Hydroxycinnamic Acid Derivatives Obtained from a Commercial Crataegus Extract and from Authentic Crataegus spp.§

Abstract Eleven hydroxycinnamic acid derivatives were isolated from a 70% methanolic Crataegus extract (Crataegi folium cum flore) and partly verified and quantified for individual Crataegus species (C. laevigata, C. monogyna, C. nigra, C. pentagyna) by HPLC: 3-O-(E)-p-coumaroylquinic acid (1), 5-O-(E)-p-coumaroyl-quinic acid (2), 4-O-(E)-p-coumaroylquinic acid (3), 3-O-(E)-caffeoylquinic acid (4), 4-O-(E)-caffeoylquinic acid (5), 5-O-(E)-caffeoylquinic acid (6), 3,5-di-O-(E)-caffeoylquinic acid (7), 4,5-di-O-(E)-caffeoylquinic acid (8), (-)-2-O-(E)-caffeoyl-L-threonic acid (9), (-)-4-O-(E)-caffeoyl-L-threonic acid (10), and (-)-4-O-(E)-p-coumaroyl-L-threonic acid (11). Further, (-)-2-O-(E)-caffeoyl-D-malic acid (12) was isolated from C. submollis and also identified for C. pentagyna and C. nigra by co-chromatography. The isolates 10 and 11 were not found in the authentic fresh specimen, indicating that they may be formed during extraction by acyl migration from the 2-O-acylderivatives. Also, 9 and 11 are described here for the first time. All structures were assigned on the basis of their spectroscopic data (1H-, 13C-NMR, MS, optical rotation).


Introduction
The monograph "hawthorn leaf and flower" (Crataegi folium cum flore) of the European Pharmacopoeia (PhEur) consists of the dried flowers and bearing branches of Crataegus monogyna Jacq.(Lindm.),C. laevigata (Poiret) D.C. (syn.C. oxyacantha Thuill.), C. pentagyna Waldst.et Kit.ex Willd., C. nigra Waldst.et Kit., and C. azarolus L. [1].Preparations based on the hydroalcoholic extracts are commonly used as rational phytomedicines for the treatment of cardiac insufficiency corresponding to class II (NYHA I-II).As for the pharmacological properties, phytochemical studies of the last three decades have been focused on flavonoids and oligomeric procyanidins [see ref. [2][3][4][5][6] and literature cited therein].In contrast to the polyphenols, the knowledge about hydroxycinnamic acids and their derivatives in the drug material was limited.Some common cinnamic acids and benzoic acids were found after acidic hydrolysis of a methanolic total extract from the callus culture of Crataegus monogyna [7].Besides some reports on chlorogenic acid (5caffeoylquinic acid), the N,N',N''-tricoumaroylspermidine has been unambiguously identified from hawthorn flowers (Crataegi flos) [7][8][9][10][11][12].More recently, 5-p-coumaroylquinic acid was also detected from Crataegus monogyna cell suspension cultures [13].The aim of the present study was to analyse the hydroxycinnamic acid derivatives (HCAs) from a commercial Crataegus extract (LI 132) in detail and to analyze them in the single Crataegus species used in the PhEur monograph.

Results and Discussion
A range of hydroxycinnamic acid derivatives (HCAs) were isolated using a combination of CC on Sephadex LH-20 and different HPLC techniques (for formulas, see Fig. 1).The individual HCAs showed typical UV absorption bands for caffeic acids (4-10, 12) at 240 and 336 nm or at 240 and 320 nm for p-coumaric acid derivatives (1)(2)(3)11).The 1 H-NMR spectra showed the typical aromatic AMX-spin system for caffeic and the AA'XX'-spinsystem for p-coumaric acid moieties, respectively.All hydroxycinnamic acid derivatives showed signals for the protons of an (E)-configurated olefinic double bond with the typical coupling constant of about 16 Hz.Acylation positions at the HCA's moieties were determined by 1 H-/ 13 C-NMR experiments according to the acylation shift of the esterfied position and chemical shift of geminal protons compared with the free quinic acid or aldonic acids, respectively [14]; they were also directly proven by HMBC experiments.The 1 H-NMR data of the quinic acid derivatives were also confirmed by spectral simulation.Here we present the complete 1 H-and 13 C-NMR data sets in MeOH-d 4 of isomeric quinic acids in Tables 1S-4S in "Supplemental Information."Concerning the problem of solvent effects in the structure dereplication of caffeoyl quinic acid, see [15].
The ESI-MS of 10 showed a m/z of 299 (M+H + ) which differs clearly from those of quinic acid derivatives.The 1 H-NMR (200 MHz) in methanol-d 4 revealed, in addition to the protons of the caffeoyl moiety, a broad signal at δ 4.2-4.3ppm, which was poorly separated at 600 MHz into the expected corresponding protons of the tertiary carbons (δ 4.24 ppm) and the two protons of the secondary carbon at δ 4.26 ppm (Table 5S).The 13 C-NMR of 10 (D 2 O) showed two tertiary carbons at δ 71.9 and 71.7 ppm, a secondary at δ 65.7 ppm, and a carboxylic carbon at δ 176.5 ppm.GC-MS of the TMS-ether of 10 showed an m/z = 658 and m/z 439 and 219 as a result of an alpha splitting between the carbons C-2 and C-3, thereby confirming the trihydroxybutyric acid structure (Table 6S).Substitution of the acyl residue was confirmed at C-4 by HMBC experiments.NMR data Sci Pharm.2014; 82: 835-846 and the optical rotation value of 10 were in agreement with the literature data [16,17].

Cpd
The 13 C-and 1 H-NMR data and the optical rotation value of compound 9 coincided with the data obtained for 2-O-(E)-caffeoyl-L-threonic acid lactone [16].However, in contrast to [16], the ESI-MS gave a relative molecular mass of 298 indicating a free caffeoyl-threonic acid.Additionally, the small 3 J H-2/3 -coupling constant of 2.4 Hz was inconsistent with a fixed trans-arrangement of the free hydroxyl functions caused by lactonisation of threonic acid [18].These results required a new assignment of the threonic acid moiety by HMBC experiments and a comparison of the NMR data with those of free L-threonic acid and the Sci Pharm.2014; 82: 835-846 synthesized L-threonic acid 1,4-lactone.Compound 9 showed no 3 J H-4/C-1 -coupling as a result of lactonisation, whereas the 3 J H-2/C-9' -coupling and thus the point of attachment of the acyl moiety was clear.On careful inspection of the 1 H-NMR data of free L-threonic acid (for data, see Table 7S), the acylation shift of the H-2 from δ 4.19 ppm to 5.28 ppm in 9 was obvious, but no similar shift for H-4 (δ 3.66, 3.61 ppm) for threonic acid or δ 3,63, 3,61 ppm for 9 was observed.In contrast, L-threonic acid lactone showed a clear acylation shift of the H-4 protons to δ 3.93 and 4.41 ppm (Table 1 and Table 7S).These results unambiguously assign the structure of compound 9 as (−)-2-O-(E)-caffeoyl-L-threonic acid.To the best of our knowledge, compounds 9 and 11 were described here for the first time.
Compounds 10 and 11 isolated from the LI 132 extract were not detected in the authentic fresh Crataegus species.Because the isolation procedure from fresh material and the LI 132 extract was identical, their presence in the LI 132 extract may be explained by the partial acyl shift during the extraction of the drug material of the 2-O-acylderivatives as shown for Chelidonium majus [16].Compound 12, which was isolated from C. submollis, was verified for C. pentagyna and origins of C. nigra by HPLC.The other species showed a very similar qualitative pattern of hydroxycinnamic acid derivatives.Only for the samples of C. laevigata, the 3-O-monohydroxycinnamic quinic acids and the aldonic acid derivatives were not observed.
Further investigations of the flowers and leaves of the different species separately and in more detail showed that the 3-O-hydroxycinnamic quinic acids were only detectable in flowers (data not shown).The contribution of the HCA derivatives of Crataegus to cardiac diseases is to the best of our knowledge not known.However, a recent systematic review and meta-analysis on chlorogenic acid and its derivatives, mainly from green coffee beans, concluded that evidence suggests that chlorogenic acid intake causes a statistically significant reduction in systolic and diastolic blood pressures [19 and references cited therein].In view of the relatively high amount of HCAs in Crataegus crude drugs of c. 1% to more than 3%, it seems worthwhile to investigate whether they participate in the therapeutical efficacy of Crataegus preparations.

General
Optical rotations were measured on a Perkin-Elmer 241 apparatus, mass spectra on a Finnigan LC-Q and Quattro LC Z (ESI-MS), and on a Finnigan MAT 8230 combined with a Varian 3400 GC using an HP-5 column (25 m) (GC-MS).The mass spectra were calibrated using a reference spectrum of sodium formate.The exact mass for 11 was determined with a Bruker micrOTOF-qII mass spectrometer coupled to a Dionex Ultimate 3000 RS UHPLC System.Separation was achieved over an RP18 column (Dionex Acclaim 120 C18, 2.2 µm, 2.1 x 100 mm) eluted by a binary water-acetonitrile gradient containing 0.1% formic acid at 0.8 mL/min, +10% acetonitrile/min beginning at 5% acetonitrile; the mass spectrum was acquired in positive mode over a m/z range of 50-1500 with a nebulizer pressure of 4.0 bar (N 2 ), dry gas flow of 9.0 L/min at 220°C (N 2 ), capillary voltage of 4500 V and 8 eV collision energy.

Isolation Procedure
An amount of 350 g of a commercial 70% methanolic extract (Crataegus LI-132) was suspended in water (700 mL) and stored for 24 h at 4°C to precipitate chlorophyll.After filtration, the remaining chlorophyll was removed by shaking with dichloromethane.The purified aqueous phase was acidified with HCl at pH 2 and successively extracted with 3 x 700 mL diethyl ether (A), 5 x 700 mL ethyl acetate (B), and 3 x 700 mL n-butanol (C).After removal of the solvents, the extracts were lyophilized to yield 3.1 g for A, 21.7 g for B, and 50.0 g for C.An aliquot (19.6 g) of the EtOAc-extract (B) enriched with phenolics was further fractionated by column chromatography over Sephadex LH-20 (510 x 55 mm) with 11.2 L 50% methanol to give 11 subfractions (I-XI), and the first 570 mL were discarded.
Compound 12 was obtained from 240 g of the air dried leaves of C. submollis by extraction with 70% methanol.After removal of methanol, the remaining aqueous phase was acidified (pH 2) with HCl and further extracted with ethyl acetate, concentrated to a brownyellow coloured extract (13.3 g).A portion (7.2 g) was fractionated (CC) over Sephadex LH-20 (400 x 25 mm) with 50% methanol.A part (270 mg) of fraction 20 (elution volume: 400-500 mL, 1.3 g) was purified by the prep.HPLC (system 3) to yield pure 12 (65 mg).

Hydrolysis of 10 and 11 and Product Analysis
The hydrolysis and analysis of free L-threonic acid were performed according to Hahn [16].Amounts of 35 mg of 11 and 10 mg of 10 were hydrolyzed enzymatically (AB enzymes EL-1/77 "Röhm-Enzym"; pH 5, 37°C, 72 h) and the optical rotation was measured after

Synthesis of γ-L-Threonic Acid Lactone
To demonstrate a missing acylation shift of 9 at position 4 (see discussion), γ-L-threonic acid lactone was synthesized from L-threonic acid lactone hemicalcium salt x 1 H 2 O (Sigma-Aldrich) by a modified method according to Angelotti [21].After converting to L-threonic acid (see above), the solution was concentrated and dried over P 2 O 5 under vacuum for five days.The resulting 1 H-and 13 C-NMR spectra were compared with the data of 11 and free L-threonic acid lactone (Table 1 and 7S).
1H-and13C-NMR data were recorded at 200 and 50 MHz, respectively, with a Varian Gemini 200 spectrometer or at 600 MHz and 150 MHz with a Varian Unity 600 (MeOH or DMSO as internal standard).Offline data processing and 1 H-NMR simulation was done with the Nuts programme package (Acorn NMR, Vermont CA).