-2010 Plus (Shimadzu, Duisburg, Germany) fitted with a FID (flame ionization detector) was used for the analysis at 250 ◦C. The carrier gas, helium, had a column flow rate of 1.24 mL/min. The temperature was held at 35 ◦C for 5 min, then increased to 210 ◦C (5 ◦C/min). The final temperature was held for 20 min. The SH-Stabilwax column with an ID of 0.25 mm, a length of 30 m, and a thickness of 0.25 µm was used to separate the compounds.
2.10. Statistical analysis
Each experiment was carried out in triplicate. The data was presented as mean ± standard deviation. SPSS for Windows (version 20) was used to carry out the statistical analysis. One-way analysis of variance (ANOVA) was applied to evaluate the data, along with Tukey’s test for multiple comparisons. The results were deemed significantly different when the p-value was less than 0.05.
3. Results and discussion
3.1. Phenolic composition
It is crucial to examine the phenolic profile of date seeds, especially with the lack of data available concerning the detailed phenolic composition of the aqueous extracts of date seeds varieties included in this research. For this, HPLC-DAD-ESI-MS was utilized, where the detection of the phenolic compounds was based on the retention time, whereas peak areas were mainly used for quantification. Table 1 presents the phenolic derivatives that were identified and quantified in the nine date pits (dry weight basis). The results revealed the presence of various phenol derivatives, e.g., proanthocyanidin, catechin, caffeic acid, and quercetin, eluting throughout the profile with varying concentrations according to the pits’ cultivar. Flavan-3-ols appeared to be the major family, which is in agreement with the available literature (Habib et al., 2014; Majid et al., 2023a). In addition, all the identified phenolic compounds presented in Table 1 were shown to be present in date seeds, as reported by previous investigations (Benmeddour et al., 2013; Platat & M Habib, 2014; Majid et al., 2023a). Overall, the sum of the obtained phenolic fractions was calculated and the values varied among varieties, ranging between 1.05 and 1.24 mg/g DM for each of Reziz and Naghal, respectively (p-value <0.05). The two predominant phenolic compounds appeared to be caffeic acids and proanthocyanidin trimers, with average concentrations of 0.58 and 0.28 mg/g DM, respectively. These were followed by proanthocyanidin dimers (0.10 mg/g), proanthocyanidin tetramers (0.09 mg/g), and catechin (0.04 mg/g). On the other hand, quercetin, with an average concentration of 0.03 mg/g DM, was the least prevalent phenolic compound. Our findings were consistent with those reported by another study which found out that proanthocyanidin trimers were the most abundant among 15 detected phenolic compounds, with a concentration of 0.613 mg/g, followed by caffeic acid (0.388 mg/g). Similar to our results, the determined amount of quercetin (0.03 mg/g), which appeared to be the least abundant phenolic component in Habib’s investigation as well, was found to be 0.034 mg/g (Habib et al., 2014). In addition, the evaluation of phenolic content revealed that total proanthocyanidin dimer concentration ranged between 0.09 mg/g (Barhi) and 0.12 mg/g (Fard and Shishi), with no significant difference. The concentration of total proanthocyanidin trimer ranged between 0.24 mg/g (Bumaan and Reziz) and 0.36 mg/g (Naghal), where p-value <0.05 showed a significant difference. It is important to note that several factors, such as climate conditions, sunlight exposure, irrigation, soil fertility, harvest time, season, maturity, storage time, and humidity, can play a crucial role in the overall composition of date seeds, including the phenolic composition (Selim et al., 2021). The analysis of total proanthocyanidin tetramer showed that its average concentration among the nine date pits was around 0.08 to 0.1 mg/g, without any significant difference. All of these values were lower than those found in previous research assessing the phenolic compounds in date seeds (Habib et al., 2014; Majid et al., 2023b). Catechin ranged between 0.04 and 0.05 mg/g, with no significant difference among varieties as well. This outcome seemed to be correlated with a study that focused on quantifying catechin concentration for the same varieties, which was found to be around 0.05 mg/g on average (Habib et al., 2014). On the other hand, the phenolic profile of three Pakistani date pits revealed an average catechin level of 1.31 mg/g, indicating that the phenolic content varies significantly depending on the seed source and cultivar (Majid et al., 2023a). As for the total caffeic acid, Reziz had the lowest value (0.56 mg/g), whereas Fard, Shishi, and Naghal had the highest value (0.6 mg/g), with great significant difference (p-value 0.01). Quercetin concentration appeared to be the same among all the different varieties (0.03 mg/g).
3.2. Antioxidant activity
Considering that natural antioxidant characteristics may be possessed by phenolic compounds, it was interesting to assess and compare the ability of date seeds to neutralize free radicals. For this, DPPH and ABTS assays were applied and results are expressed in Table 2 as Trolox equivalents (mmol TE/g sample DW). In general, date seeds extracts appeared to possess powerful antioxidant activities that varied considerably between all varieties (p-value <0.001). On average, the antioxidant activities obtained from ABTS and DPPH were compared and showed no significant difference between the two assays. This strong correlation was further confirmed with a correlation coefficient (R2 = 0.9935) (Fig. 1). Shishi and Naghal exhibited the highest DPPH inhibition (%) with TEAC values of 0.28 and 0.27 mmol TE/g DW, respectively. In contrast, Barhi and Whenetri showed the lowest TEAC (0.05 and 0.09 mmol TE/g sample DW). The results seemed similar in ABTS assay, where Naghal had the highest TEAC value (0.24 mmol TE/g sample DW), whereas Barhi had the lowest value (0.05 mmol TE/g sample DW). Naghal with the highest phenolic content exhibited the highest antiradical capacity in both assays, while Barhi, with the least phenolic compounds, showed the weakest antioxidant activity. This correlation between the phenolic content and antioxidant activity of date seeds was reported by various studies (Hamada et al., 2002; Habib et al., 2014; Radfar et al., 2019; Majid et al., 2023a). Our findings are consistent with those of the Platat & M Habib (2014) study which indicated that Barhi had the lowest antioxidant effect (0.012 mg/g) among 18 different date pits varieties. Furthermore, Naghal antioxidant capability was assessed in a study comparing between 22 date seeds and revealed a TEAC value of 0.36 mg/g vitamin C equivalent, which is similar to our obtained result for Naghal TEAC in ABTS assay (0.34 mg/g). In contrast, our results obtained for the antioxidant properties of the other varieties DM: dry matter, values are the mean ± standard error of two samples. Mean ± SD followed by the same letter, within a row, are not significantly different (p-value> 0.05). appeared to be significantly different from the findings of prior studies on the same varieties (Platat & M Habib, 2014; Rehman et al., 2017). Along with the differences in cultivar, the aforementioned factors that contribute to differences in the phenolic composition of date seeds can thereby affect their biological activity, including their antioxidant potential. To our knowledge, so far, Whenetri cultivar has not been included in previous research evaluating the phenolic content and antioxidant capacities of date seed cultivars. Nonetheless, it appears that more than one phenolic compound is involved in the reported antioxidant activities. For example, Naghal, with the highest antiradical property, seemed to have the highest level of caffeic acid (0.6 mg/g). However, Barhi and Whenetri, which both had the lowest antiradical activity, had the lowest caffeic acid concentration (0.56 mg/g). This may suggest the possible role of caffeic acid specifically in the reported antioxidant activity. Through an iron chelating mechanism and by restricting Fenton-induced oxidative damage, which is a redox reaction that generates a high amount of free hydroxyl radicals leading to cellular damage, caffeic acid is well known for its powerful antioxidant property (Gülçin, 2006; H. M. Habib, El-Fakharany, Kheadr, et al., 2022; Purushothaman et al., 2022).
3.3. Antibacterial activity
Table 3 shows the antibacterial activity of the nine date seed extracts against E. coli (Gram-negative) and L. monocytogenes (Gram-positive). Overall, all date seeds extracts exhibited significant antibacterial activities towards both strains, with stronger activity observed against the Gram-positive bacteria (L. monocytogenes) than Gram-negative (E. coli), with average zone of inhibition diameters of 22.0 and 17.5 mm, respectively. In the case of L. monocytogenes, the significantly higher TE: Trolox Equivalent, DW: dry weight Values with different alphabetical letters appeared significantly different (p-value <0.05). reduction in bacteria was seen within the zone of inhibition, where the bacterial count is close to zero at the edge of the well. Fard and Shishi had the greatest inhibitory effect against L. monocytogenes (24 mm), whereas Bumaan and Barhi had the least (20 mm). For E. coli, zone of inhibition diameters ranged between 17 and 18 mm for all varieties. According to ALrajhi et al. (2019) well diffusion method, the antibacterial activity of date seeds was classified according to four categories based on the zone of inhibition. Low antibacterial activity of date seeds was considered when zone of inhibition is ≤10 mm. Zone of inhibition >10 to 15 mm was considered a moderate activity, >15 to 20 mm was classified as strong antibacterial activity, whereas zone >20 mm was considered extremely strong. The comparison of our results to these classifications reveals strong antibacterial properties of the tested date seeds against E. coli and extremely strong antibacterial activity of almost all the varieties against L. monocytogenes . Although date pits have been found to exhibit strong antimicrobial effects, the greater effect shown in Gram-positive bacterium (L. monocytogenes) is due to a factor related to the bacterium itself. Variations in the susceptibility of bacterial strains can be attributed to differences in the bacterial strain’s cell wall structure. Generally, the antibacterial strength of phenolic compounds corresponds with the potential to cross the layer of gram-positive bacteria and interact with their peptidoglycan layer, thereby weakening cell integrity and resulting in inhibitory and/or bactericidal effects depending on the sensitivity of the bacterial strain to ionic strength and osmotic pressure. On the contrary, the outer membrane of Gram-negative bacteria contains an extra lipopolysaccharide layer which acts as a strong barrier, preventing polyphenols from binding to the peptidoglycan layer (Papuc et al., 2017). The results of the antibacterial activities of the nine date seeds could not be compared to the available literature as none of the varieties had been previously included in antimicrobial research of date seeds using distilled water as an extraction solvent and using the well method to assess the antibacterial properties. The existing data shows a wide range of antimicrobial activity of date seeds based on the bacterial strain as pointed out above, the origin and cultivar of date pits, as well as the extraction method and the antibacterial assay used. Actually, the complexity of the phenolic content has profoundly influenced biological activities, primarily antiradical and antibacterial, and is responsible for all of the variations reported between varieties, even those from the same origin. Several abiotic factors like those previously mentioned as well as extraction methodologies and experimental conditions could all contribute to the high variability of phenolic compounds among date seeds varieties (M. Al-Farsi et al., 2007; Mohamed Lemine et al., 2014).
3.4. Oil Yield and Fatty Acids Profile
Table 4 shows the composition of the nine date seeds oils extracted using the Soxhlet method and analyzed using GC–MS. The highest yield was obtained by Reziz (8.63 %), whereas Khalas yielded the lowest oil (4.69%). As can be seen in Table 4, values of total fats (g/100 g) of date seeds are very similar to the values of oil yield % and follow almost the same trend line. Overall, the two most abundant fatty acids that appeared in the nine tested date seeds oil are oleic acid and lauric acid, with average concentrations of 1.95 g/100 g and 1.63 g/100 g. In contrast, stearic acid is the least present, with an average of 0.6 g/100 g. Only a few studies have examined the fatty acid profile of date seeds, and none of the nine varieties have been previously investigated. A previous study, which indicated the presence of both saturated and unsaturated fatty acids, with oleic acid being the predominant among all fatty acids followed by lauric acid, confirms the results of our study Mrabet et al., 2020a. Around half of the fat in the majority of the date seeds was found to be oleic acid (Biglar et al., 2012). Interestingly, various studies have been conducted on oleic and lauric acids to assess their health benefits, which were found to be impressive. Both fatty acids have been found to decrease LDL cholesterol levels and scavenge free radicals, therefore lowering the risk of chronic diseases such as cardiovascular and inflammatory diseases (Cho et al., 2010; Elagbar et al., 2016; Alfhili & Aljuraiban, 2021). In addition, oleic acid has been shown to possess significant antimicrobial activity, through specifically inhibiting the growth of various Gram-positive bacterial species. Furthermore, oleic and lauric acid-rich oil was found to be highly stable (Dilika et al., 2000). Results are reported as mean ±SD DMSO: Dimethyl sulfoxide Values with different alphabetical letters appeared significantly different (p-value <0.05).
4. Conclusion
Due to their proven antioxidant and antibacterial potentials, date seeds rich in polyphenols could represent an ideal candidate for future pharmaceutical applications or functional foods to prevent and treat illnesses, along with improving human health. However, it has been shown that this potential differed according to the variety of date seeds, along with other environmental abiotic factors, which reflects the diversity reported in the phenolic profiles. Furthermore, date seeds oil appeared to include a variety of fatty acids, primarily oleic acid. The comprehensive comparison performed in this study is especially crucial when deciding on date seed varieties, whether to be used separately or in combination, to achieve a specific health goal in the manufacturing of functional foods. There is a need to conduct more research aiming to assess the safety of date seeds before producing valuable products from them, as well as to investigate the synergic effects of combinations of varieties. Ethical statement The study respects ethical statement and was not performed on animal or human. CRediT authorship contribution statement Tareq M. Osaili: Writing – review & editing, Supervision, Funding acquisition, Data curation, Conceptualization. Aseel Swaidan: Writing – original draft. Anas Al-Nabulsi: Writing – review & editing, Conceptualization. Amin Olaimat: Writing – review & editing. Susanne Neugart: Writing – review & editing. Layla Engelhardt: Writing – review & editing. Tuba Esatbeyoglu: Writing – review & editing. Mutamed Ayyash: Writing – review & editing. Leila Cheikh Ismail: Writing – review & editing, Conceptualization. Maher M Al-Dabbas: Writing – review & editing. Reyad S. Obaid: Writing – review & editing. Nada El Darra: Writing – review & editing, Supervision, Data curation. Declaration of competing interest The authors declare no conflicts of interest. Data availability No data was used for the research described in the article.
Acknowledgments
The authors thank the University of Sharjah for funding the project and Al Foah (Al Ain, UAE) for providing the samples. The authors thank Claudia Helle for her excellent technical support. This publication is partially funded by the Arab-German Young Academy of Sciences and Humanities (AGYA). AGYA draws on support from the German Federal Ministry of Education and Research (BMBF; grant no. 01DL20003). Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.afres.2024.100493.
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