Determination of the Phenolic Profile and Antioxidant Activity of Leaves and Fruits of Spanish Quercus coccifera

In this work, we report the phytochemical composition and antioxidant activity of methanol extracts of leaves and fruits (acorns) ofQuercus coccifera (kermes oak). Forty-one compounds were characterized using high-performance liquid chromatography with electrospray multistage mass spectrometry (HPLC-ESI-MS) with an ion trap mass spectrometer. A high percentage of the detected compounds were gallic acid derivatives, although some saccharides and flavonoids were also present..is phytochemical pattern is typical in Quercus species, which are rich in gallotannins. .ese compounds are partially responsible for the cardioprotective effects observed in different food samples containing them. We evaluated the antioxidant activity by ABTS and DPPH assays. In both cases, high antioxidant activity was observed, being higher in acorns than in leaves. .e high antioxidant potential of the extracts, which is related to the high total phenolic content, indicates the potential benefit of the use of this species as a source of bioactive compounds.


Introduction
Plants represent a rich source of natural compounds which are responsible for many multifunctional biological effects.In the last few years, exhaustive research is being carried out to obtain new raw materials from plants for the development of products with healthy characteristics, which help maintain or improve health and protect against chronic diseases.e genus Quercus (Fagaceae) has 450 species estimated worldwide [1] and has been widely investigated for years, not only due to their extensive use in the wine and wood industries but also for animal feeding and medicinal purposes [2].Acorns of Quercus species are a high-protein food source for a wide array of wildlife and are also used to fatten poultry and pigs [3,4].ey are not only a source of important nutrients, namely carbohydrates, proteins, fatty acids, and sterols [5,6], but also of phenolic constituents [3,6,7].On the contrary, leaves from different Quercus species, which are commonly consumed as tisanes (aqueous extracts), also contain bioactive compounds, in particular phenolics [1,8].
ese biological activities are thought to be associated, at least in part, with the presence of phenolic compounds, such as flavonoids and tannins [3,4,11,17].e phenolic profiles vary significantly among Quercus species.For instance, high levels of gentisic and chlorogenic acids, as well as of the flavonoids naringin and rutin, have been found in Quercus acuta, Quercus glauca, Quercus myrsinifolia, Quercus phylliraeoides, and Quercus salicina [18].Nevertheless, none of these compounds was detected in any other Quercus species [6].Likewise, several gallic acid derivatives have been solely found in Quercus ilex, Quercus rotundifolia, and Quercus suber [3].Despite the phylogenetic variability, flavonoids, phenolic acids, and tannins are somehow ubiquitous in all Quercus species [6].High levels of ellagitannins, a group of condensed tannins, have been reported in woods and barks of several Quercus species used in cooperages [19].Gallotannins, another essential group of tannins, have also been reported in extracts of Quercus species [1,20].Other phenolic compounds such as flavonoids of quercetin and kaempferol have been found in Quercus leaves [1].
Kermes oak (Quercus coccifera L.) is a small evergreen shrub of fewer than 2 meters, whose fruits are acorns provided with stings.Q. coccifera is the prevailing species in the evergreen sclerophyllous shrublands, which are an important part of Mediterranean rangelands.Despite its low commercial value with regard to wood production, it plays a significant role in preventing soil erosion [21], and it is used for fodder production for domestic and wild animals [22].Kermes oak acorns seem to be a highly energetic resource for small ruminants such as goats and lambs and are often compared to barley [23,24].
Several biological effects have been reported for Q. coccifera such as neuroprotective [25], antibacterial [26,27], antifungal [26], antihelmintic [28], and antioxidant [29] activity.Furthermore, previous studies have shown that Q. coccifera contains tocopherols and fatty acids [7], besides phenolic compounds such as tannins and flavonoids [27,29].In a recent article [30], the authors characterized individual phenolics in fruits and leaves of Q. coccifera, although of only seven compounds.Phenolic compounds have been shown to be responsible for many health benefits [6] and are very useful in the food industry since they can be used as dietary supplements or as preservatives instead of synthetic antioxidants such as butylhydroxytoluene (BHT) and butylhydroxyanisole (BHA), which have negative effects on human health.erefore, the leaf and acorn extracts can represent a valuable source of natural antioxidants for different applications.e extracts can be obtained by using simple extraction procedures directly from the raw material, reducing the need for additional processing stages.
Considering the variety of phenolic compounds that have been reported in different Quercus species and that the published research concerning phenolics in Q. coccifera is scarce, our work aimed at identifying the extractable phenolic compounds present in acorns and leaves from Q. coccifera and their antioxidant activity.ese data provide a better understanding of the composition of this Quercus species and can lead to further investigations regarding the valorization of its residues and the use of its biomass, within a biorefinery concept, for the production of biofuels; chemical, pharmaceutical, and care products; and bioenergy.e exploitation of leaves and acorns from Q. coccifera on an industrial scale could contribute to improving the sustainability of the agrofood chain by achieving new food products or as alternative sources of different highly-valued food ingredients.e upgrading of these products of the forest industry is an important challenge in the development of a sustainable economy and environmental friendly industrial processes.

Sample Collection and Preparation.
Plant materials were collected from different plants placed in Sierra Morena, a mountain range in the province of Jaén, Andalucía, southcentral Spain.e materials were collected in October 2016, at an approximate height of 550 meters (38 °08′01.97″N, 3 °58′30.03″W).For analysis, we separated plant materials into leaves and acorns (fully ripe) and analyzed all samples as the same batch.
For the extraction of the phenolic compounds, we carried out an ultrasound-assisted solid-liquid extraction using 5 g of sample powder and 100 mL of MeOH using a sonicator with a temperature controller (Bandelin Sonorex Digital 10P; Sigma-Aldrich, Madrid, Spain) at 35 Hz and 280 W for 60 min (room temperature).en, we filtered the extracts, eliminated the chlorophylls by adsorption on activated charcoal, and concentrated the extracts to dryness using a rotary evaporator (Buchi Rotavapor R-114) at 40 °C.
e resulting extracts were stored at 4 °C until analysis.

HPLC Analysis.
e analysis of the phytochemical profile was carried out by using HPLC-MS n .Dried extract (DE) of 5-10 mg was re-dissolved in 1 mL MeOH.After filtration through 0.45 µm PTFE membrane filters, 10 μL of each solution was injected in the chromatographic system.
An Agilent Series 1100 HPLC system (Agilent Series 1100, Agilent Technologies, Santa Clara, CA, USA) with a G1315B diode array detector was used.A reversed-phase Kinetex core-shell C 18 analytical column of 100 × 2.1 mm and 2.6 µm particle size (Phenomenex, Torrance, CA, USA) and a C 18 Security Guard Ultra cartridge (Phenomenex) of 2.1 mm i.d.placed before the analytical column were used.
e following gradients were used: initial mobile phase, 10% CH 3 CN; linear increase to 25% CH 3 CN (0-10 min); 25% CH 3 CN (10-20 min); linear increase to 50% CH 3 CN (20-40 min); linear increase to 100% CH 3 CN (40-42 min); and return to initial mobile 2 Journal of Chemistry phase and stabilization time of 7 min.e mobile phase flow rate was 0.4 mL•min −1 .e HPLC system was connected to an ion trap mass spectrometer (Esquire 6000, Bruker Daltonics, Billerica, MA, USA) equipped with an electrospray (ESI) interface.e scan range was set at m/z 100-1200 with a speed of 13,000 Da/s.e ESI conditions were as follows: drying gas (N 2 ) flow rate and temperature, 10 mL/min and 365 °C; nebulizer gas (N 2 ) pressure, 50 psi; capillary voltage, 4500 V; and capillary exit voltage, −117.3V. We used the auto MS n mode (negative and positive modes) for the acquisition, with isolation width of 4.0 m/z, and fragmentation amplitude of 0.6 V (MS n up to MS 4 ).
e analysis of the phenolic composition was performed with HPLC-ESI-MS n using negative ionization mode.

Total Phenolic and Flavonoid Contents and Antioxidant Capacity Assays.
e total phenolic (TPC) and flavonoid (TFC) contents were obtained using the Folin-Ciocalteu and aluminum chloride methods, respectively.TPC was expressed as mg of gallic acid equivalents (GAE) per g of DE.TFC was expressed as mg of quercetin equivalents (QE) per g of DE.ABTS •+ and DPPH radical scavenging activities were expressed as μmol Trolox equivalent (TE) per 100 g of DE.Detailed procedures have been previously reported [31].

Results and Discussion
Currently, there is scarce information concerning Q. coccifera, as most of the published articles regarding the Quercus genus have focused on Q. suber due to its extensive use in cork industry.For Q. coccifera, water and methanol [29] and water with mixtures of acetone, ethyl acetate, and methanol [27] have been used as extraction solvents.For Q. suber, different extraction solvents and temperature have been reported: water at 80 °C [1], 100% methanol and 80% methanol : water [12], or methanol and hexane [10].Hence, it is clear that there is no standard protocol to carry out the extraction of phenolics, as they have very different polarities.In this work, we have selected methanol-one of the most common extractants [6]-to carry out the extraction procedure.We performed an ultrasound-assisted solid-liquid extraction, whose main benefits are its simplicity and rapidity.

Phytochemical Profile.
e initial step for the characterization of the compounds consisted in the determination of the molecular weight of each compound.In the negative ion mode MS 1 spectrum, the most intense peak corresponded to the deprotonated molecular ion [M − H] − or formate adduct [M + HCOOH − H] − .e base peak chromatograms of extracts of acorns and leaves are shown in Figure 1, whereas the MS data for the detected compounds are reported in Table 1.

Tannins and Gallic Acid Derivatives.
Most of the compounds found in the extracts of Q. coccifera corresponded to tannins, which can be classified in proanthocyanidins, ellagitannins, and gallotannins (galloylglucoses).
Compounds 8 and 13 were tentatively characterized as isomers of hexahydroxydiphenyl-digalloyl-glucose based on their molecular weight and fragment ions at m/z 301 (loss of a digalloyl-hexoside residue) and 483 (loss of a hexahydroxydiphenyl residue) [20].
Compound 20, with [M − H] − at m/z 473, suffered two consecutive losses of 152 Da (galloyl moieties) to yield gallic acid at m/z 169, fragmentation that corresponded to trigallic acid.
Compound 23 exhibited the deprotonated molecular ion at m/z 615.After the neutral losses of 152 Da (galloyl) and 162 Da (hexoside), it yielded a fragment ion at m/z 301, corresponding to quercetin.Hence, we identified this compound as quercetin hexoside-gallate.
Compound 26, [M − H] − at m/z 441, suffered the neutral loss of a galloyl moiety to yield (epi)catechin at m/z 289, so we characterized it as (epi) catechin-O-gallate.
3.1.2.Flavonoids.Compound 10 was identified as catechin by comparison with an analytical standard.is compound has already been reported in several Quercus species, such as Q. acuta, Q. salicina, and Quercus resinosa, among others [6].ree kaempferol derivatives were present in the extracts.e aglycone at m/z 285 was identified by comparison with an analytical standard.Compound 38 suffered the neutral loss of 308 Da (rutinoside), whereas compound 43 displayed the neutral loss of 146 (rhamnoside) and 308 Da.
e exact nature of compound 44 could not be completely elucidated.
Compound 41 and 42 were characterized as quercetinrhamnoside-hexoside-rhamnoside isomers based on the neutral losses of rhamnoside (146 Da) and hexoside (162 Da) moieties, and the aglycone quercetin observed at m/z 301 (comparison with an analytical standard).

Journal of Chemistry
e presence of kaempferol derivatives and quercetin derivatives has previously been reported in the leaves of di erent Quercus species [1], although mono-and diglycosides were detected, not tri-glycosides, which are here reported for the rst time to the best of our knowledge.

Other Compounds.
Compound 1 was characterized as a quinic acid derivative due to the 191⟶127 fragmentation.
Compound 2 was identi ed as a disaccharide, whereas compounds 24 and 32 were tentatively characterized as saccharide derivatives due to the MS n fragment ions at m/z 179, 161, 143, 119, and 113 [37].
Compound 3 was identi ed as citric acid by comparison with an analytical standard.
Compound 14, with a deprotonated molecular ion at m/z 387, displayed fragment ions at m/z 207 and 163, characteristic of the lignan medioresinol [38].
Compound 15 was characterized as roseoside (formate adduct) due to the deprotonated molecular ion at m/z 385 and fragment ions at m/z 223 and 153 [31].

(Semi)quanti cation of Phenolics.
We performed the semiquantitative analysis of the main compounds found in leaves and acorns.Acorn extracts were rich in gallic acid derivatives.Hence, the quanti cation of these compounds was carried out using gallic acid to construct the analytical graph, using the UV chromatograph at 275 nm.For leaf extracts, we used the UV signal of analytical standards of catechin (280 nm), gallic acid (275 nm), quercetin (350 nm), and kaempferol (350 nm) to construct the calibration graphs.e results are summarized in Table 2. ese results are expressed in mg/g DE.In addition, humidity percentages were also calculated (62 ± 2% and 58 ± 3% for acorns and leaves, resp.), so that the amounts of phenolics in fresh samples could be calculated too.
To the best of our knowledge, the quanti cation of individual phenolics has not been reported for Q. coccifera.However, the results can be compared with data from other Quercus species, always keeping in mind that di erent solvents and extraction procedures have been used.

Total Phenolic Content and Antioxidant
Assays.We determined the total phenolic content (TPC) and the antioxidant activity (ABTS •+ and DPPH) of the extracts using the procedures previously reported [31].e results are depicted in Figure 2. It can be observed that very high values of TPC were observed in both acorns and leaves, although acorns had higher TPC values than leaves.High TPC values (similar to the ones observed here in leaves) have been previously reported in Q. suber [12].In a similar way to TPC, the values obtained for the ABTS •+ and DPPH antioxidant assays were also higher in acorns compared to leaves.

Conclusions
We have carried out the characterization of the phenolic pro le of methanolic extracts of leaves and acorns of Q. coccifera by using HPLC-ESI-MS n .A total of forty-one compounds were identi ed or tentatively characterized.Although some avonoids were identi ed, mainly kaempferol and quercetin derivatives, most of the compounds were condensed tannins and gallic acid derivatives.e extracts were particularly rich in gallotannins, which is in line with the reports in other Quercus species.
e phytochemical pro les of acorns and leaves were similar, although (epi) catechin dimers were only detected in leaves, and hexahydroxydiphenyl-digalloyl-glucose isomers were only present in acorns.e main compounds were quanti ed by using HPLC with UV detection.Both acorns and leaves had   high antioxidant potential, which was in agreement with the TPC values observed, particularly in acorns.
is study provides additional information concerning the phytochemical profile of this plant, which can be a valuable source of phytochemicals for the food or pharmacological industries.However, it is important to mention that these results are representative of the studied area, but samples were collected only in one year.Hence, more results are required considering that they may vary within different years or collection places.

Figure 1 :
Figure 1: HPLC-ESI/MS n base peak chromatograms (BPCs) of the methanolic extracts of acorns and leaves of Q. coccifera.

Table 1 :
Characterization of the compounds found in the methanolic extracts of acorns and leaves of Q. coccifera.

Table 2 :
Quanti cation of compounds in extracts of Q. coccifera.Results are expressed in mg/g DE.