Introduction
The date palm (Phoenix dactylifera L.) is mainly grown in arid and semi-arid regions of the world. It is popular in countries found in South Asia, America and North Africa. The global production of dates reached up to 8.5 million metric tons in 2016 with approximately 1 million metric tons of date seeds. Pakistan ranks sixth among different date producing countries.1 The fruit consists of a fleshy pericarp and the seeds comprise 10–15% of total weight of the fruit. The date seeds remain a problem for the date processing industry after manufacturing of pitted date cakes, confectionary and syrups. Ground seeds are sometimes used as base gravel on dirty roads. In Middle and South East countries, seeds are occasionally used in animal feed.2 Date seed supplemented feeds showed increased weight gain in broilers compared to controls.3 Other studies also report a positive supplemental effect on animal diet, suggesting the presence of nutritionally important constituents in seeds.4 Date seeds are a rich source of fiber compared to wheat bran and oats.5 Phytochemicals with promising health promoting potential, including phenolic compounds, phytosterols, tocopherols and squalene, have also been found in date syrup,6 grape seeds,7 blackcurrant seeds,8 mango pits9 and citrus seeds.10,11
Hence, date seeds could be a rich source of antioxidants. In vitro and in vivo studies have shown that consumption of date seeds reduces diabetes, dyslipidemia and hypertension, and obesity.12 These effects might be caused by specific components, including protein, fibers and bioactive compounds. Among all bioactive compounds, phenolic compounds have been reported to have antioxidant, anti-carcinogenic, anti-microbial, anti-mutagenic and anti-inflammatory characteristics.13,14
Previous studies have revealed the correlation between total phenolic content (TPC) and antioxidant capacity.15 Synthetic antioxidants are expensive, possibly toxic, and more immunogenic than naturally occurring phenolics. Omani date seeds showed higher TPC content and antioxidant capacity than other date seed varieties.16 However, identification and quantification of individual phenolic compounds and the antioxidant capacity of Pakistani date seeds have not been reported. Date seeds may have extractable high value-added nutritional components that could be included in functional foods. The main objectives of this study were to identify and quantity the phenolic components and to investigate antioxidant capacity using different assays.
Experimental
Chemicals
Folin–Ciocalteu’s phenol reagent, phenolic standards (caffeic acid, gallic acid, catechin hydrate, sinapic acid, chlorogenic acid, vanillic acid, p-coumaric acid and epicatechin), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,20-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), 2,20-azobis (2-amidinopropane) dihydrochloride were purchased from Fluka (Fisher Scientific, UK). All chemicals and reagents were of analytical grade purchased from Sigma-Aldrich (Dorset, UK) unless otherwise stated.
Plant material
Three types of commonly used ripe date varieties (Karbalaen, Aseel and Khuprowere) were purchased from a local market of Khairpur, Sindh. All samples were carefully checked according to their physical parameters (size, shape and weight) and stored in the refrigerator at 4°C before analysis.
Physical Parameters
A random selection of 20 date fruits from each variety was made. Chosen samples were clean and in good condition. An electrical balance was used to calculate the weight of the dates and seeds. Seed measurements (length (L), width (W), and thickness (T)) were measured with a digital calliper to an accuracy of 0.1 mm. Percent Mass was calculated by using following formula:
Mass of date seed (%)=WDSWD×100
where, WDS: weight of date seeds, WD: weight of dates.Extraction and quantification of phenolic compounds through high performance liquid chromatography
Total phenolics were extracted using a methanol water (4:1) mixture. Briefly, 2 g of ground seeds were transferred to 30 ml of solvent mixture. The extraction was carried in glass tubes, placed in orbital shaker for 4 hours and solution was then centrifuged at 2,000 g for 20 minutes. The supernatant was carefully transferred to brown glass vials to avoid light interference and was kept at −4 °C for further analysis on high performance liquid chromatography (HPLC) and antioxidants activities. Stock standard solutions of each standard (gallic acid, catechin hydrate, chlorogenic acid, epicatechin, caffeic acid, vanillic acid, sinapic acid and p-Coumaric acid) were individually prepared in methanol.
A combined standard solution was also prepared from each standard. Five different concentration standards, from 4 µg/mL to 20 µg/mL, were prepared. Phenolic compound profile was determined using a HPLC-UV (Dionex, Sunnyvale, CA) system. Separation was performed with slight modification from ACE column (250 mm, 4.6 mm, DV10-2950, UK).17 Injection volume was 20 µL. Detection was monitored over a range of 200–400 nm by a UV Detector (MWD-3000/8018650, UK).
After optimizing the proper conditions for the mobile phase, multi-gradient analysis was selected. Multi-gradient mobile phase flow was set as: 10–15% (A), 90–15% (B) for the first eight minutes, 15–20% (A), 85–80% (B) from 8 to 25 minutes and 20–25% (A), 80–75% (B) from 25 to 60 minutes. Similarly, flow rate was gradually increased from 0.3 mL/min, 0.5 mL/min and 0.6 mL/min, respectively. Data analysis was performed with Chromeleon 6.8 software (Dionex, Sunnyvale, CA).
Total phenolic content
The TPC of date seed extracts was measured with Folin-Ciocalteu’s phenol reagent.18 The absorbance of the reaction mixture was monitored at 725 nm (Jenway 6715, Spectrophotometer, UK). The TPC of each variety of date seeds was expressed in gallic acid equivalents (GAE) per gram.
Antioxidant capacity
DPPH radical scavenging assay
The DPPH assay was performed using 50 µL of date seed extract vortexed with 2 mL of 0.9 mM DPPH in methanolic solution. The mixture was kept in absence of light for an hour then the absorbance was recorded at 517 nm. The half-maximal concentration (EC50) was measured; EC50 was defined as the concentration of seed extracts required to scavenge 50% of initial DPPH. This was calculated from results plotted as absorbance versus concentration of seed extracts.19
Ferric-reducing antioxidant power assay
Ferric-reducing antioxidant power assay (FRAP) of each date seed extract was measured using reagent prepared with acetate buffer (100 mM, pH 3.5), 2,4,6-tripyridyl-s-triazine (TPTZ) (10 mM in 50 mM HCl) and ferric chloride (25 mM) in a ratio of 10:1:1. 3 mL of this reagent was then added to 75 µL of seed extract and 300 µL of deionized water. The mixture was incubated at 37°C for an hour. The absorbance was recorded at 593 nm for each mixture. The FRAP assay results were expressed in µM Fe2+ equivalents per gram.12
2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) antiradical assay
2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) reagent was prepared as per,20 this method involves the reaction of ABTS (10 mM) and potassium persulfate (2 mM) in absence of light for 24 h. The prepared 2 mL of ABTS solution and 20 µL of date seed extract were mixed. The absorbance was recorded at 734 nm. Similarly, all samples with Trolox standards were incubated. The results were expressed in Trolox Equivalents (TE) per gram.21
Oxygen radical absorbance capacity assay
Oxygen radical absorbance capacity (ORAC) assay was performed for determination of antioxidant capacity. 6-carboxyfluorescein was used as the fluorescence probe, while 2,20-azobis (2-amidinopropane) dihydrochloride as the free radical generator. Fluorescence was measured in a 96 well-plate using a BMGLabtech (Ortenburg, Germany) microplate reader. Data was recorded after every 2 minutes for 2 hours at 37 °C, excitation and emission wavelengths were 485 nm and 520 nm, respectively. Antioxidant capacity of both samples was expressed as TE per gram.22
Statistical analysis
Three replications of each experiment were performed. The results were expressed in mean values ± standard deviations. The antioxidant capacity results were subjected to test the analysis of varience (ANOVA) and least significant difference test using SPSS V25 software. A p < 0.05 value was considered significant. The Pearson correlation coefficients among TPC, individual phenolic and antioxidant capacity assays of date seeds of three varieties were calculated using Pearson’s test.
Results and discussion
Physical parameters
Date seeds showed physical differences in length, diameter, mass and total weight of the three varieties (Table 1). These characteristics differed depending on the variety, where seed length was highest for Karbalaen, followed by Aseel and Khupro. The diameter for whole date seed changed from 22.35 ± 0.87 mm (Karbalaen), 20.42 ± 0.21 mm (Aseel) and 18.36 ± 0.56 mm (Khupro). The relative percentage weights of Karbalaen, Aseel, and Khupro seeds were 13.5%, 10.7%, and 12.3%, respectively.
Table 1Physical parameters of three different date seeds varieties
| Variety | Karbalaen | Aseel | Khupro |
|---|
| Length (mm) | 92 ± 0.5 | 90.2 ± 0.24 | 90.2 ± 2.34 |
| Diameter (mm) | 22.35 ± 0.87 | 20.42 ± 0.21 | 18.36 ± 0.56 |
| Mass of date seed (%) | 7.06 ± 0.65 | 7.21 ± 0.36 | 8.05 ± 0.8 |
| Weights of seeds | 10.703 ± 0.62 | 12.32 ± 0.42 | 13.5 ± 0.21 |
Total phenolic content
The TPC of three date seed varieties ranged from 33.6 mg to 44.2 mg GAE/g (Table 2). There was a significant variance (p < 0.05) between all three varieties (Aseel, Karbalaen and Khupro). Thouri et al. found the TPC of Tunisian date seeds (values) expressed as catechin equivalents per weight of seeds.24 It should be noted that TPC values of the date varieties presented in our study are two to three-fold higher than those reported for date syrup.23
Table 2Total phenolic content and comparative antioxidant capacity of date seeds as determined by the ABTS, ORAC and FRAP assays
| Varieties | TPC (mg GAE/g) | ABTS (µM TE/g) | ORAC (µM TE/g) | FRAP (µM Fe2+/g) |
|---|
| Karbalaen | 41.3 ±1.3a | 72.4 ± 38b | 21.26 ± 24a | 14.3 ± 57a |
| Aseel | 44.2 ± 0.7a | 66.71 ± 7b | 23.32 ± 51c | 12.1 ± 34c |
| Khupro | 33.6 ± 1.1b | 41.9 ± 4c | 12.43 ± 87c | 11.8 ± 62b |
| Genotype mean | 39.7 ± 5.47 | 60.3 ± 16.2 | 19 ± 5.78 | 12.73 ± 1.36 |
Identification and quantification of phenolic compounds
HPLC chromatogram of Karbalaen and Aseel seed extract depicted eight dominant peaks (1–8) in (Fig. 1). The maximum wavelength, λmax, of compounds (1–8) is presented in Table 3. Aseel seeds showed the highest amount of total phenolics. Individual phenolic compounds detected are also shown in Table 3. Gallic acid quantity in Karbalaen, Aseel and Khupro seeds was 246 mg/kg, 210.42 mg/kg and 517 mg/kg, respectively with retention time (tR) at 3.4 min. The tR of caffeic acid was 8.8 min and was present in the lowest quantity 1.62 mg/kg, 4.69 mg/kg and 4.11 mg/kg in Aseel, Karbalaen and Khupro seed respectively. Sinapic acid was found only in Aseel (36.6 mg/kg) and Karbalaen (39.31 mg/kg) varieties with retention times at 15.3 min and 15.8 min, respectively. Sinapic acid was not detected in Khupro. The tR is shorter in Aseel than that of Karbalaen and its standard suggests that the two varieties might contain different glycosides of sinapic acid. Ethyl gallate and penta-ogalloyl-β-D-glucose could not be detected in any of the three tested varieties. However, presesence of various biofunctional components in date has been reviewed by Wadhwa et al.25 p-Coumeric acid (14.25 mg/kg and 5.33 mg/kg), epicatechin (6.98 mg/kg and 38.68 mg/kg) and vanillic acid (4.64 mg/kg and 11.52 mg/kg) were measured in Aseel and Karbalaen seeds, respectively, but not in Khupro. Among eight phenolics detected from Karbalaen seeds, two are derivatives of hydroxyl benzoic acid (gallic acid and vanillic acid) and four are cinnamic acid derivatives (caffeic acid, p-coumaric acid, synapic acid, and chlorogenic acid).
Table 3Identification and quantification of individual phenolic compounds
| Peak No. | tR (min) | Standards (R2) | λmax (nm) | Phenolics | Concentration (mg/kg)
|
|---|
| Aseel | Karbalaen | Khupro |
|---|
| 1 | 3.4 | 0.994 | 280 | Gallic acid | 210.4 | 246.08 | 572.46 |
| 2 | 4.9 | 0.998 | 280 | Catechin hydrate | 109.1 | 178.4 | 108.48 |
| 3 | 5.8 | 0.998 | 280 | Chlorogenic acid | 16.86 | 45.09 | 16.49 |
| 4 | 7.7 | 0.996 | 280 | Epicatechin | 6.98 | 38.68 | 39.07 |
| 5 | 8.9 | 0.997 | 320 | Caffeic acid | 1.62 | 4.69 | 4.11 |
| 6 | 14.1 | 0.997 | 260 | Vanillic acid | 4.64 | 11.61 | 8.98 |
| 7 | 15.3 | 0.996 | 320 | Sinapic acid | 36.65 | 39.31 | ND |
| 8 | 18.5 | 0.998 | 320 | p-Coumaric acid | 14.25 | 5.33 | 5.68 |
Catechin and epicatechin are part of the flavan-3-ol family of flavonoids. p-coumeric acid and its glycosides have been found in mango, apple and avocado fruit seeds.26 The compounds with glycosidic moieties, the use of non-glycodic or even single glycosidic residue can compromise the authentication of obtained results.27 The two hydroxyl benzoic acid derivatives were present predominantly in the Aseel, Karbalaen and Khupro seed extract, however their quantity was statistically different (p < 0.05) from each other. Hydroxycinnamic acid derivatives were the most abundant phenolic compounds in Aseel, Karbalaen and Khupro seed extracts, consistent with the results for lupin seed extracts.27 In contrast, they found flavones as predominant class of phenolic compounds in Lupinus angustifolius L. seeds, where hydroxycinnamic acids constituted only 5% of the total phenolic compounds.27 El-Rahman et al. found a lower amount of total phenolic compounds from L. albus seeds compared to the values obtained in this study.28 The abundance of catechin in date seed varieties is worth emphasizing as this type of bound phenolic compound is not usually available in most dietary seeds. Many reports have been published that link the health beneficial effects of the aforementioned phenolic compounds to anti-inflammatory, antioxidant, anticancer, hypoglycemic, anti-aging, and cholesterlemic activities.29
Antioxidant capacity of date seeds
The free radical scavenging activity of date seed extracts against ABTS (TE/g) is shown in Table 2. Aseel seed extracts showed a lower activity (66 mM TE/g) compared to Karbalaen 72 mM TE/g) and Khupro (41.9 mM TE/g). The results of the present study are much higher than previously reported antioxidant studies in dates.30,31 The free radical scavenging activity values against ABTS fell within the range reported for different leguminous extracts, including fava bean and black bean extracts (1.5 – 1.75 mM TE/g), and similar values for navy bean extracts (27–45 mM TE/g).32 Kidney bean and soya bean extract showed comparable antiradical activity against ABTH to date seed extracts.33
The DPPH antiradical activity of P. dactylifera L. kernel extracts are shown in Figure 2. A significant variation (p < 0.05) in the values of three date varieties was observed. Antioxidant capacity decreased in the order of Aseel > Karbalaen > Khupro. The EC50 of Aseel was twofold higher than Khupro. The variation in DPPH assay for date syrup of different varieties has previously been reported.34 The average DPPH antiradical activity of various Omani date species had slightly lower values than Irani date species.35 The EC50 values of date seeds are higher compared to TPC. In turn, mango seed extracts had similar TPC, and showed higher DPPH assay values. This phenomenon could be related to the chemical configuration of phenolic compounds, as bound phenolic might interfere with reaction of Folin-Ciocalteu’s reagent and phenols provide inflated TPC values.36 In addition, in bound phenolic structures, electron donating hydroxyl groups could be blocked and participate in scavenging assay. As a result, bound phenolics usually have lower scavenging potential than free phenolics.
The antioxidant capacity of date seeds extracts to reduce the TPTZ-Fe3+ complex to TPTZ-Fe2+ is shown in Table 2. The highest FRAP was found for Karbalaen seeds extracts (14.3 µM Fe2+/g), Aseel (12.1 µM Fe2+/g) and Khupro (11.8 µM Fe2+/g). Antioxidant capacity using the ORAC assay measures the total reaction between reactive species and presence of oxidants. Antioxidant capacity for Aseel, Karbalaen and Khupro were 23.26 µM TE/g, 21.32 µM TE/g and 12.43 µM TE/g, respectively. These values are considerably higher than Omani and Irani date seeds extract.37 However, ORAC values were higher compared to the edible part of dates. Al-Farsi et al. reported flesh part values between 1.13–1.75 µM GAE/g−1 for Omani dates and these were in agreement with Iranian Kharak dates.23 Antioxidant capacities of date flesh obtained by Saleh et al. were 5.7–6.6 GAE/g,16 which were higher than those published in.38 Mansouri et al. have reported the positive correlation of the antioxidant capacity and phenolic profile in date fruit from Algeria.39
Antioxidant capacity is mainly attributed to the water-soluble component’s potential radical scavenging capacity such as cinnamic acid, vinallic acid, and cumeric acid. Many factors such as temperature, soil conditions, maturity, geographic location and post harvesting treatment might be contributing to the differences in phenolic profiles and related antioxidant capacity. Fig, prune and raisin seeds antioxidant capacity values are comparable to date seeds. These values are directly proportional to the amount of phenolics present. High antioxidant capacity for flesh has not been reported with reference to ripening stages of date fruits.
Correlation analysis
The TPC of date seeds demonstrated strong correlation as shown in Table 4 with ABTS (r = 0.903, p < 0.05), ORAC (r = 0.996, p < 0.05) and FRAP were r = 0.994, p < 0.05 (Table 4). Similarly, the correlation coefficient for ABTS and ORAC was 0.937. This is because the reactions of phenolics with ORAC and ABTS and Folin-Ciocalteu’s reagent work based on electron transfer principle.27 The strong linear correlations between ORAC, ABTS and TPC have been reported in previous studies for different leguminous seeds varieties.40 In DPPH assay, antioxidants scavenge the free radicals by electron transfer yet the EC50 measured values were not significantly correlated with FRAP, ABTS and TPC (p > 0.05). The above results reveal that date seed extracts contained phenolics, which are more actively participating in FRAP, ABTS and ORAC scavenging, while other phytochemical along with phenolic constituents are also active in DPPH assay. From the dominant phenolic compounds of date seed extract, the gallic acid correlated with TPC (r = 0.937, p < 0.05), ABTS (r = 0.996, p < 0.05), ORAC (r = 0.964, p < 0.05) and FRAP (r = 0.662, p < 0.05). Similar relationships were observed for sinapic acid and vanillic acid. The correlations of antioxidant capacity of seed extracts in DPPH assay to the gallic acid, vanillic acid and sinapic acid content were non-significant (p > 0.05). Karamać et al. have reported that the content of gallic acid in lupin seed extracts was strongly correlated with TPC, however the total scavenging activity was not significant.27 The less polar p-coumaric acid of date seed extracts likely weakly affected the scavenging potential and TPC, as there was no significant correlations (p > 0.05). Previous studies also have shown a strong correlation between TPC and ABTS, ORAC and FRAP assays.40 Jing et al. reported a strong correlation between hydroxyl benzoic acid and Hydroxycinnamic acids derivatives (r = 0.996) and free radical scavenging potentials.41
Table 4Correlation coefficients (r) between the total phenolic content and the antioxidant capacity of date seeds extracts
| TPC | ABTS | ORAC | FRAP |
|---|
| Gallic acid | 0.937* | 0.996* | 0.964* | 0.662* |
| Caffeic acid | 0.077ns | 0.498ns | 0.165ns | 0.959* |
| Catechin hydrate | 0.717 | 0.348ns | 0.652* | 0.394ns |
| Sinapic acid | 0.979* | 0.972* | 0.993ns | 0.542ns |
| Chlorogenic acid | 0.719* | 0.351ns | 0.655ns | 0.391ns |
| Vinallic acid | 0.127ns | 0.312ns | 0.038ns | 0.881* |
| p-coumaric acid | 0.219ns | 0.618ns | 0.305ns | 0.990* |
| Epicatechin | 0.263ns | 0.652ns | 0.348ns | 0.995* |
| TPC | 1 | 0.903* | 0.996* | 0.358ns |
| ABTS | | 1 | 0.937* | 0.725* |
| ORAC | | | 1 | 0.439 |
| FRAP | | | | 1 |
However, Hatamnia et al. reported that TPC and EC50 of DPPH did not exhibit a significant correlation.42 Positive correlation between phenolic compounds and antioxidant actives have been reported in the different plant extracts. Zakhm-e-hayat rhizomes crude extract showed a similar trend of phenolic and flavonoid content with its antioxidant activity.43 TPCs and presence of aforementioned free and bound phenolics contribute to the overall antioxidant capacity of date seed extract. Furthermore, this study also suggests that TPCs and a single free radical scavenging assay might not be sufficient to reveal the whole picture of antioxidant capacity and as the best indicator for antioxidant capacities.