Tentative Characterization of Polyphenolic Compounds in the Male Flowers of Phoenix dactylifera by Liquid Chromatography Coupled with Mass Spectrometry and DFT

Phoenix dacylifera is an ancient palm species rich in (poly)phenols. These phenolic compounds were tentatively identified by using liquid chromatography coupled with ion spray mass spectrometry in tandem mode (LC/MS/MS) with negative ion detection. Negative identification of the compounds was based on their retention times and mass spectra in full scan mode (MS), and in different MS/MS modes. For the first time, complete hypothesis, and routs for both p-coumaroylshikimic acids (CoSA) and caffeoylshikimic acids (CSA) were suggested and confirmed by Density Fonctional Theory (DFT) study. Notably, of the 53 compounds characterized, 19 hydroxycinnamates derivatives were tentativelycharacterized in male flowers of date palm and 15 of them were recorded for the first time. In addition, five organic acids, six B-type proanthocyanidins, two anthocyanidin and 21 flavonoid derivatives have been tentatively characterized. Identification of B-type proanthocyanidins were based on the diagnostic ions resulting from heterocyclic ring fission (HRF) and retro-Diels-Alder (RDA) reaction of flavan-3-ol provided information on the hydroxylation pattern and the type of inter-flavan bond proanthocyanidins. The sequence of proanthocyanidins was detected through ions extracted from quinone methide (QM) cleavage of the inter-flavan bond.


Introduction
Phoenix dactylifera (date palm) is a native to the Middle East region over centuries, cultivated for its edible sweet fruit. Date palm male flowers and seeds are widely used in the Arab region as a tonic drink as well as an anti-diabetic [1] and anti-oxidant [2][3][4]. P. dactylifera male flowers suspension is a herbal mixture that is widely used as a folk remedy for curing male fertility in traditional medicine [5,6].
In ancient Egypt, it was used to promote women's fertility. The male flowers of the date palm are also eaten directly by people as a fresh vegetable, allegedly to enhance fertility. The previous study displayed that the extract of date palm pollen grains contains estrogenic compounds, estrone, as gonad-stimulating agents that improve male infertility and display gonadotropin activity in the rat [7]. The previous phytochemical studies on the Egyptian palm male flowers indicated the presence of cholesterol, diosgenin, estrone, estradiol, esteriol, β-amrin, β-sitosterol, and many flavonoids [8].
Hydroxycinnamates such as p-coumaric acid, caffeic acid, ferulic acid, syringic acid, sinapic acid, gallic acid and protocatechuic acid are widely spread throughout the plant families, which are combined with malic acid, anthranilic acid, shikimic acid, quinic acid and glycerol [9,10]. The isomers of caffeoylshikimic acids (3-, 4-and 5-CSA) were isolated from date of P. dactylifera as an enzymic browning substrate in dates [11]. 5-CSA was detected as the major hydroxycinnamoylshikimic acid derivative, and the 3-and 4-isomers were detected as minor components, no traces of p-coumaroylshikimic acids were detected [12].
Proanthocyanidins (PACs) are polymeric products of the flavonoid biosynthetic pathway with synonym name condensed tannins that represent one of ubiquitous groups of all plant phenols. The degree of polymerization can be used to describe the PAC molecules size [13]. PACs containing catechin, gallocatechin or afzelechin as subunits are named proanthocyanidin. PACs are found in many plant parts such as fruits, seeds, leaves and barks where they protect them against predation. Moreover, they give astringency to beverages and flavor such as fruit juices, wine and teas, and as having advantageous effects on human health.
Due to the difficulties of separation and structural complexity of hydroxycinnamates and proanthocyanidin derivatives, investigation on these compounds, in comparison with other polyphenols are limited [14,15]. Due to large number of phenolic groups and the resemblance in catechin structures, the HPLC peaks of catechin derivatives are usually unresolved [15,16]. RP-HPLC is the most communal method used in the analysis of these compounds [17].
To date, little phytochemical studies were reported for P. dactylifera male flowers [18]. The present work is focused on the analysis of the phenolic constituents of P. dactylifera male flowers as a novel rich source of phenolic acids, flavonoids and proanthocyanidins. To understand the variation of phenolic acid stereoisomers proportions, a theoretical calculation based on the Density Fonctional Theory (DFT) approach implemented in the GAUSSIAN09 series of programs were carried out.

Results and Discussion
MS experiments on LC-MS system coupled with an electrospray ion source and an ion trap mass spectrometer were performed in order to investigate the presence of different isobaric compounds and then to carry out a qualitative analysis of phenolic constituents occurring in 40% and 60% MeOH fractions from the extract of date palm male flowers. A full scan mass spectral data were obtained and reconstructed ion chromatograms (RICs) were extracted for each of the expected m/z values based on the molecular weights of the possible constituents ( Figure 1).
The ESI-MS base peak chromatogram from the male flowers shows a relatively complex mixture containing peaks of phenolic acids, flavonoids, flavones and proanthocyanidins (monomers, dimers, trimmers and tetramers). Forty-eight phenolic compounds and five other organic acids were identified using fragmentation patterns observed in tandem mass spectra (Table 1). The isomers of caffeoylshikimic and coumaroylshikimic acid have been hypothesized using MS/MS spectra. Nineteen conjugated hydroxycinnamic acids with shikimic and quininc acids, six flavan-3-ol derivatives, two anthocyandin, and 21 flavonol and flavone derivatives were tentatively identified regarding to their fragmentation.

LC-ESI-MS and DFT Analysis of Hydroxycinnamic Acid Derivatives
Hypotheses of caffeoylshikimic acids (3-, 4-and 5-CSA) and p-coumaroylshikimic acids (3-, 4and 5-p-CoSA) isomer's were illustrated ( Figure 2, Scheme 1). Moreover, DFT study was used to confirm their structures (Table 2, Figure 3 Figure 3). Caffeic acid can be esterified at positions 3, 4 or 5 on shikimic acid (dehydrated quinic acid) to give three positional isomers. The three precursor ions did not show any reproducible differences in their MS/MS spectra and contained the same main ions at m/z 179, 161 and 135, characteristic of caffeoyl moiety. On the other hand, the previous studies differentiated between them depending on the intensity of the base peak for each isomer [9].

LC-ESI-MS and DFT Analysis of Hydroxycinnamic Acid Derivatives
Generally, after collision-induced dissociation, phenolic acids produce fragmentation patterns characterized by the loss of a CO 2 (44 Da) from the carboxylic acid group. Due to this neutral loss, each tentative isomer of caffeoylshikimic acid produced the same previous studies differentiated between them depending on the intensity of the base peak for each isomer [9]. Generally, after collision-induced dissociation, phenolic acids produce fragmentation patterns characterized by the loss of a CO2 (44 Da Table 2. Relative energies (kcal·mol −1 ) and populations at 298 K in the gas phase of caffeoylshikimic acid (CSA) and p-coumaroylshikimic acid (3-p-CoSA)isomer's calculated at the B3LYP/6-31 + G(d,p) level.  Table 2. Relative energies (kcal·mol −1 ) and populations at 298 K in the gas phase of caffeoylshikimic acid (CSA) and p-coumaroylshikimic acid (3-p-CoSA)isomer's calculated at the B3LYP/6-31 + G(d,p) level. Similarly, quasi-molecular ion m/z 319 forming compounds 5 and 6 at Rt 21.13 and 22.92 min, respectively, were tentatively corresponding to p-coumaroylshikimic acid ( Figure 2, Table 1). These peaks have the same fragmentation spectra, established by the presence of ions at m/z 163, 145 and at m/z 119, which are characteristic for p-coumaroyl moiety. On the other hand, the base peak for each isomer was similar at m/z 163 but the intensity of the peak at m/z 145 showing a difference in each (Figures 2 and 4 and Scheme 1). Based on the arguments presented above, the two isomers were tentatively assigned as 3 and 5-p-coumaroylshikimic acids (3-p-CoSA and 5-p-CoSA). Structures of all these isomers were optimized by means of the DFT/B3LYP method and their local minima on the singlet PES were studied. Relative energies as well as corrections of zero point (ZPE), thermal enthalpy and free energy were gathered in Table 2 and optimized geometries are given in Figure 3. The difference in energy between the regioisomers of caffeoylshikimic acids (CSA) was 0.3 kcal·mol −1 . Similarly, quasi-molecular ion m/z 319 forming compounds 5 and 6 at Rt 21.13 and 22.92 min, respectively, were tentatively corresponding to p-coumaroylshikimic acid ( Figure 2, Table 1). These peaks have the same fragmentation spectra, established by the presence of ions at m/z 163, 145 and at m/z 119, which are characteristic for p-coumaroyl moiety. On the other hand, the base peak for each isomer was similar at m/z 163 but the intensity of the peak at m/z 145 showing a difference in each (Figures 2 and 4 and Scheme 1). Based on the arguments presented above, the two isomers were tentatively assigned as 3 and 5-p-coumaroylshikimic acids (3-p-CoSA and 5-p-CoSA). Structures of all these isomers were optimized by means of the DFT/B3LYP method and their local minima on the singlet PES were studied. Relative energies as well as corrections of zero point (ZPE), thermal enthalpy and free energy were gathered in Table 2 and optimized geometries are given in Figure 3. The difference in energy between the regioisomers of caffeoylshikimic acids (CSA) was 0.3 kcal·mol −1 . The isomers 3-CSA and 5-CSA were more stable than isomer 4-CSA by 0.3 kcal·mol −1 . Moreover, there were similarity differences in energy between the isomers of p-coumaroylshikimic acids (p-CoSA) ( Table 2). The relative free energy for each series of regioisomers has been calculated. The population of each isomer was carried out in the gas state at 298 K by using the Boltzmann population. The 3-CSA and 3-p-CoSA isomers were the most prevalent (67% and 76% respectively). The 5-CSA and 5-p-CoSA isomers population are 28% and 15%, respectively. We concluded that all regioisomers were existed, but the more prevalent regioisomers were (3-CSA and 3-p-CoSA).

LC-ESI-MS Analysis of Proanthocyanidins Derivatives
The obtained m/z values evidenced the presence of six flavan-3-ol derivatives belonging to catechin and gallocatechin series. Proanthocyanidins are formed from the condensation of monomeric units and may differ in the position and configuration of their monomeric linkage. ESI-MS fingerprint obtained from P. dactylifera extract exhibited a complex mixture containing molecular ions for proanthocyanidin monomers, dimers, trimmers and tetramers.
The two deprotonated ions at m/z 289 with different retention times resulted from the diastereoisomers (catechin and epicatechin). MS/MS spectrum pattern of the ion peaks at m/z 289 exhibited the major product ions at m/z 109, 125, 137, 151, 165, 179, 205, 245, 247 and 271. The product

LC-ESI-MS Analysis of Proanthocyanidins Derivatives
The obtained m/z values evidenced the presence of six flavan-3-ol derivatives belonging to catechin and gallocatechin series. Proanthocyanidins are formed from the condensation of monomeric units and may differ in the position and configuration of their monomeric linkage. ESI-MS fingerprint obtained from P. dactylifera extract exhibited a complex mixture containing molecular ions for proanthocyanidin monomers, dimers, trimmers and tetramers.
The two deprotonated ions at m/z 289 with different retention times resulted from the diastereoisomers (catechin and epicatechin). MS/MS spectrum pattern of the ion peaks at m/z 289 exhibited the major product ions at m/z 109, 125, 137, 151, 165, 179, 205, 245, 247 and 271. The product ion at m/z 271 ion was derived from the loss of a water equivalent (18 Da) and the product ions at m/z 245 and 247 were due to the loss of 42 and 44 Da (HC≡C-OH, CH 2 =CH-OH). The product ion at m/z 165 [M − H-125] − may be resulted after the elimination of the ring A from it by the HRF fission [11]. The ion with m/z 125 [M − H-165] − confirmed the HRF fission. The ion product at m/z 137 was resulted from an RDA of ring C that was confirmed by the presence of product ion at m/z 151 [11].  [11]. The ESI-MS/MS spectral data of afzelechin (at m/z 273) and gallocatechin (at m/z 305) derivatives were similar of catechin and epicatechin [24].
In Table 1, the spectra of date palm male flowers PAC dimers are represented by the two B-type compounds with quasi-molecular ions at m/z 577 and 593 [23,24]. Their MS/MS spectra elucidated that the relative abundance of major product ions (e. g., 287, 289, 305, 407, 409, 417, 421, 425, 433, 439, 441, 449) varied, indicating that they may represent different isomeric forms, arising from the different linkage of monomeric flavan-3-ol units [11]. After neutral losing of a 152 Da, the deprotonate peak ion at m/z 577 give the product ion at m/z 425 due to the cleavage of ring B from the flavan-3-ol through RDA reaction of ring C. Loss of 152 Da indicates that ring B of the top unit has two hydroxyl groups. The product ion at m/z 425 was due to loss of 18 Da (H2O), most likely from the free 3-OH, to give a stable product ion at m/z 407 (RDA). The m/z 451 ion was formed after HRF on the top unit of the dimer. HRF on the base unit of the dimer was not suitable for the same reason as RDA. Loss of 126 Da indicates that the A ring of the top unit has a 1,3,5-trihydroxybenzene structure. Moreover, formation of the diagnostic product ion at m/z 451 was due to the cleavage of two hydroxyl moieties at the position 3′ and 4′ of the B ring. The top unit of this dimer was tentatively identified as catechin derivative. Because the chirality of C-3 on the flavan-3-ols cannot be differentiated by MS, catechin elucidates either catechin or epicatechin. The connection sequence of this dimer has been derived to be catechin-catechin [24][25][26].  After neutral losing of a 152 Da, the deprotonate peak ion at m/z 577 give the product ion at m/z 425 due to the cleavage of ring B from the flavan-3-ol through RDA reaction of ring C. Loss of 152 Da indicates that ring B of the top unit has two hydroxyl groups. The product ion at m/z 425 was due to loss of 18 Da (H 2 O), most likely from the free 3-OH, to give a stable product ion at m/z 407 (RDA). The m/z 451 ion was formed after HRF on the top unit of the dimer. HRF on the base unit of the dimer was not suitable for the same reason as RDA. Loss of 126 Da indicates that the A ring of the top unit has a 1,3,5-trihydroxybenzene structure. Moreover, formation of the diagnostic product ion at m/z 451 was due to the cleavage of two hydroxyl moieties at the position 3 and 4 of the B ring. The top unit of this dimer was tentatively identified as catechin derivative. Because the chirality of C-3 on the flavan-3-ols cannot be differentiated by MS, catechin elucidates either catechin or epicatechin. The connection sequence of this dimer has been derived to be catechin-catechin [24][25][26] The flow rate of sheath gas (N 2 ) was 70 (arbitrary units) and the flow rate of auxiliary gas (N 2 ) was 10 (arbitrary units). The scan range was m/z 150-2000, the duration for each injection was 150 ms and the number of micro-scan was two. The first scan step was a full-scan mass spectrum to obtain data on anions in the specified scan range. The collision energy of the second scan step (MS/MS) was 35%. The resulting product ions [M − H] − are examined in a second mass measurement step (MS 2 ) [13,28].

DFT Study
Relative stabilities of these isomers were optimized using the DFT approach implemented in the GAUSSIAN09 series of programs [29]. A 6-31 + G(d,p) basis set was employed for each atom. The potential energy surfaces (PES) was described by using the B3LYP hybrid functional [30]. The analytic gradients were employed in order to optimize the geometries of the isomers. In order to identify the local minima and to estimate the corresponding zero-point vibrational energy (ZPE) [31,32], the frequencies of the isomers were calculated. The thermodynamic quantities (relative energies, corrections of zero point energy, thermal enthalpy, and free energy differences) at 298 K in the gas phase of caffeoylshikimic acids (3-, 4-and 5-CSA) and p-coumaroylshikimic acids (3-, 4-and 5-p-CoSA) were done. The relative populations of the different isomers were calculated from Boltzmann's distribution using the calculated values of Gibbs free energy: P i P j = e G • j −G • j k B T , where P n is the probability of state n, G • n is the free energy of state n with n = (i, j), k B is the Boltzmann constant, T is the temperature of the system.

Conclusions
The analytical procedure elucidated in this work proved a rapid, simple method to simultaneously extract phenolic acids, proanthocyanidins and flavonoids. Oligomers and polymers detection is important for plant scientists; however, the constituent units and the connection sequence of low oligomers (DP < 5) identification are crucial for the nutritionists who are interested to study their possible human health and bioavailability effects. This qualitative analysis provided a valuable fingerprint of the main metabolites occurring in P. dactylifera male flowers. This is the first phytochemical analysis has been done from male flowers. The varieties of the structures of B-type proanthocyanidin, monomers, dimers, trimmers and tetramers were revealed by HPLC-ESI-MS/MS studies. The identification of B-type linkages using LC-MS/MS alone eliminates a number of tedious separation steps. The data obtained in our research shows that P. dactylifera male flowers could be a good source of hydrolysable proanthocyanidins, flavonoids glycosides and phenolic cinnamate derivatives, therefore it can be considered as a rich source of catechin and gallocatechin derivatives. Qualitative analysis results confirmed that male flowers of P. dactylifera obtained during flower production could represent an interesting source of phenolic compounds, with respect to the high variety of compounds showed by the metabolic profiling analysis presented in this experimental work.