UPLC-ESI-Q-TOF-MS/MS Characterization of Phenolics from Crataegus monogyna and Crataegus laevigata (Hawthorn) Leaves, Fruits and their Herbal Derived Drops (Crataegutt Tropfen)

Crataegus species are medicinal plants naturally growing in Europe, Asia and the north of Africa. The plant extracts have been used for a long time in traditional medicine for the treatment of cardiovascular diseases. Many natural health product, including tablets, teas, and aqueous extracts are made from Crataegus species. These products are currently marketed as an alternative therapy for New York Heart Association (NYHA I-III) heart failure. But further studies suggested the use of the plant extracts for various other cardiovascular diseases including hypertension, hyperlipidemia, arrhythmia and angina. Thus, due to the important role that hawthorn plays in medicine and human health, we have investigated qualitatively the phytoconstituents of C. monogyna and C. laevigata , leaves, fruits and their herbal derived drops (Crataegutt Tropfen) using UPLC-ESI-Q-TOF-MS/MS and HPLC-ESI-MS n . A total of 113 compounds were identified, characterized or tentatively assigned on the basis of their accurate mass data generated by Q-TOF-MS, MS/MS, MS n fragmentation patterns, retention behaviors, or by comparison with commercial reference standards and literature data. The identified constituents belonged to chlorogenic acids, phenolic acids, proanthocyanidins, flavonoid glycosides, flavonoid aglycones and derivatives, and other compounds. To our knowledge 63 of the identified phytoconstituents were not reported previously in Crataegus species and two of them for the first time in nature. Additionally, it is important to highlight that this is the first comprehensive study of the bioactive phenolic compounds of C. monogyna and C. laevigata , leaves, fruits and the herbal derived drops (Crataegutt Tropfen).


Introduction
Crataegus species also known as hawthorn (Rosaceae) are small trees and shrubs naturally growing in Europe, Asia and the north of Africa. Many natural health products, including tablets, teas, and aqueous extracts are made from Crataegus species. Crataegus is described in various pharmacopoeia including German Pharmacopoeia and Chinese Pharmacopoeia. Several reports suggested that hawthorn and the herbal derived drops (Crataegutt Tropfen) could be used as an alternative therapy for various cardiovascular diseases, such as hyperlipidemia, hypertension, arrhythmia, angina and New York Heart Association (NYHA) class I-III heart failure [1,2]. Thus, confirmed the use of hawthorn flowers, leaves and berries, alone or in combination traditionally in Europe for the treatment of a variety of ailments including high blood pressure and heart disorders [3]. However, the Complete German Commission E Monographs declares that Crataegus monogyna and Crataegus laevigata can be used to treat cases of cardiac failure [4].
Polyphenols are natural compounds characterized by a high structural diversity, and many of them are essential components in our diets. They are found only in plants and certain fungal species and are not synthesized by humans or animals [5]. Currently, polyphenols are seen as secondary metabolites characterized by a high range of physiological functions [5]. Several studies have shown that the high consumption of polyphenols have protective effects against cancer and inflammatory diseases [6]. The anti-inflammatory effects of phenolic compounds have been attributed mostly to their antioxidant activity presumably scavengers the reactive oxygen and nitrogen species in vivo [7,8]. However, the antioxidant hypothesis is under intense scrutiny [9]. It is also reported that the phenolics have anti-tumor, antidiabetic, anti-mutagenic and anti-HIV properties [10,11]. Moreover, there are several studies suggesting benefits of polyphenols intake for reducing the risks of cardiovascular problems, skin diseases, asthma, wound healing, protect from drug toxicity and UV radiations [10,12]. Although dietary plants polyphenols have been associated with several beneficial health effects for humans, their bioavailability is still under discussion [13,14].
A number of analytical techniques had been used such as liquid chromatography with diode array detection (HPLC-DAD) and liquid chromatography coupled to electrospray ionization multistage mass spectrometry (LC-ESI-MS n ) to identify different classes of phytoconstituents in hawthorn species including proanthocyanidins, phenolic acids, flavonoid glycosides, flavonoid aglycones, triterpene acids, sterols and chlorogenic acids [3,[15][16][17][18]. Herein, the aim of this study was to improve the knowledge of the methanol: water (2:1) mixture extract of the fruits and leaves of C. monogyna and C. laevigata and the traditionally derived drops (Crataegutt Tropfen)

UPLC-Q-TOF-MS/MS
The UPLC system (Agilent infinity 1260 series, Germany) was incorporated a binary pump, an auto sampler (G1367E), degasser (G1322A) and a DAD detector (1315D) with a light-pipe flow cell (recording at 280 and 320 nm). This was coupled to Ultra-Highresolution-Quadropole-Time-of-Flight (UHR-Q-TOF) (Bruker Impact HD, Bruker DaltoniK GmbH, Bremen Germany) equipped with an ESI source operating on Auto-MS/MS mode. The analysis was achieved in the negative ion mode in a mass range from m/z 50-1200. The ESI source parameters were: capillary voltage 4.5 KV; nebulising gas pressure 1.8 Bar; drying gas temperature 200.0°C, drying gas flow 9.0 L/min; Funnel 1RF 250.0 Vpp; transfer time 50.0 μs; and pre-pulse storage 2.0 μs. The MS data were analyzed through Data Analysis 4.2 software (Bruker Daltonics, Bremen, Germany). Internal calibration was achieved with 10 mL of 0.1 M sodium formate solution injected through a six port valve prior to each chromatographic run. Calibration was done using the High Precision Calibration (HPC).

UPLC and HPLC
The UPLC separation was achieved on a Polaris reverse phase C18 amide (RF-C18-A), 150 length x 2 mm inner-diameter, particle size 3 µm column (Agilent, Germany). Solvent A was water : formic acid (1000 : 0.05 v/v) and solvent B was methanol. Solvents were delivered at a total flow rate of 0.2 mL/min. The gradient profile was from 10% B to 80% B linearly in 70 min followed by 10 min isocratic and a return to 10% B at 90 min and 10min isocratic to re-equilibrate. The injection volume was 2 µL.
The HPLC separation was achieved on a 250 length x 3 mminner-diameter column containing 5 µm C18 amide, with a 5 mm x 3 mm-inner-diameter guard column (Varian, Darmstadt, Germany). Solvent A was water : formic acid (1000 : 0.05 v/v), and solvent B was acetonitrile. Solvents were delivered at a total flow rate of 0.5 mL/ min. The gradient profile was from 6% B to 80% B linearly in 70 min followed by 10 min isocratic and a return to 10% B at 90 min and 10 min isocratic to re-equilibrate. The injection volume was 5 µL [22,23].

UV Irradiation
UV irradiation experiments were performed as previously reported [24].

Results and Discussion
A reversed phase UHPLC-MS method was developed, allowing separation of 113 peaks in methanolic Crataegus extracts. Compound assignment was carried out for the identified phytoconstituents along with their m/z experimental and calculated, error (ppm), MS/MS fragments and molecular formula is presented in Table 1. Chemical structures of the identified phytoconstituents are shown in Figure 1. All identified compounds displaying a mass error of below 2 ppm thus confirming their elemental composition. Only three compounds errors were higher than 2 and below 5 ppm but their MS/MS and tandem MS data were with agreement with molecular formulas and suggested structures. Moreover, in this study not all identified compounds were fragmented in the Q-TOF. Consequently, the tandem MS fragmentation obtained from the LC-MS n was considered using a quadruple ion trap MS detector ( Table 2). The UV chromatograms at 280 nm are shown LC-ESI-MS n as a powerful analytical techniques. In this contribution, we have tested the methanolic extract of the plant materials in particular, because it known to be more effective in extraction of low molecular weight phenolic compounds. The obtained results may contribute to a better understanding of influence of hawthorn phenolics on biological, nutritional and medicinal prosperities.
Recently, the improvement of ultra-high-pressure pump systems and small size filling substances leads to improve resolution, greater separation, high peak efficiency and reduced solvent consumption and running time compared with usual HPLC. In this regard, the combination of UPLC with MS allows a better separation, identification and characterisation of bioactive compounds in medicinal plants and complex mixtures. Consequently, the mass analyser Q-TOF-MS combines the high efficiency of TOF analysis in both MS and tandem MS (MS/MS) manners, providing high mass accuracy and better sensitivity for both precursor and fragment ions [19,20].
On numerous occasions we have shown that use of modern analytical instrumentation frequently merits reinvestigation of well characterized medicinal or dietary plant material. Using improved resolution and sensitivity on occasions large number of previously overlooked secondary metabolites can be readily identified. For this reasons we decided to re-investigate Crataegus as one of the best and most intensity studied medicinal plants.

Experimental Chemicals and standards
All the chemicals (analytical grade) and authentic standards of polyphenols were purchased from Sigma-Aldrich, Applichem, HWI analytic, and Phytolab (Germany).

Plant materials and the herbal drops
Leaves and fruits of C. monogyna and C. laevigata were freshly collected from a garden in Jacobs University, Bremen, Germany. The apple (Malus domestica) fruits were purchased from a local market in Bremen, Germany. The herbal drops (Crataegutt Tropfen, Schwabe, Karlsruhe) was granted from a pharmacy in Bremen, Germany.

Sample preparation
The sample preparation was achieved as previously described [21]. Fresh hawthorn samples (10 g of each) were freeze dried with the liquid nitrogen and crushed by a mortar. Then, the samples were extracted with methanol:water (2:1) mixture by sonication for 30 min and filtered through a Whatman no. 1 filter paper. The solvents were removed by evaporation in vacuo and the extracts were stored at −20°C until required, thawed at room temperature, dissolved in methanol (50 mg/10 mL of methanol), filtered through a membrane filter (0.45 μm) and used directly for UPLC-ESI-Q-TOF-MS/MS and HPLC-ESI-MS n . The herbal drops (Crataegutt Tropfen, 94 mg/mL, extracting agent: Ethanol 45%) was diluted 10 times in methanol and directly analyzed.   in Figure S 1 in the Supporting Information. Furthermore, compounds were also compared with authentic reference standards, structured under the same experimental conditions. Taking into account the data previously reported in literature, the flavonol glycosides were allowed further structure assignment and differentiated by using suggested rules and identification criteria previously reported [25].
In the following section structure assignment of selected compounds is illustrated. Full assignment arguments are provided in the Supporting Information.

Characterization of phenolic acids and phenolic acid glycosides
Eight phenolic acids and seven phenolic acid glycosides were identified in the plant extracts and the herbal drug.
Characterization of phenolic acids: Compounds 1, 44, 106 and 108 with retention times (t R ) of 2.9, 32.1, 3.4, 3.9 min and m/z of 191, 163, 133, and 175 were identified as quinic acid, p-coumaric acid, malic acid and ascorbic acid, respectively (Table 1), by comparing their retention times and fragmentation behaviour to the MS/MS spectral data of the corresponding phenolic acids authentic standards. Peaks 92, 98, 105 and 107 (t R 4.2, 7.6, 11.6 and 3.9 min) were assigned as citric acid, salicylic acid, syringic acid and pyruvic acid, respectively. The structural identification of these compounds was based on a comparison of their MS/MS and MS n data (Table 1 and 2) with those reported in literature [26][27][28]. Phenolic acids have previously been identified and quantified in Crataegus species [29][30][31].

Characterization of phenolic acids glycosides
Compound 91 (t R 9.0 min) was identified as protocatechuic acid O-hexoside with a pseudomolecular ion [M−H] − of 315.0772 (Table  1). It produced daughter ions at m/z 153.0148 corresponding to protocatechuic acid after the neutral loss of the hexoside group and at m/z 109.0288 by the neutral loss of a hexose moiety followed by the neutral loss of CO 2 (44 Da).

Characterization of benzyl alcohol-hexose-pentose
One benzoic acid derivative was detected at t R of 21.6 min and m/z of 401.1455 (C 18 H 25 O 10 ) in the plant extracts and regarded as benzyl alcohol-hexose-pentose 23 based on the MS/MS data, which provided a daughter ion at m/z 269.1041 (C 13 H 17 O 6 ) resulted from the neutral loss of pentose unit. Moreover, the compound showed a mass error below 1 ppm thus confirming its elemental composition. This compound was already mentioned in the literature [33,37].  Table  2) due to the subsequence neutral loss of the methyl group. It produced the MS 3 base peak at m/z 300 [ellagic acid−2H] − . Similar ellagic acid derivatives were already mentioned in the literature [38]. To the best of our knowledge, compound 85 was not previously reported in nature.

Characterisation of flavonoids and flavonoids glycosides
Flavonoids are derived from the shikimate pathway in plant kingdom. They have a basic structure consisting of two aromatic benzene rings separated by an oxygenated heterocyclic ring. Several compounds from different flavonoid classes, such as flavonols, flavanones, flavones and others have been characterized and identified in hawthorn and the herbal drops samples. Crataegus species analyzed in this study showed C-glycosides and O-glycosides flavonoids isomers as represented in Table 1 (Table 1 and 2). Compound 48 was characterized as luteolin 8-C-glucoside based on the comparison of the retention time and the fragmentation patterns with the authentic standard. From the literature and from our experiments, we have found that the flavonoids glycosylated with galactoside units are less polar than the flavonoids glycosylated with glucoside units [34,35]. Based on the above argument, the earlier eluted isomer (t R 33.4 min) was assigned as luteolin 8-C-galactoside 47. Luteolin and luteolin glycoside derivatives were already reported in the literature in Crataegus species [31,42]. In addition to the apigenin C-glycosides, we also identified two apigenin O-glycoside derivatives, namely, apigenin 7-O-rutinoside 74 and apigenin 7-O-glucoside 76 (Table 1) Table 2). These fragments, together with the precursor ion at m/z 611.1615 and the molecular formula C 27 H 31 O 16 pointed to an eriodictyol di-Chexoside. Eriodictyol has been previously isolated from C. microphylla [44]. Compound 45 was present in all investigated samples.  Table 2). This compound has been recently reported by De Rosso et al. in hybrid grapes [53]. It is worth mentioning that this compound has been detected in Crataegus for the first time.  (Figure 4). These compounds showed the same fragmentation patterns. In their MS n they showed ions corresponding to the aglycone kaempferol (Table  2) [54] by the neutral loss of (rhamnosyl-hexoside) [M-H-308] -. Therefore they were suggested to be kaempferol -O-(6-O-rhamnosyl-hexoside) isomers. We have found that flavonol-7-O-glycosides elute first followed by flavonol-3-O-glycosides [25]. Based on the order of elution and the similarity of the fragmentation behaviors, isomers 57 and 59 were assigned as kaempferol 7-O-(6-O-rhamnosyl-glucoside) and kaempferol 3-O-(6-O-rhamnosyl-glucoside), respectively. For further evidence, the UV spectrum, the MS fragmentation and the retention time of compound 59 were compared with the kaempferol 3-O-(6-O-rhamnosyl-glucoside) authentic standard. This compound has been previously recorded in quince (Cydonia oblonga) fruits [21]. Considering the elution order in the RF-C18-A column, isomer 58 was regarded as a glycosylated isomer of compound 59 and hence assigned as kaempferol 3-O-(6-O-rhamnosyl-galactoside) [34,35]. Recently, we have detected this compound in I. coccinea [25]. Although kaempferol glycosides derivatives were already mentioned in the literature in Crataegus species [31,55], these compounds have been reported here for the first time.  (Table 1) and data from the literature [57]. The presence of the fragment ion at m/z 315.0502 (C 16 H 11 O 7 ) indicates the neutral loss of a hexosyl moiety and the presence of the aglycone isorhamnetin ( Figure  3D).  Table 1 and 2) [60]. In addition, the aglycone myricetin was already reported in a recent study in C. cuneata [61]. To our knowledge this compound has been reported in hawthorn for the first time.  (Table 1) and the information obtained from the literature [62].

Characterisation of phloretin derivatives
The analysis in TOF-MS mode also showed the presence of phloretin derivatives in Crataegus species and the herbal drug (Crataegutt Tropfen). Compound 81 (t R 53.9 min; m/z 435.1297; C 21 H 24 O 10 ) was positively identified as phloretin 2'-O-glucoside (phlorizin) based on the comparison of the retention time and the fragmentation patterns with the authentic standard (  (Table 2) and the data obtained from literature [63]. As far as we know, phlorizin and phloretin have never been reported in Crataegus species before.

Characterisation of anthocyanidins, proanthocyanidins and their derivatives
Anthocyanidins, proanthocyanidins and their derivatives are wellknown phytoconstituents in Crataegus species. Our findings boosted and were in line with previous studies [15,64,65].
Characterization of epicatechin derivatives: Peak 30 (t R 25.8 min) at m/z 289.0716 was identified as epicatechin by comparison of UV spectrum and retention time with a commercial standard.
Compound 20 at m/z 451.1241 (t R 18.3 min) with molecular formula C 21 H 23 O 11 was considered as (epi)catechin C-hexoside relying on its MS and MS/MS fragmentation pattern ( Figure 3F) and the data obtained from literature [66].
Characterization of B-type proanthocyanidins and their derivatives: B-type proanthocyanidin oligomers were also detected in the herbal medicine and Crataegus samples. They showed UV spectra with λ max 280 nm, characteristic of proanthocyanidins. Thus, peaks 25 (Table 1). Thus, this dimer was suggested to (epi) afzelechin-(epi)catechin. This compound has been previously reported in literature and its fragmentation path way was described [67]. It is worth noting that peak 46 has been reported in Crataegus for the first time.
At m/z 739.1880 two compounds (17,18) were detected at retention times of 17.3 and 21.8 min and were proposed as proanthocyanidin dimers having hexose groups attached to them, showed by the acceptable data of MS (Table 1), fragmentation pattern of MS 2 /MS 3 ( Table 2) and the data obtained from literature [68]. Moreover, these peaks represented the characteristic flavan  Figure 5). Consequently, compounds 17 and 18 were assigned as (epi)catechin-4,8′-(epi)catechin C-hexoside isomers. Nevertheless, assignment of positions and identity of the hexose units was not possible. According to our knowledge, these isomers have never been reported in Crataegus species before.
We have calculated identical molecular formula (C 45 H 36 O 18 ) for peaks 87-90 at m/z 863 (Table 1). They eluted between 30 and 40 min in the developed reverse phase chromatographic method. These compounds were tentatively assigned as trimeric A-type proanthocyanidins with (epi)catechin monomeric units. The suggested structures and fragmentation pathways of A-type proanthocyanidin oligomers have been discussed in previous studies [21,23,25,67].
Characterization of cyanidin derivatives: The detected precursor ion at m/z 287.0561 (C 21 H 21 O 11 + ), which correspond to peaks 84 (t R 62.6 min) was assigned as cyanidin based on the acceptable data of MS with the daughter fragment ions of MS/MS (Table 1) in addition to the information previously reported in the literature [69].
With m/z 449.1082 three peaks with identical molecular formula C 21 H 21 O 11 + were detected at retention times of 27.0, 38.5 and 56.8 min. These compounds showed a neutral loss of hexose moiety resulting in a fragment ion at m/z 287.0554, which corresponds to cyanidin (Table  1). Therefore, they were suggested as cyanidin O-hexoside isomers (31)(32)(33). Based on the order of elution these peaks were tentatively identified as cyanidin 7-O-glucoside 31, cyanidin 3-O-galactoside 32 and cyanidin 3-O-glucoside 33 [25,34,35,70]. Although compounds 32 and 33 were already mentioned in the literature in hawthorn [70], compound 31 has been reported here for the first time ( Figure 3G).
Peak 19 (t R 17.6 min) revealed a deprotonated molecule at m/z 325.0927 and MS/MS fragment ions at m/z 163.0401 and m/z 119.0503 corresponding to the neutral loss of a hexosyl moiety [M−H − 162] − followed by the neutral loss of carbon dioxide molecule (CO 2 ). In line with the previous reported MS data [32], this compound was assigned as p-coumaric acid O-hexoside ( Figure 3H). Assignment of the position of the hexose (glucose or galactose) was not possible because of the lack of a commercial standard.
At m/z 335.0773 (C 16 H 16 O 9 ) one peak was detected at retention time of 29.7 min in the EIC. It generated MS 2 fragment ions at m/z 179, 173, 135 and 191 (Table 2). Based on this fragmentation pattern this compound was tentatively assigned as 3-O-caffeoylshikimic acid 41, apart from the ion at m/z 191, interestingly, it showed however similar MS 2 and MS 3 spectra if compared to the MS 2 and MS 3 spectra of 3-O-caffeoylshikimic acid previously reported [21,78].
With retention time of 22.2 min and m/z of 515.1407 (C 22 H 28 O 14 ), a further small peak (10) was observed. Compound 10 exhibited fragmentation patterns identical to the 5-O-(3'-O-Caffeoyl glucosyl) quinic acid (8) and we assumed that it might be a cis isomer of compound 8. For confirmation of this isomer, the extract of C. monogyna leaves was irradiated with UV light at 245 nm for 60 min. After irradiation, we found that the cis isomer in the chromatogram as peak with significantly increased intensities if compared to trans isomer from the original plant extract, which confirmed the presence of the cis-5-O-(3'-O-Caffeoyl glucosyl)quinic acid 10 [24]. We have reported the presence of cis derivatives of chlorogenic acids earlier in L. henryi, I. coccinea, quince fruits, coffee leaves, Carlina acaulis, Helianthus tuberosus, Symphyotrichum novae-angliae and Rudbeckia hirta, [21,22,24,25,79]. It is important noting that caffeoylquinic acid glucosides have been reported in hawthorn for the first time.

Conclusion
Crataegus species are medicinal plants used extensively in traditional medicine for the treatment of cardiovascular diseases, arrhythmia and hypertension. Using UPLC-ESI-Q-TOF-MS/MS and HPLC-ESI-MS n , a total of 113 compounds were tentatively identified. To the best of our knowledge 63 of them are described for the first time

Characterisation of acylated quinic acid derivatives
Several mono and di-acylated quinic acid derivatives including chlorogenic acids and chlorogenic acids glycosides were also detected in fruits and leaves of C. monogyna and C. laevigata and the traditionally derived drops (Crataegutt Tropfen).  (Table 1) by comparison with their commercial standards. 3,5-di-O-caffeoylquinic acid has been previously reported in C. monogyna [56], while 3,4-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid have been reported here for the first time.
Four caffeoylquinic acids (2)(3)(4)(5), four p-coumaroylquinic acids (12)(13)(14)(15)(16) and one feruloylquinic acid were identified in TOF-MS mode using accurate mass measurements and MS/MS fragmentations (Table 1) in addition to identification keys previously cited as 3-O-caffeoylquinic in Crataegus species and two for the first time in nature. In this context, the obtained results indicate that Crataegus species are rich source of phenolic constituents including simple phenolic acids, chlorogenic acids, proanthocyanidins, flavonoids and flavonoids glycosides.
The current study clearly emphasis the need for re-investigation of medicinal plants using powerful state of the art analytical instrumentation, providing additional information on minor secondary metabolites previously not obtainable.