ABSTRACT
Date palm (Phoenix dactylifera) fruit is an important food in the Middle East. Some date palm cultivars like the Ajwa is revered for its health-promoting properties. Phytochemicals like phenolic compounds are in part responsible for such health benefits. However, detailed information on these compounds is lacking. The present work aimed to investigate phytochemical components of Ajwa cultivar using UHPLC-ESI-QTOF-MS/MS in three different extraction solvents (aqueous, methanol–water, acetone–water) and three different extraction durations (2, 5 and 24 h). The proposed method provided tentative identification of 169 bioactive compounds out of which 44 (polyphenols and other phytochemical compounds) were successfully identified from three different extracts and three different extraction durations. Twenty-one compounds never previously reported in the Ajwa cultivar were identified. Aqueous-based extraction solvent and 24-h extraction duration yielded most phytochemical compounds.
Introduction
Date palm, Phoenix dactylifera from Arecaceae family, is an important food crop in the Middle East. It is primarily cultivated for its sweet edible fruit which can be eaten fresh or processed into various products like vinegar, syrup, drink, powder and confectionery (Al-Shahib and Marshall, Citation2003; Salah et al., Citation2010). Other parts of the date palm like leaves and timber may also be useful as traditional building material (Riahi et al., Citation2009; Zabar and Borowy, Citation2012). More than 2000 cultivars have been developed over centuries from distinctive regions across the Middle East and the Mediterranean. The Ajwa cultivar, developed and cultivated only in Madinah, Saudi Arabia is one of the most expensive and highly revered cultivar in the market mostly due to religious and ethnomedical belief of it as functional food for health (Yasin et al., Citation2015). Evidence is emerging for the cardioprotective effects of Ajwa date (Al-Yahya et al., Citation2016; Khan and Siddiqui, Citation2017). Anticancer effects of Ajwa date have been demonstrated in rodent models (Khan et al., Citation2018) and cancer cell lines for breast (Khan et al., Citation2016), prostate (Mirza et al., Citation2018) and colon (Eid et al., Citation2014) cancers. Other effects include liver (Sheikh et al., Citation2014), gut (Eid et al., Citation2014), tissue (Ragab et al., Citation2013) and kidney (Ali et al., Citation2011) protection. Ajwa date is also effective against pathogens like fungi (Belmir et al., Citation2016). However, few investigators studied nutritional and phytochemical contents of Ajwa date. Assirey (Citation2015) studied nutritional composition of different date cultivars and demonstrated that Ajwa date contained the highest amount of minerals compared to other cultivars like Mabroom, Shalaby, Safawy, Khodari, Brni, Anabarah, Suqaey, Sukkari and Labanah. Eid et al. (Citation2013) demonstrated the superiority of Ajwa cultivar compared to Barni and Khalas cultivar in terms of phenolic content using liquid chromatography mass spectrometry analysis on methanol extract. Zhang et al. (Citation2013) confirmed the bioactivity of Ajwa date using antioxidant and anti-inflammatory assays. However, little is known about the phytochemical components of Ajwa date responsible for its therapeutic effects. Thus, detailed and extended profiling of the phytochemicals of Ajwa date using high sensitive tools is necessary. Mass spectrometry coupled to high-performance liquid chromatography (HPLC–MS/MS) has been increasingly used in the structural characterization of complex matrices and has been proven to be the tool of choice to identify phenolic compounds (Abu-Reidah et al., Citation2015). Different extraction techniques (Wang and Weller, Citation2006), solvents (González-Montelongo et al., Citation2010) and duration (Chew et al., Citation2011) may influence the amount and type of biochemical component yield. Using a potent analytical technique like ultra high-performance liquid chromatography electrospray time of light mass spectrometry (UHPLC-ESI-QTOF-MS/MS) we investigated the phytochemical composition of Ajwa date on different extraction solvents and duration.
Materials and Methods
Chemicals
Methanol (CH4O) of analytical grade was purchased from John Kollin Corporation (Midlothian, UK). Acetone (C3H6O) of analytical grade was purchased from R&M Chemicals (Semenyih, Malaysia) 0.1 formic acid, 5 mM ammonium formate and acetonitrile were of UHPLC-QTOF-MS/MS grade.
Sample Preparation
Ajwa dates (500 g) were purchased from a local supplier Syarikat Abdul Ghaffar Sdn. Bhd. (Penang, Malaysia) who imports from Madinah, Saudi Arabia. The flesh was washed and dried at room temperature for 24 h and then kept at room temperature until further use.
Extraction
Aqueous Extract
Aqueous extract was prepared according to the protocols of Vayalil (Citation2002) and El-Arem et al. (Citation2014) with slight modifications. 40 g of Ajwa date flesh were ground with pestle and mortar and soaked in 120 mL distilled water then left at room temperature for either 2, 5 or 24 h. The mixtures were filtered using sterile cotton gauze followed by Whatman® no. 1 filter paper (Whatman Int. Ltd., Maidstone, UK) and the filtrates collected and kept at −80°C until further use.
Methanol–water Extract (80%)
Methanol–water extract was prepared according to the protocols of Singh et al. (Citation2012) and Odeh et al. (Citation2014) with slight modification. Forty grams of Ajwa date flesh were ground with mortar and pestle and dissolved in 120 mL methanol–water (80%), then left at room temperature for either 2, 5 or 24 h. The solutions were filtered using sterile cotton gauze followed by Whatman® no.1 filter paper (Whatman Int. Ltd., Maidstone, UK). The filtrates were collected then concentrated using IKA® RV10 rotary evaporator (IKA-Werke GmbH, Staufen, Germany) and JEIO TECH VE-11 electrical aspirator (JEIO TECH Co. Ltd., Seoul, Korea) to approximately 20 mL. The extracts were kept at −80°C until further use.
Acetone–water Extract (70%)
Acetone–water extract was prepared according to the protocols of Williams et al. (Citation2014) and Aïssa et al. (Citation2015) with slight modification. 10 g of Ajwa date flesh were ground with pestle and mortar and dissolved in 100 mL of acetone–water (70%). The solution was left at room temperature for either 2, 5 or 24 h. The solutions were filtered using sterile cotton gauze followed by Whatman no.1 filter paper (Whatman Int. Ltd., Maidstone, UK). The filtrates were collected then concentrated using IKA® RV10 rotary evaporator (IKA-Werke GmbH, Staufen, Germany) and JEIO TECH VE-11 electrical aspirator (JEIO TECH Co. Ltd., Seoul, Korea) to approximately 20 mL. The extracts were kept at −80̊C until further used for analysis.
UHPLC-ESI-QTOF-MS/MS Analysis
All the extracts were subjected to UHPLC-ESI-QTOF-MS/MS analysis conducted by Advanced Chemistry Solutions Sdn. Bhd., Petaling Jaya, Malaysia. The analysis was carried out on the Eksigent® 110 Ultra-High-Performance Liquid Chromatography system (AB Sciex Pte. Ltd., Biopolis, Singapore) coupled with Sciex TripleTOF® 5600 + high resolution accurate mass tandem mass spectrometry (AB Sciex Pte. Ltd., Biopolis, Singapore). All samples were filtered with Merck Millex® 0.45-µm nylon syringe filter (Merck & Co., Kenilworth, New Jersey, USA) before injection into LC-MS/MS. 10 µL of sample was injected via the Eksigent® 110 UHPLC autosampler (AB Sciex Pte. Ltd., Biopolis, Singapore) and compound separation achieved with a reverse phase Phenomenex® Synergy RP C-18 column (100 × 2.0 mm × 3 µm) (Phenomenex Inc., California, USA). Sample elution was carried out using gradient elution comprising solvent A [water: 0.1% formic acid (Merck & Co., Kenilworth, New Jersey, USA): 5 mM ammonium formate (Merck & Co., Kenilworth, New Jersey, USA)] and solvent B [acetonitrile (Merck & Co., Kenilworth, New Jersey, USA): 0.1% formic acid (Merck & Co., Kenilworth, New Jersey, USA): 5 mM ammonium formate (Merck & Co., Kenilworth, New Jersey, USA)]. The following gradient was used for the chromatography separation: 5% B to 95% B from 0.01 to 20 min, hold for 5 min and back to 5% B in 0.1 min and re-equilibrated for 5 min. The Sciex TripleTOF ® 5600 + high resolution accurate mass spectrometer (AB Sciex Pte. Ltd., Biopolis, Singapore) was used to obtain the mass spectrum. The ionization source used was Sciex patented Duo Spray source with electrospray ionization (ESI) in negative mode. Ionization voltage used was 5500kV with 500°C source temperature. For the identification of phytochemical compounds, the mass scan range was set from m/z 100 to 1200 with information-dependent acquisition (IDA) of mass fragment spectrum. The mass fragmentation spectrum is an important requirement to ensure identification of the compounds. Further identification was done via chemical formula finding based on high resolution accurate mass data obtained. Cross-referencing with existing publications as well SCIEX’s natural products database.
Result and Discussion
In the present work, bioactive composition from the aqueous-based extracts (aqueous, methanol–water and acetone–water) of Ajwa date palm fruit was carried out using UHPLC-ESI-QTOF-MS/MS in negative ionization mode. The method tentatively detected 169 phytochemical compounds, of which 44 were successfully characterized (). In addition, 21 new compounds were detected for the first time in Ajwa date palm fruit (sinapic acid hexoside, p-coumaroyl hexose, p-hydroxybenzoylhexose, protocatechuic acid hexoside, chrysoeriol, chrysoeriol hexosyl sulfate, luteolin hexoside, luteolin rhamnosyl hexoside, luteolin rhamnosyl dihexoside, dihydrokaempferol hexoside, isorhamnetin rhamnosyl hexoside, isorhamnoetin-3-o-glucoside, chrysoeriol rhamnosyl hexoside, kaempferol-3-o-glucoside-7-o-rhamnoside, afzelin-o-gallate, eriodictyol xyloyl deoxy hexose, pinene-ol-o-glucoside, trihydroxy-9,14-octadecadienoic acid, trihydroxy-octadecenoic acid, hydroxy octadecadienoic acid and gluconic acid). The compounds detected in this work were tentatively characterized by means of MS data, together with the interpretation of the observed MS/MS spectra in comparison with those found in the literature and internal database.
Organic Acids
Malic acid, gluconic acid and ascorbic acid were detected. Malic and ascorbic acids have been previously reported (Hamad et al., Citation2015).
Phenolic Acids and Derivatives
In the present work, 14 phenolic acid derivatives were identified, and 11 of which were hydroxycinnaminic acids and 3 were hydroxybenzoic acids. The 11 hydroxycinnaminic acids were caffeic acid, caffeic acid hexoside, caffeic acid hexoside conjugate, caffeoyl acid, O-caffeoyl shikimic acid, O-dicaffeoyl shikimic acid, O-dicaffeoyl shikimic acid acetate, sinapic acid hexoside, ferulic acid, chlorogenic acid and p-coumaroyl hexose. The three hydroxybenzoic acids were p-hydroxybenzoylhexose, protocatechuic acid hexoside and gallic acid. Caffeic acid, gallic acid, ferulic acid and chlorogenic acid have already been reported by Saleh et al. (Citation2011); Hamad et al. (Citation2015); Ahmed et al. (Citation2016a).
Flavonoid Derivatives
A total of 24 flavonoid derivatives were detected. Out of this eight were flavones which included apigenin pentosyl hexoside, apigenin 7-O-neohesperidoside, chrysoeriol, chrysoeriol hexosyl sufate, luteolin, luteolin hexoside, luteolin rhamnosyl hexoside, luteolin rhamnosyl dihexoside. 15 were flavonols which included quercetin, quercetin rhamnosyl dihexoside, quercetin 3-hexose, quercetin 7-O-hexoside-3-O-rhamnoside, isoquercetin, isoquercetin acetate, isoquercetin sulfate, dihydrokaempferol hexoside, rutin, isorhamnetin rhamnosyl hexoside, isorhamnetin-3-O-glucoside, chrysoeriol rhamnosyl hexoside, kaempferol-3-O-glucoside-7-O-rhamnoside and afzelin-O-gallate. Only 1 flavanone which included eriodictyol xylol deoxy hexose. Quercetin, luteolin, rutin, iso-quercetin and apigenin have all been reported previously (Ahmed et al., Citation2016a; Hamad et al., Citation2015; Saleh et al., Citation2011).
Terpenoids
For the first time, one terpenoid was detected as pinenol-O-glucoside.
Fatty Acids
For the first time, three fatty acids were detected in Ajwa date palm fruit which include trihydroxy-9,14-octadecadienoic acid, trihydroxy-octadecenoic acid and hydroxy octadecadienoic acid.
UHPLC-ESI-QTOF-MS/MS
The present study used UHPLC-ESI-QTOF-MS/MS, the combination of liquid chromatography and mass spectroscopy is a highly sensitive and accurate technique compared to HPLC alone. The ionization method (electrospray ionization) which was used in the present study has the ability to produce multiply charged molecular ions in comparison to other methods like MALDI (Matrix assisted laser desorption) which leads to formation of single charged molecular ions. Additionally, the present study also used triple quadrupole time-of-flight tandem mass 10 spectrometry (QTOF-MS/MS) to analyze individual component and this technique adds specificity to the given analysis. Date fruit extract was initially analyzed in both negative and positive ion modes (Farag et al., Citation2014). Our study utilized negative ion mode [M-H]- due to better sensitivity and more observable peaks especially for polyphenolic compounds (Farag et al., Citation2014). The identification of phytochemical compounds was based on five different parameters: retention time, molecular weight of each compound, m/z [M-H]-, molecular formula and fragmentation pattern. Representative chromatograms of LC-MS/MS data from two extraction samples of Ajwa date fruit in the present study were shown in . The chromatograms showed identification of O-dicaffeoyl shikimic acid (MS peak at m/z 497.1) which had retention time at 4.671 min and caffeic acid (MS peak at m/z 179) which had retention time at 4.178 min.
Effects of Different Extraction Solvents on Identified Compounds
In all the three extracts, extraction duration of 24 h qualitatively yielded most compounds. Although all three solvents were aqueous based, pure aqueous extract yielded more bioactive compounds which include phenolic acids and flavonoids compared to methanol–water and acetone–water-based extraction. This finding is in agreement with other study which has demonstrated that water is a good solvent for extracting phenolic compounds from apple pomace (Reis et al., Citation2012). Similarly, other studies have also found that the yield of phenolic compounds from aqueous extract was superior than pure organic solvent extract (Do et al., Citation2014). This indicated that water is the best solvent to extract phenolic acids and flavonoids compared to methanol–water and acetone–water extracts. Besides glycated compounds, sulfated compounds were detected. Though not usually found in natural products, it has been reported in dates (Farag et al., Citation2014). In the present study, 11 of bioactive compounds were successfully characterized in water extract which included apigenin pentosyl hexoside, gluconic acid, malic acid, afzelin-o-gallate, protocatechuic acid hexoside, p-hydroxy benzoyl hexose, dihydrokaempferol hexoside, isoquercetrin acetate, isorhamnetin rhamnosyl hexoside, eriodictyol xyloyl deoxy hexose and p-coumaroyl hexose (). Whereas, 10 bioactive compounds were successfully characterized in methanol–water (80%) including luteolin, sinapic acid hexoside, caffeoyl acid, ferulic acid, gallic acid, quercetin, quercetin-7-O-hexoside-3-O-rhamnoside, quercetin-3-hexose, apigenin-7-O-neahesperidoside and caffeic acid hexoside conjucate () as well as 8 bioactive compounds appeared in acetone–water (70%) which included ascorbic acid, caffeic acid, kaempferol-3-O-glucoside-7-O-rhamnoside, chlorogenic acid, hydroxy actadecadienoic acid, pinenol-O-glucoside, O-dicaffeoyl shikimic acid and luteolin rhamnosyl dihexoside (). On the other hand, nine compounds were identified in all three solvents like O-dicaffeoyl shikimic acid, isoquercetin sulfate, isoquercetin, trihydroxy octadecenoic acid, chrysoeriol, O-caffeoyl shikimic acid, chrysoeriol rhamnosyl hexoside, chrysoeriol hexosyl sulfate and luteolin hexoside (). Whereas three compounds such as luteolin rhamnosyl dihexoside, trihydroxy-9,14-octadecadienoic acid and rutin appeared only in each of aqueous and acetone–water extracts (), only one compound (caffeic acid hexoside) was detected in each aqueous and methanol–water extracts () and two compounds like isorhamnetin-3-O-glucoside and quercetin rhamnosyl dihexoside were found only in each of methanol–water and acetone–water extracts (). The proposed method in this study also showed that methanol–water extract has more phytochemical compounds compared to acetone–water extract. Solvent such as methanol has a superior extraction power compared to acetone or ethyl acetate when they are used to extract polar compounds from several medicinal plants (Miliauskas et al., Citation2004). Polarity of extraction solvent may play an important role in extracting polar compounds. In terms of polarity, methanol is more polar than acetone (Sadek, Citation2002). Besides that, the yield of extraction also depends on pH, temperature, extraction time and composition of the sample (Do et al., Citation2014). The extraction yield decreased when using absolute organic solvents.
Conclusion
The study found date of Ajwa cultivar rich in phytochemicals. Polyphenols could be major phytochemicals identified in this studied date. Interestingly, 21 compounds were identified using UHPLC-ESI-QTOF-MS/MS for the first time. Further works are needed to quantify these compounds. Extraction of phytochemicals using aqueous-based solvents for 24 h exhibited high yield.
Additional information
Funding
References
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