1. Introduction
Date palm (
Phoenix dactylifera L.) is a tropical plant that is grown in Middle Eastern and North African countries [
1]. The date fruit is an essential component of the diet and has important social, environmental, and economic significance in Saudi Arabia. The Kingdom of Saudi Arabia is the leading date producer, with an annual production exceeding 1.5 million tons, which represents 17% of the total global production [
2]. There are more than 5000 different varieties of dates worldwide, of which around 400 cultivars are grown in Saudi Arabia [
3,
4].
Several researchers have reported that dates are rich sources of carbohydrates, energy, vitamins, and minerals [
5]. The top-quality date varieties, such as Sukkari, Ajwa, and Barhi, are consumed in the Rutab stage. In contrast, middle- and low-quality varieties are used to produce date paste, jam, syrup, vinegar, and other products [
6]. Date seeds (also called stones, kernels, or pits) represent 10–15% of the ripe date weight, based on maturity, variety, and the quality of the date [
7]. It has been demonstrated that date seeds contain several functional components, such as dietary fiber, minerals, vitamins, fatty acids, and amino acids [
8]. They are also a rich source of bioactive compounds such as carotenoids, polyphenols, flavonoids, and tocopherols [
9,
10,
11]. Hilary et al. [
12] reported that date seeds contain about 51.1 g of polyphenolic compounds per kg, higher than other poly phenol-rich foods like flaxseed, tea, and grapes. Similarly, Habib et al. [
13] observed a high total polyphenol content of 4768.87 mg GAE/100 g in date seed. The cinnamic acid derivatives and benzoic acid hydroxylated derivatives were the predominant phenolic acids in date seeds [
14,
15], while rutin, catechin, quercetin, luteolin, and kaempferol were the main flavonoids [
16,
17]. These natural antioxidants’ presence in date seeds is reported to provide protection effects against many oxidative-stress-induced diseases, such as inflammation, hyperlipidemia, and diabetes [
18,
19,
20].
In vitro and in vivo studies on date seeds have shown their effective antioxidant properties. Abuelgassim et al. [
21] reported that date seed extracts had high scavenging activity against ABTS, DDPH, and hydroxyl radicals. Date seeds have also shown higher antioxidant properties than date flesh, making them a viable natural source of antioxidants [
22]. Oral administration of date seed extracts was found to lower oxidative stress damage and improve the defense systems of the organs [
23,
24]. Recently, Alahmadi and Banayah [
25] reported the hypolipidemic and hypoglycemic effects of date seed extract in diabetic rats. They attributed the lipid- and glucose-lowering actions to the antioxidant properties of the date seeds. The antioxidant properties of date seeds may be due to poly phenolic compounds that act as reducing agents, free radical scavengers, and hydrogen donors, which may explain their efficiency in treating different conditions [
26].
The antioxidant properties and polyphenolic compounds of date seeds were reported to be affected by many factors, such as genetic diversity, fertilizer and soil type, water availability, maturity stages, storage conditions, the solvent system, and extraction methods [
1,
27]. The date industry generates thousands of discarded by-products, such as date pomace and seeds, that are rich with bioactive compounds. New aspects of using these by-products to produce high-nutritional-value food products have recently attracted interest. Therefore, the aim of this study is to chemical composition and validate the extraction conditions of bioactive compounds from seeds of Ruthana, Barhi, and Qatarah date varieties by using different solvent systems.
4. Discussion
This study was conducted to assess the proximate composition, phytochemical content, and antioxidant properties of Barhi, Ruthana, and Qatarah date seeds. Furthermore, the optimization of phenolic compounds and antioxidants’ extraction by using different solvent systems was also performed. During the ripening stages of the date (Kimri, Khalal, Rutab, and Tamr), the moisture content decreased from 20% at the Kimri stage to about 6–12% at the Tamr stage [
37]. The moisture contents analysis showed 12.4, 6.6, and 7.6% for Barhi, Ruthana, and Qatarah, respectively. Our findings agree with those reported for Emirati date seeds [
38,
39], Moroccan date seeds [
40], and Omani date seeds [
10]. The variation in the moisture content among the cultivars might be due to the climate conditions and the relative humidity of the atmosphere. Nevertheless, the low moisture content of the seeds limits the growth of bacteria and allows for better preservation [
41]. Carbohydrates are the main component of date seeds, and their quantity varies with different varieties [
42]. The carbohydrate contents of the Barhi, Ruthana, and Qatarah date seeds were 70.0, 74.6, and 73.6%, respectively. These results agree with those found by [
38], who analyzed six date cultivar pits (seeds), namely Mabroom, Lulu, Khalas, Fard, Khodari, and Abu Maan, and reported that the total carbohydrate content ranged from 70.14 to 78.70% but was lower than that of the Omani date seeds (83.14–86.89%), namely Shahal, Mabseeli, Um-Sellah [
10], and Tunisian date seeds (Allig and Deglet Nour) (ranged between 81.0% and 83.1%) [
43]. Interestingly, date seeds contain a significantly higher amount of protein and fat compared with date flesh [
44,
45]. Date seeds were found to contain a significant amount of fat; it ranged from 6.3% for the Ruthana to 8.7% for the Barhi variety. This agrees with that of [
44,
45], who reported the fat content of various Saudi date seeds, including Barhi, ranging from 7.83 to 8.69%. The date seed fat analysis revealed relatively high percentages of fatty acids, tocopherols, phenolic compounds, phytosterols, and carotenoids with excellent thermal and oxidative stability and a lower acidity content (0.5%) compared to soybean oil (0.86%), sunflower oil (1.4%), and palm Olean7 (4–5.5%), making it convenient for use in cosmetic and food applications [
46]. The protein content of the date seeds did not significantly vary among the cultivars. The protein content of the seeds reported in this study were higher than those of the Tunisian date variety [
47]. Date seed protein is reported to contain the most essential amino acids and has a higher concentration of sulfur amino acids than soybean, peanut, and cottonseed [
48]. The differences in chemical composition could be due to the variability in the cultivars being studied, environmental conditions, agronomic practices, use of fertilizer, postharvest treatments, and storage conditions.
Researchers from different regions have investigated date seeds’ phytochemical compounds and their biological activities. Date seeds were reported to be a rich source of biologically active compounds such as phenolic compounds, tocopherols, carotenoids, and phytosterols that are responsible for several pharmacological activities, such as hypolipidemic, antidiabetic, anti-inflammatory, anticancer, antioxidant, and antimicrobial activities [
18,
49]. In this study, the Ruthana seed showed a significantly high TPC, followed by Barhi and Qatarah. The total phenolic content (TPC) ranges from 13.0 to 93.357 mg GAE/g. The 100% ethanol showed the highest phenolic content, while water had the lowest. This finding agrees with the results reported by [
20,
50], who assessed the efficiency of different solvent systems and found that ethanol is the most effective solvent for extracting phenolic compounds from date seeds, whereas, other researchers [
16,
18] observed that 50% and 70% acetone were more efficient in extracting phenolic compounds from date seeds. The TPC of the 100% methanol extract of the date seeds found in this study is higher than that reported for 14 varieties of Iranian date seeds [
11] but was lower than that of the methanolic extract of some Tunisian date seeds [
49]. These results revealed that it is hard to find a certain solvent system that extracts all phenolic compounds; therefore, employing an organic solvent alone or water can be more efficient in extracting most of the phenolic components of date seed samples. Similar to the TPC, Ruthana showed a significantly high total flavonoid content (TFC) while Qatarah showed the lowest. The results showed that 100% methanol was the most effective solvent, followed by the mixture of methanol:ethanol:water (40:40:20) and methanol 50% for the extraction of the TFC from date seed varieties, whereas water was the least efficient for TFC extraction. The TFC values of the date seeds were in agreement with those reported by [
16] for the Saudi date seeds but higher than those of the Tunisian date seed varieties [
40]. The high flavonoid yield of the mixture could be attributed to the high polarity of the solvent, which favors the extraction of flavonoids [
49].
The Ruthana seed exhibited the highest TPC and TFC, ranging from 53.64 to 93.36 mg GAE/g and 21.89 to 59.89 mg CE/g, respectively, depending on the solvent system compared to the others. These values are higher than those of the Ajwa seed TPC and TFC, which range from 31.55 to 39.32 mg GAE/g and 18.4 to 29.56 mg CE/g, respectively [
50], but lower than those of the Sukkari date seed, which contained 98.1 mg GAE/g phenolic and 70 mg CE/g flavonoids [
16]. Several factors, including the date variety, geographic location, soil, sunlight, analytical and extraction methods, as well as the solvent system, might be responsible for the variation in the phenolic and flavonoid contents of the date seeds. The phenolic compounds in seeds are affected by the ripening stages of dates; they increase in the Rutab stage and decline progressively as the dates mature to the Tamr stage, and this was attributed to their embryo-protective effect, while at the Tamr stage, these compounds are broken down [
11,
31].
Carotenoids are major phytochemicals that act as colorants, vitamin precursors, and antioxidants. In the seed, carotenoid presence is necessary for the synthesis of abscisic acid, a phytohormone that regulates plant growth and development and significantly affects plant defense against bacteria and fungus. Carotenoids were found to protect against the lipid peroxidation of the membranes during seed aging and loss of viability [
51]. Both the total carotenoids and anthocyanin levels contribute to the antioxidant activity of date seeds. The total carotenoid content (TCC) of the three date seeds reported in this study is higher than that of the Emirati date seed varieties [
52]. The total anthocyanin content (TAC) of the three date seed varieties is in agreement with a previous study that revealed the TAC in Deglet Nour seed [
53] but is higher than the TAC values of some Tunisian date seed varieties [
47]. Similar to the other bioactive compounds, carotenoid and anthocyanin content were reported to vary according to cultivars, ripening stages, storage conditions, and postharvest processing [
54].
Date seed was reported to contain higher antioxidant activity than date fruit [
44]. The antioxidant activity of the seeds was reported to increase until the Rutab stage and then decrease in the Tamr stage due to a decrease in phenolic compounds, mainly because of polyphenol oxidase activity [
55]. In plant extracts, the antioxidant activity is determined by using DPPH free radical scavenging activity and TRP, which are associated with the presence of antioxidant molecules that neutralize free radical chains by donating a hydrogen atom. This study found significant antioxidant activity differences among the tested cultivars, similar to the phenolic and flavonoid content. A strong correlation between the phenolic components and the antioxidant activity of the date seeds was observed in this study. The Ruthana seeds had the highest antioxidant activity values (81.88% for the DPPH and 60.29 mg AAE/g for the TRP), which contain more polyphenols and flavonoids whereas the Qatarah seeds, had the lowest phenolic compounds and antioxidant activity (42.07% for the DPPH and 33.27 mg AAE/g for the TRP). Several studies revealed an association between the antioxidant activity of the samples and their phenolic content: the higher the levels of the total phenolic content and flavonoids, the greater the antioxidant activity [
47,
50,
56]. Our results revealed that the antioxidant capacity of the date seeds was strongly and significantly correlated with the phenolic content (r = 0.718 for the DPPH and r = 0.788 for the TRP) and flavonoid contents (r = 0.748 for the DPPH and r = 0.743 for the TRP).
Moreover, similar to phenolic compounds, the antioxidant activity of extracts is found to be affected by the solvent system used. The mixture extracts showed the highest antioxidant activity as assayed by the DPPH or TRP, while water extracts showed the lowest values. Our data agree with those reported by [
50], who found that antioxidants are more dissolved in polar solvents and their aqueous mixtures due to the wide range of polyphenolic compounds that can dissolve in them compared to water. These results indicate that solvent polarity increases the extraction efficiency of the antioxidant compounds and antioxidant activity compared to water or a pure solvent.