Identification of Phenolic Compounds from Hancornia speciosa (Apocynaceae) Leaves by UHPLC Orbitrap-HRMS

Apocynaceae is a botanical family distributed mainly in tropical and subtropical regions of the world. In Brazil, they comprise about 90 genera and 850 species, inhabiting various types of vegetation. Within this large botanical family, the genus Hancornia is considered monotypic, with its only species Hancornia speciosa Gomes. Antihypertensive, antidiabetic, and antiviral activities are described for this species. Despite having been the target of some studies, knowledge of its chemical composition is still limited. In this study, the phenolics of H. speciosa leaves were analyzed using ultra-high performance liquid chromatography (UHPLC) coupled to Orbitrap high-resolution mass spectrometry (HRMS). As a result, 14 compounds were identified viz. protocatechuic acid, catechin, and quercetin, and another 14 were putatively identified viz. B- and C-type procyanidins, while just one compound remained unknown. From the identified compounds, 17 are reported for the first time viz. coumaroylquinic acid isomers and eriodyctiol. The results show that Hancornia speciosa can serve as source of valuable phenolics.


Introduction
Apocynaceae is a botanical family that includes between 3700 and 5100 species, which are distributed almost all over the world, but mainly in tropical and subtropical regions. Approximately 90 genera and 850 species of Apocynaceae occur in Brazil, inhabiting various types of vegetation. Within this large botanical family is the monotypic genus Hancornia, with the species Hancornia speciosa Gomes [1].
Popularly known as "mangabeira", H. speciosa is a native Brazilian fruit tree that is distributed from the north to the south of the country. The phytogeographic domains are the Amazon Rainforest (mainly Pará and Amapá States), Caatinga, Central Brazilian Savanna (cerrado), and Atlantic Rainforest [2]. The fruit name "mangaba" comes from the indigenous language Tupi-guarani and means "good fruit for eating". The tree is medium-sized (2 to 10 m tall) with an irregular canopy and a twisted, highly-branched trunk. The branches are smooth and reddish, exuding latex over its entire length [3,4].

Results
The constituents of the ethanolic extracts of H. speciosa were determined by UHPLC/MS/MS. Ethanol was selected as the extractive solvent because the previously described pharmacological activities were based on an ethanolic extract [13][14][15]. In a pilot study using an ion-trap machine, both positive and negative modes were analyzed. Visualization of both chromatograms revealed more intense and well-resolved chromatographic peaks in the negative compared to the positive ion mode. This is in line with the observation that H. speciosa is a species rich in phenolic compounds [19], which are supposed to ionize better and have a characteristic mass spectral behavior in the negative mode [22]. Therefore, profiling of the phenolics present in the ethanolic extract of H. speciosa leaf, negative electrospray ionization (ESI) mode with a UHPLC run of 15 min was adopted as method of choice. Figure 1 shows the base peak chromatogram obtained under conditions described in detail in the Materials and Methods section. Compounds were identified through co-injection with reference samples or on the basis of fragmentation patterns compared with literature data. Thus, it was possible to identify 28 compounds, while one compound remained unknown. Table 1 compiles the data of the suggested compounds. Chemical structures of these compounds are given in Figure 2. Out of the 28 identified compounds, 15 are described here for the first time for H. speciosa. Compound 1, eluted at 0.35 min, was identified on the basis of the ion obtained [M − H] − at m/z 191.0559. Literature ascribes this exact mass to the structure of quinic acid [23]. This compound was previously reported in the leaves of H. speciosa [15]. Compound 2 at 0.68 min showed a mass spectrum with a [M − H] − peak at m/z 315.0716. Examination of mass data banks showed these data to be consistent with the structure of protocatechuic acid hexoside. This finding was reinforced by the analysis of the fragments formed from this ion at m/z 153 originating from hydrolysis with the loss of an osidic moiety of 109 mass units, giving rise to the fragment m/z 153 after loss of CO2, which is in line with literature data for the suggested structure [24]. This compound is reported for the first time in H. speciosa.
Compound 3 at 0.85 min showed a mass spectrum with the [M − H] − peak at m/z 153.0193, consistent with the structure of protocatechuic acid. This finding was reinforced by co-injection of a standard sample of this compound, which is reported for the first time in H. speciosa. At least two compounds co-eluted at 1.30 min; the ion at m/z 343.1027 did not match any compound listed in the databases (compound 4), and the other at m/z 577.1341 was consistent with the deprotonated molecule of B-type procyanidin (compound 5). Likewise, the mass spectra from the peaks at 2.88 and 3.56 min had similar molecular ions and fragmentation patterns as compound 5, which led us to believe that compounds 9 and 13 were also B-type procyanidins. These compounds have already been reported in infusions of the bark of H. speciosa by direct injection [25].
The peaks at 1.64 and 3.36 min showed a molecular ion at m/z 289.0712, and were assigned to the deprotonated molecule of (+)-catechin (compound 6) and (−)-epicatechin (compound 10), respectively, according to the retention time of their corresponding standards. For both molecules, the main fragment produced in the MS 2 spectra was the [M -H − CO2] − ion (m/z 245). This dominant fragmentation behavior when using Orbitrap has been previously described [22]. In addition, MS 2 spectra revealed two other fragments, m/z 205 generated from a scission in the B ring, and m/z 179 from the loss of a catechol moiety.  [23]. This compound was previously reported in the leaves of H. speciosa [15]. Compound 2 at 0.68 min showed a mass spectrum with a [M − H] − peak at m/z 315.0716. Examination of mass data banks showed these data to be consistent with the structure of protocatechuic acid hexoside. This finding was reinforced by the analysis of the fragments formed from this ion at m/z 153 originating from hydrolysis with the loss of an osidic moiety of 109 mass units, giving rise to the fragment m/z 153 after loss of CO 2 , which is in line with literature data for the suggested structure [24]. This compound is reported for the first time in H. speciosa.
Compound 3 at 0.85 min showed a mass spectrum with the [M − H] − peak at m/z 153.0193, consistent with the structure of protocatechuic acid. This finding was reinforced by co-injection of a standard sample of this compound, which is reported for the first time in H. speciosa. At least two compounds co-eluted at 1.30 min; the ion at m/z 343.1027 did not match any compound listed in the databases (compound 4), and the other at m/z 577.1341 was consistent with the deprotonated molecule of B-type procyanidin (compound 5). Likewise, the mass spectra from the peaks at 2.88 and 3.56 min had similar molecular ions and fragmentation patterns as compound 5, which led us to believe that compounds 9 and 13 were also B-type procyanidins. These compounds have already been reported in infusions of the bark of H. speciosa by direct injection [25].
The peaks at 1.64 and 3.36 min showed a molecular ion at m/z 289.0712, and were assigned to the deprotonated molecule of (+)-catechin (compound 6) and (−)-epicatechin (compound 10), respectively, according to the retention time of their corresponding standards. For both molecules, the main fragment produced in the MS 2 spectra was the [M -H − CO 2 ] − ion (m/z 245). This dominant fragmentation behavior when using Orbitrap has been previously described [22]. In addition, MS 2 spectra revealed two other fragments, m/z 205 generated from a scission in the B ring, and m/z 179 from the loss of a catechol moiety.  The peaks at 3.45 and 3.62 min showed a deprotonated molecule at m/z 337.0923, and were tentatively assigned to coumaroylquinic acid isomers (compounds 11 and 14). This finding was supported by its product ion spectrum, which showed peaks at m/z 191, 173, and 163, obtained through heterolytic scissions and loss of water, which is in agreement with literature data [26]. These isomers are reported for the first time for H. speciosa.
Compound 12 at 3.53 min showed a molecular ion peak at m/z 865.1958, which was assigned to a C-type procyanidin. Additionally, this deprotonated molecule showed a fragment ion at m/z 577 due to a homolytic scission, corresponding to the dimer unit, and at m/z 695, originating from a retro-Diels-Alder fragmentation and loss of water. These data corroborate the literature data for the suggested chemical structure [25]. This is the first report of a C-type procyanidin in an H. speciosa extract.
The peak at 3.65 min had a molecular ion at m/z 609.1453, and was assigned to rutin (compound 15). This identification was supported by the base peak in its MS 2 spectrum (m/z 301), corresponding to the loss of a rutinoside moiety, and on the basis of the co-injection of a reference compound. Rutin has already been reported in H. speciosa leaves [15]. Compound 16 at 3.70 min showed a mass spectrum with a molecular peak at m/z 463.0887, consistent with the structure of quercetin hexoside. The peaks at 3.45 and 3.62 min showed a deprotonated molecule at m/z 337.0923, and were tentatively assigned to coumaroylquinic acid isomers (compounds 11 and 14). This finding was supported by its product ion spectrum, which showed peaks at m/z 191, 173, and 163, obtained through heterolytic scissions and loss of water, which is in agreement with literature data [26]. These isomers are reported for the first time for H. speciosa.
Compound 12 at 3.53 min showed a molecular ion peak at m/z 865.1958, which was assigned to a C-type procyanidin. Additionally, this deprotonated molecule showed a fragment ion at m/z 577 due to a homolytic scission, corresponding to the dimer unit, and at m/z 695, originating from a retro-Diels-Alder fragmentation and loss of water. These data corroborate the literature data for the suggested chemical structure [25]. This is the first report of a C-type procyanidin in an H. speciosa extract.
The peak at 3.65 min had a molecular ion at m/z 609.1453, and was assigned to rutin (compound 15). This identification was supported by the base peak in its MS 2 spectrum (m/z 301), corresponding to the loss of a rutinoside moiety, and on the basis of the co-injection of a reference compound. Rutin has already been reported in H. speciosa leaves [15]. Compound 16 at 3.70 min showed a mass spectrum with a molecular peak at m/z 463.0887, consistent with the structure of quercetin hexoside. Like rutin, the deprotonated molecule also had a product ion at m/z 301 due to the loss of a hexose moiety. Quercetin 3-β-D-glucoside standard was co-injected, and had a similar retention time. Hence, we can suggest that the hexose moiety was bound to position C3 of quercetin. Following the elution order, compounds 17 and 18 were tentatively assigned as quercetin pentoside

Discussion
The present work reports a detailed chemical characterization of the ethanolic extract of H. speciosa leaves, using UHPLC coupled to HRMS in negative mode. Phenolic compounds comprise the majority in the composition of the studied plant, and these compounds are probably correlated to some of the pharmacological activities previously described in the literature. Our finding of phloretin and phlorizin can especially be seen as support for the proposed antidiabetic activity of the species. These compounds are SGLT1/2 inhibitors, and are found in apples or other health foods. They are suggested to influence glucose perception and signaling in humans [29]. In addition to phloretin/phlorizin, the flavonols quercetin, kaempferol, and rutin-identified in H. speciosa extract-can play a role in the serum glucose balance. The aglycone flavonoids reduce the activity of the duodenal maltase [30], while rutin beyond that is able to retard deterioration from symptomatic diabetes to death and stabilize body-weight [31]. Altogether-and in addition to the presence of cyclitols (see above)-the presence of these phenolics in H. speciosa can be seen as support for the proposed antidiabetic activity of this plant and its potential use for treatment of diabetes mellitus.
The previously reported antihypertensive effect of H. speciosa ethanolic leaf extract [14,32] can be explained by the high level of phenolics in this type of extract, as shown in the present study. The intake of this class of secondary metabolites can contribute to the treatment of hypertension via the stimulation of several mechanisms of action that reduce blood pressure [33,34]. More specifically, rutin-the major peak in the HPLC fingerprint of this extract-was shown to contribute to the antihypertensive property of H. speciosa by the induction of endothelium-dependent vasodilation in rat superior mesenteric arteries [35]. Similar to the chemical composition of the leaves shown herein, the aqueous fruit extract of the same species showed the phenolics rutin and chlorogenic acid as prominent constituents. The fruit extract and the trunk latex exhibited anti-inflammatory activity in animal models [36,37]. Additionally, the latex showed significant angiogenic activity and a potential osteogenic effect, and had no cytotoxic or genotoxic effects on life systems [19,38,39]. Chemical analysis of H. speciosa latex resulted in the identification of naringenin-7-O-glucoside, chlorogenic acid, catechin, and procyanidin [19]. The ethanolic extract of H. speciosa bark was efficient in the prevention of gastric ulcers in rodents, together with anti-Helicobacter pylori effects [10]. It may be speculated that the here-described hydroxyl fatty acids for which antibiotic behavior is reported further contribute to this activity [40].
The fruit pulp of H. speciosa is rich in minerals, viz. potassium, copper, zinc, and iron; ascorbic acid and phenolics, viz. catechin, rutin, chlorogenic, vanillic, coumaric, and rosmarinic acids. The absence of toxic effects in in vivo assays-in addition to the antimutagenic properties of this fruit-highlight its potential as a functional food [41]. All of these findings support the widespread applicability of H. speciosa in the food and pharmaceutical industry to produce nutraceuticals and herbal drugs for varied treatments.
In the present work, 28 compounds were detected via UHPLC-HRMS in H. speciosa ethanolic extract. Phenolics such as rutin (15), quinic (1), and chlorogenic (7) acids have been widely reported in the bark and leaf extracts of this species [20,21,25]. A direct flow injection into the ESI source of a mass analyzer of the bark infusion of H. speciosa led to the tentative identification of (epi)catechin and procyanidins (dimers to hexamers of (epi)catechin) [25]. Herein, (+)-catechin (6) and (−)-catechin (10) could be confirmed in the leaf extract because of the use of a separation step (UHPLC) and the co-injection of reference standards, which were not included in the aforementioned method. Procyanidins viz. dimers (5, 9, and 13) and a trimer (12) of (epi)catechin could be annotated in the ethanolic leaf extract in the same way as they were in the bark infusion study [25]. Derivatives of caffeic acid, quercetin, and kaempferol are commonly found in H. speciosa leaf extracts [15,42]. The identification of their aglycone forms in the present work was supported by the co-injection of reference standards.
Although phenolics are widely identified in species belonging to the Apocynaceae family [43,44], most of its genera is known to have monoterpene indole alkaloids as the major class of secondary metabolites [45][46][47][48]. Hancornia seems to be an exception; no alkaloids have been described for this species. Despite these phytochemical differences, H. speciosa and other Apocynaceae plants share some pharmacological activities-e.g., antidiabetic [49], antibacterial [50], cytotoxic [51], and anti-inflammatory [52] properties.
The flavones luteolin (23) and apigenin (24)-reported for the first time in H. speciosa-and the flavonol quercetin (22) are some of the most efficacious plant flavonoids for cancer chemoprevention [53]. In general, plants rich in phenolic acids and flavonoids show a broad range of therapeutic properties that can contribute to a reduction in the incidence of chronic health problems, such as cancers, diabetes, and cardiovascular diseases [54][55][56].

Plant Material and Sample Preparation
Leaves of H. speciosa were collected in November 2015 in the municipality of Santa Rita, Paraíba State, Brazil. Identification of the plant species was validated by Professor Dr. Leonardo de Melo Versieux, from the Federal University of Rio Grande do Norte (UFRN), and a voucher specimen (UFRN20382) is currently deposited at the Herbarium of UFRN.
The collected material was air-dried in an air-forced oven at a continuous temperature of 40 • C for 72 h and ground in a mechanical mill prior to extraction. About 300 g of the powder were macerated in ethanol:water (70:30 v/v) at room temperature for 7 days under constant stirring. The suspension was filtered, and the solvent was removed under reduced pressure in a rotary evaporator at 40 • C. The extract was freeze-dried, and 10 mg were then homogenized with 1.5 mL of 100% methanol using an ultrasonic bath for 10 min. Extracts were then vortexed vigorously and centrifuged at 12,000 g for 5 min. For UHPLC-MS analyses, 1 mL of the supernatant was aliquoted and placed on a C18 cartridge (100 mg) preconditioned with water and methanol. The sample was then eluted using 1 mL of methanol and directly injected into the UHPLC system.

UHPLC-MS Analysis
The negative ion high-resolution ESI and collision-induced dissociation (CID) MS n spectra were obtained from an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a heated electrospray ion source (negative spray voltage of 3 kV, capillary temperature of 300 • C, source heater temperature of 400 • C, Fourier Transform Mass Spectrometer (FTMS) resolution of 30.000). Nitrogen was used as a sheath and auxiliary gas. The MS system was coupled to a UHPLC system (Dionex UltiMate 3000, Thermo Fisher Scientific) equipped with a RP-18 column (particle size 1.9 µm, pore size 175 Å, 50 × 2.1 mm ID, Hypersil GOLD, Thermo Fisher Scientific; column temperature 40 • C) and a photodiode array detector (220-600 nm, Thermo Fisher Scientific). The mobile phases were H 2 O (A; Fluka Analytical, LC-MS Chromasolv) and CH 3 CN (B; Fluka Analytical, LC-MS Chromasolv) with 0.2% formic acid by using the following binary gradient at a flow rate of 400 µL/min: 0 to 1 min, isocratic 95% A, 5% B; 1 to 1.5 min, linear from 5% to 70% B; 1.5 to 8.5 min, linear from 70% to 100% B; 8.5 to 10 min, isocratic 100% B; and 10 to 15 min, isocratic 5% B. The injection volume was 2 µL. The CID mass spectra (buffer gas: helium) were recorded using normalized collision energy (NCE) of 35%. The instrument was externally calibrated by the Pierce ESI negative ion calibration solution (product No. 88324) from Thermo Fisher Scientific. The data were evaluated using the software Xcalibur 2.7 SP1.

Identification of Compounds in H. speciosa Extract via UHPLC-MS
The limit of detection (LOD) for every peak to be considered as caused by a compound was the signal-to-noise ratio (s/n) of 3:1. Compounds were characterized by their UV/Vis spectra (220-600 nm), retention time, full mass spectra, and MS n fragmentation patterns, and by comparison to spectra of reference compounds and literature data. Accurate m/z values with their predicted molecular formula (error less than 5 ppm) were used for putative identification after database searches, such as in NIST, mass bank, Reaxys, SciFinder, and phytochemical dictionary database (CRC, Wiley). MS 2 fragments were compared with compounds listed in the MassBank (http://www.massbank.jp/) or using in silico fragmentation in MetFrag (http://msbi.ipb-halle.de/MetFragBeta/). Identifications were confirmed with authentic compounds whenever available in-house.

Conclusions
The chemical characterization of the extract of H. speciosa leaves was achieved using a rapid and sensitive method-namely, UHPLC Orbitrap-HRMS. This technique allowed us to identify twenty-eight compounds, while just one remained unknown. From the identified compounds, seventeen were described for the first time for the plant species, of which the hydroxyl fatty acids found support for antibiotic claims, and the two phenylpropanoids phloretin and phlorizin and the flavonols quercetin and kaempferol support the proposed antidiabetic properties. The results demonstrate the potential of H. speciosa as a source of valuable phenolics. The knowledge obtained is of importance for future studies and possible uses of this plant, allowing a better analysis of cultivars or standardization of extracts. Further studies are needed to analyze the variation in the phenolic content of H. speciosa cultivated under distinct conditions, like regional, seasonal, and time of harvest influences.