Abstract: Background and Objective: A number of studies have been published from a pharmaceutical and medicinal point of view, focusing on phenolic compounds and their biological activities in various date palm fruits from different date growing countries based on aqueous or alcoholic extracts. The present study was performed to identify the volatile constituents of date palm fruits cultivated in the Kingdom of Saudi Arabia at the besser, rutab and tamr maturation stages, as well as evaluating the total phenolic content in the volatile extracts and their antioxidant activity. Materials and Methods: Four varieties of date palm fruits (Phoenix dactylifera L.) cultivated in Saudi Arabia Khalas, Sokary, Seqah and Khenazy were analyzed for their aroma volatiles, total phenolic compounds and antioxidant activity at three maturation stage, besser, rutab and tamr. Results: The 71 aroma constituents were identified using the dynamic head space technique during the maturation stages. Alcohols were the major class of compounds present with 2.6-81.31% followed by carbonyls with 1.69-83.97% in Seqah rutab stage where esters accounted 2.66-49.89% of detected species. Total phenolic compounds were highest in the Khenazy rutab stage (9.23 μg GAE/100 g), which is in agreement with the radical scavenging activity (IC50 1.45 μg mL–1). Conclusion: The data obtained revealed that, the date palm cultivated in Saudi Arabia are rich in phenolic and antioxidant constituents and probably have use as nutraceutical and functional food additives.
INTRODUCTION
Date palm fruit (Phoenix dactylifera L.) is considered as high value product, that is produced in many arid regions of the world such as Middle Eastern countries especially, The Kingdom of Saudi Arabia, Iran, Algeria, Tunisia and Egypt. However, during the last three centuries, cultivation of dates has been introduced into South Africa, South America, Australia and North America. Dates have played significant roles in the economy, society and environment of the countries which cultivated this crop1. The Kingdom of Saudi Arabia which is one of the top three producers in the world, produce over 1 million tons of dates annually from about 23 million date palms over 400 cultivars, accounting for 17% of the total global production2,3.
Many researchers have reported the nutritional value and main chemical composition of dates e.g., Al-Shahib and Marshal4 who showed the following proportions in date palm fruits, carbohydrate (70-80%), fat (0.2-0.5%), protein (2.3-5.6%) dietary fiber (6.4-11.5%), minerals (0.1-916 mg/100 g date) and vitamins such as C, B1, B2, A, riboflavin and niacin. In addition, several investigations have been published from a pharmaceutical and medicinal point of view, focusing on phenolic compounds and their biological activities in various date palm fruits from different date growing countries based on aqueous or alcoholic extracts5-7. In spite of the importance of flavor as a key factor in consumer satisfaction and further processing of the fruits, only a few studies concerning aroma and volatiles of dates could be found in the literature. Jaddou et al.8 extracted 38 compounds from an Iraqi Zahdi cultivar by vacuum distillation, whereas, Max et al.9 and Torres et al.10 have reported 36 and 25 aroma compounds respectively in three Tunisian dates cultivars extracted by headspace pentane method. More recently, El Arem et al.11 identified 69 compounds in some Tunisian cultivars at different stages of maturation (besser, rutab and tamr) after extraction using Headspace Solid Phase Micro extraction/Gas Chromatography-Mass Spectrometry (HS-SPME/GC-MS). However, nothing could be found in the literature concerning volatiles or their bioactivity for dates cultivated in the Arab peninsula or the Kingdom of Saudi Arabia. During maturation, dates pass through many stages in which the chemical composition as well as the aroma can vary. Compounds are continuously synthesized and the total composition changes qualitatively and quantitatively during ripening12.
The aim of the present study was to identify the volatile and aromatic constituents of date palm fruits cultivated in the Kingdom of Saudi Arabia at the besser, rutab and tamr maturation stages, as well as evaluating the total phenolic content in the volatile extracts and their antioxidant activity.
MATERIALS AND METHODS
Plant materials: Four different varieties of date palm fruit (Khalas, Sokary, Seqah and Khenazy) at different ripening stages (besser, rutab and tamr) were collected from the Station of research and agricultural experiments in Dirab, Faculty of Science of Food and Agriculture, King Saud University, Riyadh, KSA, during the 2013 harvest season. Uniformed fruits, free of defects were selected and stored at -20°C until analysis. Three replicate analysis were carried out for each type of date.
Chemicals and reagents: Diethyl ether and methanol were purchased from (Fisher Chemicals). Mixtures of n-alkanes C6-C26, authentic compounds, sodium bicarbonates, anhydrous sodium sulfate, linoleic acid (>99%), Tween 40, β-carotene (>97%), Folin-Ciocalteu reagent for total phenolic compounds, 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and gallic acid were obtained from Sigma Aldrich Chemical Co. (St. Louis, MO, USA).
Extraction of volatile aroma compounds: The edible part of date palm fruits (100 g) was pitted, crushed and cut to small pieces with a sharp knife and dry-blended for 3 min in a blender (Moulinex, France). The tissue of date palm fruits was rapidly juiced and then the volatiles were isolated by using a dynamic headspace system (DHS). The samples were purged for 3 h with nitrogen gas (grade of N2>99.99%) at a flow rate 100 mL min–1. the headspace volatiles were swept into cold traps containing diethyl ether and held at -10°C. The volatile extracts were dried over anhydrous sodium sulfate for 1 h and then reduced to 1 mL on a rotary evaporator (Heidolph, Germany).
Gas chromatographic-mass spectrometric analysis (GC/MS): The analysis was carried out by using a coupled gas chromatography Hewlett-Packard model (5890) and mass spectrometric detector (Hewlett-Packard5970). A fused silica capillary column DB-5 (60 m×0.32 mm, id) was used. The oven temperature was maintained initially at 50°C for 5 min. and then programmed from 50-250°C at a rate of 4°C min–1. Helium was used as the carrier gas, at a flow rate of 1.1 mL min–1. The injector and detector temperatures were 220 and 250°C, respectively. The ionization voltage was 70 eV and mass range m/z 39-400 amu. The identification of the compound was based on matching with the mss spectra library (NIST library version 2005), comparison with spectra of authentic compounds and published data13. The relative percentage of the oil constituents was calculated from the GC peak areas. A linear retention was calculated for each compound using the retention times of a homologous series of C6-C26 n-alkanes13.
Antioxidant activity assays
β-Carotene bleaching assay: The antioxidant activity was determined by a β-carotene/linoleic acid system as described by Jayaprakasha et al.14 in comparison to tert-butylhydroquinone (TBHQ). The absorbance was measured at 470 nm over a 60 min period.
DPPH radical scavenging assay: The potential antioxidant activities of volatile extracts were assessed according to Hatano et al.15 in comparison to synthetic antioxidant used in food industry, TBHQ. The absorbance was measured at 517 nm using spectrophotometer (Evolution 300 Thermo UV-VIS), all tests were run in three replicates and averaged.
Determination of the total phenolic contents: The total phenolic content of the volatile extracts was determined using Folin–Ciocalteu reagent according to a modified method of Singleton et al.16 with gallic acid as the standard. The reaction mixtures were incubated in a thermostat at 45°C for 45 min before the absorbance at 765 nm was measured.
Statistical analysis: Statistical analyses were performed using SPSS software version 16 (SPSS Inc., Chicago, IL, US). The variation in the results is expressed as mean±standard deviation (Mean±SD). The significance of the difference between samples was determined using t-test. The difference was regarded significant when p<0.05, where p is a level of significance.
RESULTS
Aroma volatiles of different date palm fruits varieties, Khalas, Sokary, Seqah and Khenazy, at different maturation
stages, are reported in Table 1 and Fig. 1. Seventy one compounds were identified and classified into alcohols, esters, carbonyls (aldehydes and ketones), acids and hydrocarbons. The identified volatile compounds comprised about 90.04-99.15% of the total aroma compounds.
Twenty five alcohols were detected and constituted the major qualitative class of identified volatiles at 2.6-81.3% in all varieties and maturation stages. These were followed by 18 carbonyls (aldehydes and ketones) representing 1.69-83.97% of the detected species, 16 esters comprising 2.66-49.89% of the volatiles identified and finally 5 terpenes, 3 fatty acids and 4 long-chain hydrocarbons at 0-4.03, 0-23.84 and 0-5.19%, respectively.
According to Table 1 and Fig. 1, increases in alcohols could be noted in Khalas cultivar from the besser stage to the tamr stage (32.84-69.5%) as well as in Sokary (64.86-70.01%) and in khenazy (66.4-72.93%). In the Seqah variety, the besser stage had 35.41% of alcohols, which dropped dramatically to 2.6% in the rutab stage, before raising again to 35.91% in the tamr stage. The 2-Hexen-1-ol was the predominant alcohol identified, ranging from 0.8% in Khalas besser to 77.02% in the rutab stage of Khenazy, followed by 2-phenylethanol which found at 21.51% in besser stage of Khalas, while 1-hexanol was ranged from 1.45% in Khalas rutab stage to 3.88% in Sokary at tamr stage.
The concentrations of esters decreased from 30.36% at the besser stage to 16.21% in the tamr stage of Khalas cultivar and the changes in the Khenazy variety were from 19.78-17.24%. A slight increase was noted in the Sokary variety (16.89-19.32%) and a dramatic increase was seen in the Seqah cultivar from 13.9-49.89%. Ethyl nonanoate characterized besser stage in all cultivars, especially in Khalas (6.5%), in addition to another predominant fatty acid ester, hexadecenyl acetate which found at 21.32% in besser stage Khalas and 11.86% in tamr stage Seqah but was absent in other cultivars.
| Fig. 1: | Concentrations of different volatile classes along different maturation stages in date palm cultivars |
| Table 1: | Volatile constituents of date palm fruits at different maturation stages |
aPercentage obtained by MS peak area normalization, bRetention indices (kovats index). *Varying degree of the result is expressed as mean±standard deviation (Mean±SD) |
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| Table 2: | Antioxidant activity assays and total phenolic content of date palm fruit verities during different maturation stages* |
*Results are Mean±SD (n = 3), amg gallic acid equivalents (GAE)/100 g dry weight (DW), bEfficient concentration (IC50): Amount of antioxidant needed to decrease the initial DPPH-concentration by 50%, cAntiradical efficiency: 1/EC50 |
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In addition to the aromatic aldehydes especially dihydroxy benzaldehyde and cyclocitral which found only in the rutab stage of Seqah (3.35 and 3.59%) with floral and fruity notes (Table 1), other aliphatic aldehydes were detected in some maturation stages, mainly 2-methylbutanal, heptanal, undecanal and dodecanal. Generally, carbonyls ranged from 1.69-10.86% in various maturation stages of the date cultivars under investigation, except for Seqah at the rutab stage (83.97%), due to the presence of 2-pentadecanone (76.01%).
Seqah variety at tamr stage was found to be the richest in saturated hydrocarbons especially n-hexadecane (4.42%), while no hydrocarbons could be detected in the rutab stage of sokary.
In addition to 9-hexadecenoic acid which represented a major component among the volatiles of Khalas besser, hexanoic acid was detected in besser stage of the Sokary with 7.1% and in rutab stage of Khenazy with 1.12%.
The determination of total phenolic content was based on the absorbance values of the various extract solutions after reaction with Folin-Ciocalteu reagent and compared with standard solutions of gallic equivalents as described in section 2, 5 and 3. The highest amount of total phenolics was in Khenazy at rutab stage followed by both Khalas and Sokary at rutab stage, while the lowest was in Seqah rutab stage.
In the present study, the relative antioxidant abilities of the date palm fruit extracts at different maturation stages were investigated through two in vitro models, antioxidant capacity by β-carotene linoleate system and radical scavenging activity using, DPPH method (Table 2). It was found that, the rutab stage of the Khenazy variety had the highest radical scavenging activity (IC50 = 1.45 μg mL–1), followed by the Khalas type at rutab stage (2.01 μg mL–1) and the rutab stage of Sokary (2.95 μg mL–1), however, the rutab stage of the Seqah variety had the lowest radical scavenging activity (IC50 = 5.44 μg mL–1). The β-carotene linoleate assay showed similar results to those found in DPPH assay (Table 2), where the rutab stage of the Khenazy variety again had the highest radical scavenging activity (0.689) in comparison to the other varieties at different maturation stages.
DISCUSSION
This study discovers the relation between maturity stages and the using of date palm fruits cultivated in Kingdom of Saudi Arabia, as a nutraceutical food based on their sensory compounds and phenolic content. It believed that, it will encourage the researchers to study and explore extensively the date palm fruits cultivated in arab peninsula. Stages of maturity have been shown to affect the aroma volatile constituents and the phenolic content of date palm fruits as well as their antioxidant activity. Alcohols have been found as the dominant class among the volatiles identified followed by carbonyls and esters. El Arem et al.12 reported that, alcohols are the most important class formed during ripening as lipid oxidation products. Along with terpenes, alcohols are responsible for the herbaceous, fruity, citrus, floral and fungal aromas of dates17. With respect to the predominant alcohols identified in different cultivars at various stages, 2-hexen-1-ol, 1-hexanol and phenylethanol are probably contribute to the green, herbal and floral aroma notes of the dates fruits, respectively17,18.
The concentrations of terpenes were found to be inversely proportional to the amounts of alcohols formed during all maturation stages (Table 1, Fig. 1). Terpenes e.g., borneol with a piney, camphor-like odor and dihydrocarveol with a floral and fruity aroma were detected only in the Seqah maturations stages, while cyclocitral, citronellol and β-ionone could be identified on other cultivars18.
Esters are characterized by fruity, pineapple and apple odours17. Ethyl acetate and methyl propionate are the dominants of this class and seem to be similar for Degla, Horra and other varieties cultivated in Morocco and Tunisia as reported by Harrak et al.19 and El Arem et al.12. Detection of ethyl nonanoate in besser stage of all studied cultivars, is in agreement to the findings of El Arem et al.11 while the presence of hexadecenyl acetate as a dominant ester in Khalas besser and Seqah tamr, is in accordance to Torres et al.10, who reported the formation of such esters as being due to lipid metabolism. Generally, like other volatile classes and as Max et al.9 stated, the concentration of esters increased at the expense of alcohols (Fig. 1), since volatile esters are formed by esterification of alcohols during fruits ripening20.
Aliphatic aldehydes are responsible for malt, aldehydic, green, citrus and sweet aroma notes especially in early maturation19. Short-chain aldehydes e.g., 2-methyl and 2-ethylbutanal were decreased over ripening, while the concentrations of other longer aldehydes e.g., 4-heptenal and dodecanal were found to be increased during ripening especially in Khalas, Sokary and Seqah varieties. Other aldehydes e.g., octanal, nonanal and tridecanal were reported by Jaddou et al.8, El Arem et al.12 and Harrak et al.19, in other cultivars e.g., Zahdi in Iraq, Aziza, Bouskri and Jihel in Morocco and Allig, Degla, Gosbi and Horra in Tunisia.
Long-chain hydrocarbons increased considerably through different maturation stages, which is in accordance to El Arem et al.12. Jaddou et al.8 and Harrak et al.19 were reported the presence of hydrocarbons on different date cultivars, however, these compounds are not among the impact flavor of dates.
Hexanoic acid which has a pleasant fatty note reported by Burdock18, seems to be the most important among fatty acids class from the sensory point of view. Many fatty acids were reported by Harrak et al.19 e.g., C6, C8-C12, C14, C16, C18, C18:2 and C20, however, only C6 and C16:1 were identified in the varieties under investigation.
Wu et al.21 have been suggested that date fruit may contain a higher level of total phenolic content than among other fresh and dried fruits. According, we have determined the amount of phenolic compounds in the tested extracts of different date varieties (Table 2). The results showed that the date palm fruits varieties under investigation had a similar level of phenolic content (2.47-9.23 mg GAE/100 g DW) to those of Algerian and Iranian cultivars22-24. However, Al-Farsi et al.25 reported higher levels between 172 and 246 mg gallic acid equivalents/100 g fresh weight in Omani dates.
The antioxidant activity of the date palm fruits at different maturation stages were related to the phenolic contents of the species under investigation, whereas the rutab stage of Khenazy, Khalas and Sokary were presented the highest activity. The potential antioxidant activity recorded for the date palm fruits varieties under investigation at different maturation stages (1.45-5.44 μg mL–1), favors their possible application as a nutraceutical food and natural antioxidant. Findings of the antioxidant activity are in agreement with Vayalil26, who reported that, the amount of fresh extract required to scavenge 50% of superoxide radicals was equivalent to 0.8 mg mL–1 in the riboflavin photoreduction method and of 2.2 mg mL–1 for 50% hydroxyl radical scavenging activity in the deoxyribose degradation method. The antioxidant activity of the different date varieties under investigation depended on their phenolic content which is in accordance with Biglari et al.23. Presence of higher amounts of phenolic content in Khenazy, Khalas and Sokary rutab stage suggested their responsibility for the higher antioxidant activity of such varieties, which encourage the use of date fruits at various maturation stages as functional food ingredient.
CONCLUSION
The Khenazy, Khalas and Sokary varieties at rutab stage are good and easily accessible source of phenolic compounds in comparison with other maturation stages, which is in agreement with the antioxidant activities recorded for all samples under investigation. The 71 aroma volatiles identified in date palm fruits could be classified into alcohols, esters, carbonyl compounds, terpenes carboxylic acids and long-chain hydrocarbons. Qualitative and quantitative differences in compounds responsible for aroma were noted across maturation stages. While alcohols are the dominant species, there is an inverse relationship between concentrations of these and other classes of compounds present.
ACKNOWLEDGMENT
This project was funded by the National Plan for Science, Technology and Innovation (MAARIFAH), King Abdulaziz City for Science and Technology, Kingdom of Saudi Arabia, Award Number (11-AGR 1600-2).