Wild grown red and yellow hawthorn fruits from Tunisia as source of antioxidants

2.1 Samples

2 kg of Crataegus azarolus and Crataegus monogyna fruit samples was recollected in September 2009 from Kef en Nsour (Jendouba) northwestern Tunisia, located at 36° 33′ 2″ N latitude, 8° 25′ 29″ E longitude and 606 m altitude. Fruits were immediately transported after recollection to our laboratory. Fruits were peeled with a sharp knife, and the three parts: peel, pulp and seeds were lyophilized and stored at −20 °C until analyzed.

2.2 Standards and reagents

Analytical grade phenolic standards: phloroglucinol, gallic, protocatechuic, OH-benzoic, vanillic, caffeic, syringic, p-coumaric, sinapic, 3-(2′,5′-dimethoxybenzoyl) propionic (DMB propionic), homovanillic, homogentisic, ferulic and chlorogenic acids; phloridzin, catechin, epicatechin, quercetin, quercetin-3-O-glucoside, kaempferol, were purchased from Sigma–Aldrich Química S. A. (Madrid, Spain). Solvents used were HPLC-grade and were obtained from Merck (Darmstadt, Germany). Methanol, ethanol, chloroform and chlorhydric acid were supplied by Prolabo (Madrid, Spain).

2.3 Preparation of the phenolic extracts

The lyophilized peel, pulp and seed were processed separately; Approximately 2 g of dried parts from each Crataegus species was extracted three times by 15 ml of methanol/acidified water HCl 1.5 N, during 30 min in an ultrasonic bath (FALC Instruments, Italy) (Khanizadeh et al., 2008). The extracts were centrifuged and the three methanolic supernatants were combined and the methanol was removed in vacuo. The aqua resultant extracts were lyophilized until dried. Finally, 5 ml of ultrapure water (Millipore Milli-Q water purification system) was added into the dried extracts and filtered through a 0.45 μm membrane-filter.

2.4 HPLC–UV Analysis of Phenolics

A sample of 20 μL of the different supernatants above-obtained was analyzed using an Agilent 1200 Series liquid chromatography with a quaternary pump and a photodiode array detector (DAD) and an Ultrabase C18 column (5 μm; 4.6 mm × 150 mm) which was set thermostatically at 25 °C. Solvents used to analysis were acetic acid 2.5% (A), HPLC-grade acetonitrile (B), ultra-pure water (C) and acetic acid 2.5% HPLC-grade acetonitrile (90:10) (D) at a flow rate of 0.5 mL min⁻¹. Elution was performed as previously described by Pallaufa et al. (2008).

2.5 HPLC–MS analysis

In order to confirm the identity of the phenolic compounds that could not be done by HPLC–UV, additional analysis was carried out using HPLC with mass spectrometry detection Agilent 1100 Series liquid chromatography equipped with an API source and employing an ESI (electrospray ionization) interface. The HPLC system was connected to a DAD and a simple quadruple G1946D Q-LC/MS. Sheath as well as auxiliary gas was a mixture of helium and nitrogen. The capillary voltage was 3 V and the capillary temperature 180 °C. Solvents used were ultrapure water (A), HPLC-grade acetonitrile (B), formic acid 1% (C) and formic acid 1% HPLC-grade acetonitrile (90:10) (D) at a flow rate of 1 mL min⁻¹. Elution program start with 100% C, the gradient was the following: from 100% C to 100% D in 3 min, from 100% D to 1% B in 4 min, isocratically 1% B in 3 min, from 1% B to 12% B in 20 min, from 12% B to 50% B in 5 min, isocratically 50% B in 5 min, from 50% B to 100% C in 2 min (Pallaufa et al., 2008). Spectra were recorded in the positive ion mode and the MS detector was programmed to perform a series of consecutive scan: full scan from m/z 150 to 1500.

2.6 Phenolic identification and quantification

Chromatograms were recorded at 280, 330, 370 and 520 nm. Phenolic compounds were identified by their UV spectra recorded with a diode array detector and by LC–MS. Some of these phenolics have been previously identified with authentic markers, and others were identified by their MS spectra and their corresponding daughter MS² fragments. Anthocyanins were quantified at 520 nm as cyanidin 3-glucoside, flavonols at 360 nm as quercetin-3-O-glucoside, hydroxycinnamic acid derivatives around 320 nm as chlorogenic, acid and flavan-3-ols at 280 nm as catechin.

3.1 Antioxidant peaks identification

The method coupling high-performance liquid chromatography (HPLC) with diode-array detector (DAD) and electrospray ionization mass spectrometry with an ion trap analyser was optimized for the separation, identification and characterization of phenolic acids, flavonoid glycosides flavonoid aglycones and anthocyanins by data of the retention time, λ_max, pseudomolecular ion, main fragment ions in MS².

The Phenolic chromatographic profile at 280, 320, 370 and 520 nm of the two species of hawthorn is shown in Figs. 2 and 3. Four classes of phenolic compounds were identified, mass spectrometry analysis revealed that peaks PA1 to PA4 corresponded to different phenolic acids, A1 and A2 represented the anthocyanins glucoside, while peaks P1 to P4 were assigned to proanthocyanidins. The symbolized picks F1 to F8 belong to the flavonols and flavones subclasses.

Close

Figure 2

HPLC/MS chromatograms of different parts of C. monogyna.

Export to PPT

Close

Figure 3

HPLC/MS chromatograms of different parts of C. azarolus.

Export to PPT

3.2 Identification and structure characterization of phenolic acids and derivatives

The major phenolics compounds of Crataegus species studied by LC–MS in positive ionization mode are presented in Tables 1 and 2. Four phenolic acids are present in Crataegus corresponded to hydroxycinnamic acids derivatives such as 5-O-caffeoylquinic acid (PA1), chlorogenic acid (PA3) and p-coumaric acid (PA4). According to their UV spectra (λ_max between 314 and 328 nm) and pseudo molecular ions [M − H]⁺ (m/z at 355, 355 and 165, all of them yielding a product ion at m/z 193, due to the deprotonated quinic acid) (Rodrigues et al., 2012). Hydroxybenzoic acid represented by protocatechuic acid peak PA2 at 8.6 min (pseudomolecular ion [M − H]⁺ at m/z 155).

3.3 Identification of proanthocyanidins

The flavan-3-ols with a different degree of polymerization (i.e., catechins and proanthocyanidins) were other relevant flavonoids found in Crataegus species, especially, fruit extracts of the C. monogyna samples (Tables 1 and 2). The flavan-3-ols profiles, showed the presence of four peaks respectively symbolized in chromatograms by P1, P2, P3 and P4. The major positive ions containing structural information for peaks P1 to P4 m/z were respectively; 291, 579, and 867. Peak P4 was identified as (−)-epicatechin by comparison of its UV spectra and retention time with a commercial standard. Signal at m/z 579 (peak P2) is associated with B-type procyanidin dimer. Peaks P1 and P3 (same pseudomolecular ion [M − H]⁺ at m/z 867) were assigned to two procyanidin trimer C1 and trimer C2 containing two B-type interflavonoid linkages.

The obtained fragmentation patterns of the condensed tannins (Peaks P1–P3) are 697, 579, 427, 409, 289 and 291. They may be the assumption of two fragmentation mechanisms for these types of compounds. Either by retro Diels–Alder fragmentation; loss of 152 amu followed by dehydration; loss 18 amu and loss of 122 amu not characterized correspond to C₆H₂O₃. Also by cleavage of the C–C bonds of the interflavonoid linkages by loss of a monomeric unit [M − H-291]⁺ (Friedrich et al., 2000).

Results show considerable variations in the contents of these compounds between red and yellow fruits extracts. (−)-epicatechin is the monomer presents in all fruits parts of both Crataegus species extract, while the condensed tannins are present only in red fruit extracts.

3.4 Identification of anthocyanins

The anthocyanin profiles obtained for Crataegus species were very different. Only two broad peaks (A1 and A2) detected around 517 nm were assigned to anthocyanin especially in red Crataegus fruits (peel extract), respectively at Rt = 13.1 and 16.8 min. Identification of individual anthocyanin was performed by comparison with standards, retention times, UV/vis, mass spectral data and the mass of the sugars bound to the aglycons and the specific fragmentation patterns of the compounds. The bound sugar moieties consist of hexoses with a mass unit of 162 (glucose) and pentose with a mass unit of 132 (arabinose).

Cyanidin aglycone mass is 287, the order of elution of the glycosides on the C18 column is glucoside before the arabinoside. The MS data of the molecular and product ions of compounds A₁ and A₂ (Fig. 4) were consistent with tow constituents of Crataegus anthocyanins previously reported: cyanidin-3-O-glucoside (449/287) and cyanidin-3-O-arabinoside (419/287).

Close

Figure 4

MS spectra, ion nomenclature and major fragments from red Crataegus peel extract.

Export to PPT

The red color of Crataegus fruit was coherent with the presence of the two major anthocyanin pigments, however, in the other parts of fruits and the yellow specie, the anthocyanin was absent.

3.5 Identification of flavonols

Mass spectrometric methods can be used to obtain information on the carbohydrate sequence and the aglycone flavonols. Their identities were assigned based on their retention times (Rt), maximal UV wavelength (λ_max), pseudomolecular ions and MS² spectra, releasing fragments corresponding to the losses of sugar. Based on bibliographical sadies, fragmentation of O-glycosylated flavonoids produced the cleavage of the glycosidic bond with protons rearrangement resulting in the formation of the genin. The removing of monosaccharide residue is demonstrated by the loss of sugar. (Wolfender et al., 2000; Cuyckens and Claeys, 2004). In none of them the identity of the sugar and positions of location of the substituents could be established.

Furthermore, the pseudomolecular ions [M − H]⁺ of the identified flavonols compounds have respectively at m/z 627, 449 and 465. Major diagnostic fragments of flavonols aglycone identification are those involving the cleavage of two C–C bonds of the C-ring giving two fragment ions which provide information about the number and type of substituents. Therefore the product ions, [aglycon–H]⁺, were detected at m/z 303. In Fig. 5 are shown fragmentations of flavonols peaks. F1, F3 (same pseudomolecular ion [M − H]⁺ at m/z 627) corresponding to quercetin-3,7-O-digalactoside, quercetin-3,7-O-diglucoside. Peaks F4 and F5 also with identical molecular ions at m/z 449, thus, these peaks were tentatively assigned respectively as quercetin-3-O-galactoside and quercetin-3-O-glucoside based on their fragmentation pattern and relative fragment ion abundances. Similar reasoning was applied for the assignment of peaks F7 and F8 ([M − H]⁺ at m/z 465), releasing typical MS² fragments ions, but with different characteristics UV and different retention time, these peaks were associated with kaempherol-3-O-galactoside and kaempherol-3-O-glucoside.

Close

Figure 5

MS spectra and fragmentation pattern of the identified flavonols and flavones in Crataegus fruits extract.

Export to PPT

3.6 Identification of flavones

The C-glycosylated flavones were also found in Crataegus fruits species. Peaks F2 and F6 showed a pseudomolecular ion [M − H]⁺ respectively at m/z 595 and 433 (Fig.5). Their identities were assigned based on their pseudomolecular ions and MS² spectra, releasing fragments corresponding to the losses of rhamnosylhexosyl (−146 to 162 uma) to obtain the Luteolin aglycon at m/z 287. This disaccharide should be either a rutinoside = Glc-Rha (m/z 308) or a Gal-Rha (m/z 308) linked to the luteolin aglycone by either Glc or Rha sugar. The fragment corresponding to the losses of a hexosyl (glucose = −162 uma) giving an apeginin aglycon at m/z 271 (Ferreres et al., 2003). Therefore, these peaks were assigned to luteolin-7-O-rutinoside and apigenin-7-O-glucoside.

3.7 Quantification of antioxidants in Crataegus fruits species

The quantification of Phenolic compounds present in the fruit extracts was performed by comparison with the standard curves appropriate in each case. The individual phenolic compounds were divided into five subclasses such as phenolic acids, anthocyanins, proanthocyanidins, flavonols and flavones. Cyanidin-3-O-glucoside was used as the standard for anthocyanin quantification, quercetin-3-O-glucoside for flavonols, (−)-epicatechin and (+)-catechin for proanthocyanidins. The phenolic contents in the different parts of each Crataegus fruits species are summarized in Table 3.

Hawthorn fruit extracts are a good source of antioxidant. Being flavonols the most abundant group they represent 73.38% and 67.59% followed by tannins with a percentage 21.31 and 13.05 of the total composition, respectively in red and yellow fruits. The lower phenols content correspond to the flavones family (0.27% C. monogyna and 7.16% C. azarolus). Anthocyanin pigmentations were present only in red fruits.

Chlorogenic acid and (−)-epicatechin are the most abundant phenolic compounds identified in all fruit parts of both Crataegus species with the highest levels are localized in peel fraction. Chlorogenic acid and its isomer 5-CQA, are widely recognized to be an antioxidant for human LDL (Nardini et al., 1995). It is also known as a scavenger for reactive species of oxygen and nitrogen (Kono et al., 1997).

Other compounds are specific to each fruit parts of the two species such as hyperoside which is identified only in red peel fruit, while p-coumaric acid is specific for the yellow seed fruits.