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High-performance counter-current chromatography separation of Peucedanum cervaria fruit extract for the isolation of rare coumarin derivatives Krystyna Skalicka-Woźniak*, Tomasz Mroczek, Ewelina Kozioł Department of Pharmacognosy with Medicinal Plant Unit, Medical University of Lublin, 1 Chodzki Str, 20-093 Lublin, Poland *
Corresponding author at Department of Pharmacognosy with Medicinal Plant Unit, Medical
University of Lublin, 1 Chodzki Str., 20-093 Lublin, Poland Email address: [email protected] (dr K. Skalicka-Woźniak) Tel.: +48817423807, fax: +48817423809
Keywords: coumarins, counter-current chromatography, dihydrooroselol derivatives, Peucedanum cervaria,
Abstract For the first time, rare major and minor compounds from fruits of Peucedanum cervaria were isolated. High-performance counter-current chromatography with two different solvent systems, heptane/ethyl acetate/methanol/water (3:2:3:2 and 2:1:2:1, v/v) was successfully used in the reversed-phase mode. A scale-up process from analytical to semi-preparative in a very short time was developed. The structures of isolated compounds were evaluated by highperformance liquid chromatography with diode array detection and electrospray ionization mass spectrometry, gas chromatography with mass spectrometry, and one- and twodimensional dihydrooroselol
NMR
spectroscopy.
(compound
B),
(8S,9R)-9-(3-Methylbutenoyloxy)-O-acetyl-8,9(8S,9R)-9-(2-methyl-Z-butenoyloxy)-O-acetyl-8,9-
Received: 28-Sep-2014; Revised: 22-Oct-2014; Accepted: 31-Oct-2014 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jssc.201401072.
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dihydrooroselol (edultin, compound C) and (8S,9R)-9-acetoxy-O-(2-methylbutyryl)-8,9dihydrooroselol (compound D) were obtained using heptane/ethyl acetate/methanol/water (2:1:2:1, v/v) in the less than 40 min. The method yielded 4.6 mg of a mixture of compounds B and C (11:89) and 3.7 mg of compound D. These amounts were obtained from the crude extract (0.5 g) in a single run. Although the compounds are known, their isolation by countercurrent chromatography and the analysis of their relative stereochemistry by two-dimensional NMR spectroscopy have been performed for the first time. Additionally, heptane/ethyl acetate/methanol/water (3:2:3:2, v/v) led to the isolation of oxypeucedanin (1.2 mg; compound A). This is the first time that angular dihydrofuranocoumarin was isolated from plant extract by counter-current chromatography.
1. Introduction CCC is a form of LLE in which either a centrifugal or gravitational force is used to retain one liquid phase in a coil or train of chambers, while a second, immiscible phase is passed through, making contact with the first phase [1]. The technique is an all-liquid method, which relies on the partition of a sample between two immiscible solvents to achieve separation [2]. In HPCCC, to obtain phase distribution and retention of the stationary phase, the specific planetary motion is used. Thanks to the centrifugal force, mixing and settling zones occur, where separation takes place [3]. The most important step in HPCCC is selecting the proper solvent system. Facility in scale up allows for the best separation without wasting of solvents and can provide satisfactory amounts of pure compounds [4]. Plant extracts are mixtures of a large number of active constituents with similar structures and widely different polarities, thus the separation and structural identification are particularly important for driving pharmaceutical development. CCC, having series of advantages mentioned above, find broad application in the isolation of natural products. Possibilities of implementation of different elution modes including stepwise elution, extrusion elution, reverse elution, recycling elution and its combination in two dimensions, allowing the establishment of routine methods and the enhancement of separation efficiency and let to separate both minor and major compounds with different polarities and high purities in single run [5–7].
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Plants belonging to genus Peucedanum (Apiaceae) have great value in traditional medicine. They were used in ancient times for the treatment of sore throat. They were also known as being effective in the treatment of epilepsy, joint pain, respiratory and gastrointestinal disorders [8–11]. In recent times, the antiviral, antidiabetic, and anticholesterol activities were confirmed [12–14]. These various bioactivities were the reasons for searching for the constituents responsible for the pharmacological action. Many bioactive compounds have been isolated so far from species belonging to the genus Peucedanum. The coumarin derivatives are the most important [15], among which also novel compounds (alsaticol and alsaticocoumarin) or very rare (notoptol, ledebouriellol, and divaricatol) were purified [16, 17]. All of these mentioned derivatives were obtained by the application of CCC. The aim of present study was to separate both the major and minor coumarin derivatives from a non-polar extract of the fruits of Peucedanum cervaria (L.) Lap., which could not be identified because of the lack of available standards. Four compounds were isolated and identified. According to the available literature, there is no report on the use of CCC for the separation and purification of dihydrooroselol derivatives from plant source. Additionally, a well-known furanocoumarin, oxypeucedanin, was isolated in a very short time. The optimal method was transferred easily from analytical to semi-preparative scale. Although isolated dihydrooroselol derivatives are known, they are very rare, and analysis of their relative stereochemistry by 2D NMR spectroscopy has been performed here for the first time. Tumor promotor-induced phenomena in vitro was described for dihydrooroselol derivatives [18], thus, to make quantities of those active molecules accessible for further extensive pharmacological study, it is important to find the efficient separation technique.
2.
Materials and methods
2.1
Apparatus A commercially available Spectrum high-performance counter-current chromatograph
(HPCCC) from Dynamic Extractions (Slough, UK) was used in this study. The apparatus was equipped with polytetrafluoroethylene multilayer coils for both analytical and semipreparative scale analysis. The analytical coil of 0.8 mm bore had a capacity of 22 mL, and a 1 mL sample loop was used. For semi-preparative purposes, a coil with a volume 136 mL and 1.6 mm bore, and a 6 mL sample loop was used. The chromatograph was connected to a This article is protected by copyright. All rights reserved.
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Sapphire UV detector and Alpha 10 pump (ECOM, Prague, Czech Republic). The apparatus was run at the maximum rotation speed 1600 rpm to obtain the maximum centrifugal force of 240 g field.
2.2 Reagents All organic solvents used to obtain the biphasic system and the dichloromethane used for the preparation of the extract from the plant material were of analytical grade and purchased from POCh Gliwice. Water was purified using a Millipore laboratory ultra pure water system Millipore, Simplicity (France). Methanol for HPLC was purchased from J.T. Baker, Germany. Oxypeucedanin standard was provided by ChromaDex (Irvine, USA).
2.3 Plant material Fruits of Peucedanum cervaria L. were collected from the Medicinal Plant Garden (Medical University of Lublin) in October 2010. Plant material was identified by specialist in botany, and the voucher specimens No. 14/4–5 are deposited with the Department of Pharmacognosy, Medical University, Lublin. The plant material was dried and immediately milled. The extract was obtained using the following procedure: prepared fruits P. cervaria (50 g) were placed in a round-bottomed flask with reflux condenser and extracted with dichloromethane (200 mL) by heating to boiling point of the solvent for 30 min. The procedure was repeated three times. Extracts were mixed and evaporated to obtain crude oily sample (5.92 g).
2.4 Selection of two phase system The composition of the two-phase solvent system was selected according to the partition coefficient (K) and peak resolution of the target compounds. Different ratios of heptane, ethyl acetate, methanol, and water (HEMW) were tested. To find a suitable volume ratio, the partition coefficient was determined by HPLC analysis. Four mixtures of HEMW solutions were made according to the method described by Garrard [1]. About 1 mg of crude sample was added to a test tube containing 2 mL of each phase of tested two-phase solvent systems. The tubes were shaken for 1 min. Then equal volumes (500 μL) of upper and lower phases were evaporated to dryness separately. The residues were dissolved in methanol (1 mL) before HPLC analysis.
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2.5 Solvent system preparation HEMW mixtures for the CCC purification were prepared in a separation funnel and thoroughly equilibrated at room temperature. The two phases were separated shortly before use. Upper and lower phases were collected in labeled bottles and sonicated for 30 min. The upper phase was used as a stationary phase and the lower phase as a mobile phase in a reversed-phase system.
2.6 Separation procedure Selection of optimal conditions for separation was carried over firstly in analytical, then in semi-preparative, scale. A constant temperature was established during the experiment equal 30°C. The multilayer coil was first filled with upper stationary phase. Then the rotation speed was set to 1600 rpm, and the mobile phase was introduced with flow speed 1 mL/min for analytical and 6 mL/min for semi-preparative scale. When there was no more stationary phase eluting from coil, the equilibrium was reached. The volume of displaced stationary phase allowed us to calculate the retention of the stationary phase on the coil. In this condition sample solution was injected. 80 mg of crude dichloromethane extract of the fruits of P. cervaria was dissolved in solvent system (1 mL, 0.5 mL upper and 0.5 mL lower phase—analytical purpose), whereas a sample (500 mg) were dissolved in biphasic system (6 mL) for semi-preparative purpose. The effluent from the tail end of the column was continuously monitored with a UV absorbance detector at 320 nm. 1 min fractions were collected, which were evaporated, dissolved in methanol, and analyzed with HPLC with diode array detection (DAD).
2.7 HPLC–DAD–ESI-TOF-MS, GC–MS and NMR analysis and identification of HPCCC peak fractions All analyses were performed using HPLC coupled with DAD and ESI-TOF-MS. A 1200 Agilent HPLC system consisting of a binary pump, membrane degasser, thermostatted column compartment, autosampler, DAD and 6210 MSD TOF mass spectrometer was used. An HPLC–DAD system was used for analysis of crude extract and all collected fractions, which were separated on Zorbax Eclipse XDB C18 stainless-steel column (250 mm × 4.6 mm) packed with 5 μm by use of a stepwise mobile phase gradient prepared from methanol (A) and water (B). The gradient was: 0 min, 50% A in B; 5 min, 60% A in B; 25 min, 80% A in
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B; 30–40 min, 100% A. The flow rate was 1 mL/min, the column temperature was 25°C, and the injection volume was 10 μL. Fractions containing pure compounds were subjected to HPLC–DAD–ESI-TOF-MS analysis, performed using the analytical column Zorbax Stable Bond RP-18 (150 mm x 2.1 mm, 3.5 m). The following gradient was applied: solvent A: 60% acetonitrile in water (+ 5 mM ammonium formate with 0.1% HCOOH), solvent B: 90% acetonitrile in water (+ 5 mM ammonium formate with 0.1% HCOOH); 0–8 min: isocratic 100% A, then 8–30 min: 0–80% B, 30–35 min: isocratic at 80% B, and 35–38 min: 80 to 0% of B. The total analysis time was 38 min with 12 min post-time; injection volume was 10 μL, DAD spectra were recorded from 200 to 400 nm; ESI-TOF-MS spectra were recorded using the following parameters: m/z 100–1000 amu, ESI source: (+)-positive mode, gas temp. 350°C, N2 flow rate 10 L/min, nebulizer pressure: 35 psig, skimmer 65 V, Octopole 1RFVpp 250 V, fragmentor: 215 and 260 V. GC/MS was performed with a Shimadzu GC-2010 Plus GC instrument coupled to a Shimadzu QP2010 Ultra mass spectrometer. Compounds were separated on a fused-silica capillary column ZB-5 MS (30 m, 0.25 mm) with a film thickness of 0.25 μm (Phenomenex). The following oven temperature program was initiated at 50°C, held for 3 min, then increased at the rate of 5°C/min to 250°C, and held for 15 min. The mass spectrometer was operated in the electron-impact (EI) mode, the scan range was 40–500 amu, the ionization energy was 70 eV, and the scan rate was 0.20 s per scan. Injector, interface and ion source were kept at 250, and 220°C, respectively. Split injection (1 μL) was conducted with a split ratio of 1:20, and helium was used as carrier gas at 1.0 mL/min flow rate. The retention indices were determined in relation to a homologous series of n-alkanes (C8–C24) under the same operating conditions. Compounds were identified using a computer-supported spectral library [19], mass spectra of reference compounds, as well as MS data from the literature [20,21]. A 600 MHz Bruker spectrometer was used for 1D NMR (1H, 175 MHz for 13C NMR spectra in CDCl3) and 2D NMR spectroscopy [correlation spectroscopy (COSY), heteronuclear
multiple-quantum
correlation
(HMQC),
heteronuclear
correlation (HMBC), nuclear Overhauser effect spectroscopy (NOESY)].
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multiple-bond
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3 Results and discussion 3.1 Selection of HPCCC conditions To achieve an efficient resolution of the target compounds, four different CCC solvent systems were examined. K values, expressed as the concentration of target compounds in the upper stationary phase divided by that in the lower mobile phase. A suitable solvent system is one in which the target components have a K value between approximately 0.2 and 5 [1]. Results, which have been published so far, indicated mixtures of HEMW as the most suitable and the most often used [16, 17, 22]. Nevertheless some authors successfully replaced alkanes with light petroleum ether [15, 23] and also mixtures of chloroform, methanol and water have been applied for purification of simple coumarins and some chromones derivatives [24]. Thus, to find suitable condition for separation of target compounds HEMW solvent systems with different volume ratios were tested for the HPCCC separation, as well as other mixtures published so far. The best separation conditions were achieved when different HEMW systems were applied. When the HEMW solvent system with the volume ratio of 5:2:5:2 was used, all target compounds were eluted before 20 min, which did not give satisfactory separation, as the target compounds had higher affinity to the mobile phase. Increasing the polarity from 5:2:5:2 v/v to 2:1:2:1 v/v led to an increase in K values from 0.85, 0.83 and 1.18 to 1.50, 1.43 and 1.87 for compounds B, C and D, respectively, and efficient separation was obtained in less than 37 min. Further increasing the polarity of the tested solvent system to 3:2:3:2 slightly changed the K values of compounds B, C and D, keeping the separation time almost the same, but the purities of the isolated compounds were significantly less. The main difference was that the more polar solvent system allowed for the isolation of compound A, which in previous experiments was eluted almost with the front of the mobile phase. Finally, a two-phase HEMW system (2:1:2:1 v/v) was used for the efficient isolation of compounds B, C, and D, while the system 3:2:3:2 v/v led to the isolation of the more polar compound A. Retention of the stationary phase was 80 and 81%, respectively. All experiments were performed first on analytical scale, and then readily transferred to a semipreparative scale. The results of increasing sample concentration were also tested on an analytical scale. As the sample concentration was increased from 15–80 mg/mL, resolution of target compounds was satisfactory. With a higher concentration, precipitation of the extract was
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observed, therefore 80 mg/mL was chosen as the maximum and optimum concentration for separation.
3.2 HPLC, MS and NMR analyses of fractions The HPLC chromatogram of the crude dichloromethane extract from the fruits of P. cervaria is presented in Fig. 1I. UV-DAD spectral analysis suggests that many of the constituents are coumarin-like. Fractions collected from the HPCCC run were analyzed (60 fractions each time) and the target compounds were detected as follows. When the HEMW (2:1:2:1 v/v) system was used, the mixture of compounds B and C (4.6 mg, with compound C being the major component present at 89% by HPLC peak area) was isolated between 26–28 min, while compound D was eluted between 35–36 min (3.7 mg). Those target compounds were detected also in fractions 25, 29–32 (compounds B + C) and 34 and 37 (compound D), but their purity was much lower than expected. To isolate compound A, more polar HEMW system (3:2:3:2 v/v) was applied which led to pure compound A between 19–20 min (1.2 mg). HPLC chromatograms of the isolated compounds, together with their UV-DAD spectra, are presented in Figs. 1 II–IV. The HPCCC chromatogram of crude extract with HEMW (2:1:2:1 v/v) is shown in Fig. 2. The HPCCC chromatogram with HEMW (3:2:3:2 v/v) is shown in Fig. S2’. The structures of the isolated compounds were confirmed by HPLC–DAD–ESI-TOFMS, GC–MS, and 1D and 2D NMR spectroscopy. In this way, various hyphenated chromatographic and mass spectrometric techniques together with 1D and 2D NMR spectroscopy were utilized to determine the structures of the isolated compounds. It has been done as it is described below, also including relative stereochemistry determination. Compound A, as a known furanocoumarin, was identified on the basis of comparison of DAD, ESI-TOF-MS and NMR spectra together with available standard and literature data [25–27]. The DAD spectrum of compound B showed prominent bands at 205 and 322 nm with a small bathochromic shift (in comparison with D), indicating a possible angular type of furanocoumarin [28]. The ESI-TOF-MS spectrum of compound B in the positive ion mode exhibited the sodium adduct of the molecular ion [C21H22NaO7]+ at m/z 409.1263 (theoretical mass 409.1258; 1.3 ppm error) and the major peak at m/z 227.0706 for C14H11O3+ (theoretical mass 227.0703; 1.3 ppm error). This indicated the possible presence of two esterified acid chains at C8 and C9. The structure and localization of these two acidic residues was This article is protected by copyright. All rights reserved.
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confirmed by 1D and 2D NMR spectroscopy, as well as by GC–MS. Using the GC–MS method, the presence of acetic and 2-(or 3)-methylcrotonic acids was confirmed by the presence of characteristic product ions at m/z 43 and 83, respectively A [18]. The complete structure was established by extensive 1D and 2D NMR spectroscopy. In the 1H NMR spectrum three pairs of doublets were present for protons 3–4, 5–6, and 8–9. They were the following: 3–4 (6.25 and 7.63 ppm, J = 9.6 Hz), 5–6 (7.43 and 6.87 ppm, J = 8.4 Hz), and 8– 9 (5.22 and 7.02 ppm, J = 6.9 Hz). Thus, the structure of an angular dihydrofuranocoumarin with a cis-orientation of the two esterified residues was confirmed. It was in agreement with previously published paper on this type of furanocoumarin [18, 29]. The COSY and HMQC spectra were used to confirm these findings, while HMBC and NOESY spectra allowed establishment of the localization of the acetic and 3-methylcrotonic acid residues, at C-8 and C-9, respectively. Surprisingly, the proton at C-9 gave a prominent cross-peak not with the C=O signal of the acetic acid ester (as in compound D), but with the C=O at =164.1 ppm of the 3-methylcrotonic acid ester in the HMBC spectrum. 3-Methylcrotonic acid ester (Osenecioyl-) was established by the presence of a broad singlet at 5.60 ppm, together with two methyl singlets at 2.02 and 1.92 ppm. The singlet (3H) of the acetyl moiety was at 2.26 ppm. In this way, compound B was determined as (8S,9R)-9-(3-methylbutenoyloxy)-Oacetoxy-8,9-dihydrooroselol, previously described in Ref. [18]. Using HMBC and NOESY techniques the different localization of the acetic acid residue in comparison with compound D was again demonstrated. HMBC correlations for compound B are presented in Fig. 3. The DAD spectrum of compound C showed a similar UV spectrum to compound B indicating a possible angular type of furanocoumarin [28]. The ESI-TOF-MS spectrum of compound C in the positive mode exhibited the same futures as for compound B, indicating an isomeric structure. It indicated also the possible presence of two esterified acid chains at C-8 and C-9. The structure and localization of these two acidic residues was confirmed by 1D and 2D NMR spectroscopy, as well as by the GC–MS method. Using GC–MS, the presence of acetic and 2-(or 3)-methylcrotonic acids were confirmed by the presence of characteristic product ions at m/z 43 and 83, respectively [18]. The complete structure was established by extensive 1D and 2D NMR spectroscopy. In the 1H NMR spectrum three pairs of doublets were also present, respectively for the protons 3–4, 5–6, and 8–9. They were the following: 3–4 (6.25 and 7.64 ppm, J = 9.6 Hz), 5–6 (7.44 and 6.88 ppm, J = 8.4 Hz), and 8–9 (5.31 and 7.10 ppm, J = 7.0 Hz). Thus, the structure of an angular dihydrofuranocoumarin with a cis This article is protected by copyright. All rights reserved.
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orientation of the two esterified residues was again confirmed. It was similar to previously published papers on this type of furanocoumarin [18, 29]. Also, the COSY and HMQC spectra were used to confirm these findings, while the HMBC and NOESY spectra allowed establishment of the region-placement of the acetic and 2-methylcrotonic acid residues, at C-8 and C-9, respectively. Surprisingly, the proton at C-9 gave a prominent crosspeak not with the C=O signal of the acetic acid ester (=170.53 ppm; like for compound D) but with C=O at 165.93 ppm of the 2-methylcrotonic acid ester in the HMBC spectrum. 2Methyl-Z-crotonic acid ester (O-angeloyl-) was proved by the presence of a quartet at 6.09 ppm (J = 7.2 Hz) coupled with the methyl doublet at 1.99 ppm with the same coupling constant. It was also apparent in the NOESY spectrum, as the 2-methyl signal at 1.85 ppm gave a cross-peak with an unsaturated proton at 6.09 ppm. The singlet (3H) of the acetyl moiety was at 2.04 ppm. In this way, compound C was determined to be (8S,9R)-9-(2methyl-Z-butenoyloxy)-O-acetoxy-8,9-dihydrooroselol, also known as edultin [18]. Using HMBC and NOESY techniques different localization of acetic acid residue in comparison with compound D was established. HMBC correlations for compound C are presented in Fig.4. The DAD spectrum of compound D showed prominent bands at 202 and 320 nm, indicating a possible angular type of furanocoumarin [28]. The ESI-TOF-MS spectrum of compound D in the positive ion mode exhibited the sodium adduct of the molecular ion [C21H24NaO7]+ at m/z 411.1412 (theoretical mass 411.1414; 0.55 ppm error) and the major peak at m/z 227.0706 for C14H11O3+ (theoretical mass 227.0703; 1.3 ppm error). The proposed structure of the major product ion at m/z 227.0706 is shown in Fig. 5. It indicated the possible presence of two esterified acid chains at C-8 and C-9. The structure and regio-placement of these two acidic residues was confirmed by 1D and 2D NMR spectroscopy and GC–MS. Using GC–MS, the presence of acetic and 2-methylbutyric acids was confirmed by the presence of characteristic product ions at m/z 43 and 85, respectively [18]. The complete structure was established by extensive 1D and 2D NMR spectroscopy. In the 1H NMR spectrum once again three pairs of doublets were present, respectively for the protons 3–4, 5–6, and 8–9. They were the following: 3–4 (6.25 and 7.65 ppm, J = 9.6 Hz), 5–6 (7.45 and 6.87 ppm, J = 8.4 Hz), and 8–9 (5.28 and 7.00 ppm, J = 6.8 Hz). Thus, the structure of the angular dihydrofuranocoumarin with a cis orientation of the two esterified residues was once more confirmed. It was also in agreement with the This article is protected by copyright. All rights reserved.
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previously published literature on this type of furanocoumarin [18, 29]. Also, the COSY and HMQC spectra were used to confirm these findings, while the HMBC and NOESY spectra allowed for the establishment of the regio-localization of the acetic and 2-methylbutyric acid residues, at C-9 and C-8, respectively. The proton at C-9 gave a prominent cross-peak with the C=O signal of the acetic acid ester (C-1’’, =170.58 ppm) in the HMBC spectrum, but not with the C=O at 174.72 ppm (C-1’, for 2-methylbutyric acid ester). The 2-methyl orientation of the acidic residue at -8 was confirmed using the NOESY technique for the 2methylbytyric residue. The singlet (3H) of the acetyl moiety was at 2.05 ppm. In this way, the compound
D
was
determined
as
(8S,9R)-9-acetoxy-O-(2-methylbutyryl)-8,9-
dihydrooroselol, previously described from Peucedanum oreoselinum (L.) Moench. [29]. Although, it is known compound, its complete relative stereochemistry together with 2D NMR spectra have now been performed for the first time. HMBC correlations for compound D are presented in Fig. 6. Before, to isolate dihydrooroselol derivatives from a non-polar extract from fruits of Angelica edulis, time-consuming conventional isolation over silica gel, followed by prepHPLC were applied [18]. Edulisin III was separated previously from fruits of Peucedanum oreoselinum Moench. in two steps, chromatography on silica gel and further recrystallization of obtained sediments [29]. Oxypeucedanin, a known and widely-distributed furanocoumarin, and has been purified previously by time-consuming (4–8 h) CCC [30, 31]. The currently proposed method decreased the separation time to 21 min.
3.3 LC–DAD–ESI-TOF-MS, GC–MS, and NMR data of identified compounds All data of identified compounds can be found in the Supporting Information.
4. Conclusions This is the first time that angular dihydrofuranocoumarin has been isolated from a plant extract by the application of CCC. (8S,9R)-9-(3-Methylbutenoyloxy)-O-acetyl-8,9dihydrooroselol
(compound
B),
(8S,9R)-9-(2-methyl-Z-butenoyloxy)-O-acetyl-8,9-
dihydrooroselol (edultin, compound C), and (8S, 9R)-9-acetoxy-O-(2-methylbutyryl)-8,9dihydrooroselol (compound D) were isolated, and a linear scale-up process from analytical to semi-preparative HPCCC has been successfully used. Although, the compounds are known,
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their relative stereochemistry has been analyzed by 2D NMR spectroscopy for the first time. The present study indicates that HPCCC is a powerful technique for the preparative separation and purification of bioactive components from plant materials and provides a significant advantage over the traditional separation techniques.
Acknowledgments This study was supported by grant no. N N405 617538 from the National Science Centre in Krakow, Poland. The authors have declared no conflict of interest.
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[13] Lee, S.O., Choi, S.Z., Lee, J.H., Chung, S.H., Park, S.H., Kang, H.C., Yang, E.Y., Cho, H.J., Lee, K.R. Arch Pharm. Res. 2004, 27, 1207–1210. [14] Movahedian, A., Zolfaghari, B., Sajjadi, S.E., Moknatjou, R. Clinics 2010, 65, 629–633. [15] Liu, R., Feng, L., Sun, A., Kong, L. J. Chromatogr. A 2004, 1057, 89–94. [16] Skalicka-Woźniak, K., Mroczek, T., Garrard, I., Głowniak, K. J. Chromatogr. A 2009, 1216, 5669–5675. [17] Skalicka-Woźniak, K., Mroczek, T., Garrard, I., Głowniak, K. J. Sep. Sci. 2012, 35, 790–797. [18] Mizuno, A., Takata, M., Okada, Y., Okuyama, T., Nishino, H., Nishino, A., Takayasu, J., Iwashima, A. Planta Med. 1994, 60, 333–336. [19] Konig, W.A., Joulain, D., Hochmuth, D.H. Mass Finder 4.0, 2008. [20] Joulain, D., Koenig, W.A., The atlas of spectral data of sesquiterpenes hydrocarbons E.B.Verlag, Hamburg, 1998. [21] Stein, S.E. NIST Chemistry WebBook, NIST Standard Reference Database 69, P.J. Linstrom and W.G. Mallard (Eds.), National Institute of Standards and Technology, Gaithersburg MD, 2011, 20899. [22] Qiu, H., Xiao, X., Li, G. J. Sep. Sci. 2012, 35, 901–906. [23] Hou, Z., Xu, D., Yao, S., Luo, J., Kong, L. J Chromatogr B, 2009, 877, 2571–2578. [24] Sun, A., Feng, L., Liu, R. J Liq. Chromatogr. Relat. Technol, 2006, 29, 751–759. [25] Liu, R., Li, A., Sun, A. J. Chromatogr. A 2004, 1052, 223–227. [26] Gökay, O., Kühner, D., Los, M., Götz, F., Bertsche, U., Albert, K. Anal. Bioanal. Chem. 2010, 398, 2039–2047. [27] Naseri, M., Monsef-Esfehani, H.R., Saeidnia, S., Dastan, D., Gohari, A.R. Asian J. Chem. 2013, 25, 1875–1878. [28] Hadaček, F., Müller, C., Werner, A., Greger, H., Proksch, P. J. Chem. Ecol. 1994, 20, 2035–2054. [29] Lemmich, E., Lemmich, J., Nielsen, B.E. Acta Chem. Scand. 1970, 24, 2893–900. [30] Wei, Y., Xie, Q., Fisher, D., Sutherland, I.A. Chromatographia 2009, 70. 1185–1189. [31] Wei, Y., Ito, Y. J. Chromatogr. A, 2006, 1115, 112–117.
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Fig 1. The HPLC chromatogram of the crude dichloromethane extract from Peucedanum cervaria fruits (I) together with isolated compounds with their UV-DAD spectra: oxypeucedanin (compound A, II), (8S,9R)-9-(3-methylbutenoyloxy)-O-acetyl-8,9dihydrooroselol (compound B, III), (8S,9R)-9-(2-methyl-Z-butenoyloxy)-O-acetyl-8,9dihydrooroselol (compound C, III) and (8S,9R)-9-acetoxy-O-(2-methylbutyryl)-8,9dihydrooroselol (compound D, IV). HPLC conditions: column: Zorbax Eclipse XDB C18 (250 mm x 4.6 mm, 5 μm); mobile phase: methanol/water (methanol: 0 min 50%; 5 min 60%; 25 min 80%; 30–40 min 100%); flow rate: 1 mL/min, temperature: 25°C, detection wavelength: 320 nm.
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Fig. 2. HPCCC chromatogram of the crude extract using HEMW (2:1:2:1). Other conditions are given in the text.
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Fig. 3. Chemical structure and HMBC correlations for (8S,9R)-9-(3-methylbutenoyloxy)-Oacetyl-8,9-dihydrooroselol (compound B);
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selected HMBC correlations.
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Fig. 4. Chemical structure and HMBC correlations for (8S,9R)-9-(2-methyl-Z-butenoyloxy)O-acetyl-8,9-dihydrooroselol (edultin, compound C);
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selected HMBC correlations.
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Fig. 5. The proposed structure of the major product ion m/z 227.0703 of compounds B–D observed in ESI-TOF-MS spectrum in positive mode.
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Fig. 6. Chemical structure and HMBC correlations for (8S,9R)-9-acetoxy-O-(2methylbutyryl)-8,9-dihydrooroselol (compound D);
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selected HMBC correlations.
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High-performance counter-current chromatography separation of Peucedanum cervaria fruit extract for the isolation of rare coumarin derivatives Krystyna Skalicka-Woźniak*, Tomasz Mroczek, Ewelina Kozioł Department of Pharmacognosy with Medicinal Plant Unit, Medical University of Lublin, 1 Chodzki Str, 20-093 Lublin, Poland *
Corresponding author at Department of Pharmacognosy with Medicinal Plant Unit, Medical
University of Lublin, 1 Chodzki Str., 20-093 Lublin, Poland Email address: [email protected] (dr K. Skalicka-Woźniak) Tel.: +48817423807, fax: +48817423809
Keywords: coumarins, counter-current chromatography, dihydrooroselol derivatives, Peucedanum cervaria,
Abstract For the first time, rare major and minor compounds from fruits of Peucedanum cervaria were isolated. High-performance counter-current chromatography with two different solvent systems, heptane/ethyl acetate/methanol/water (3:2:3:2 and 2:1:2:1, v/v) was successfully used in the reversed-phase mode. A scale-up process from analytical to semi-preparative in a very short time was developed. The structures of isolated compounds were evaluated by highperformance liquid chromatography with diode array detection and electrospray ionization mass spectrometry, gas chromatography with mass spectrometry, and one- and twodimensional dihydrooroselol
NMR
spectroscopy.
(compound
B),
(8S,9R)-9-(3-Methylbutenoyloxy)-O-acetyl-8,9(8S,9R)-9-(2-methyl-Z-butenoyloxy)-O-acetyl-8,9-
Received: 28-Sep-2014; Revised: 22-Oct-2014; Accepted: 31-Oct-2014 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jssc.201401072.
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dihydrooroselol (edultin, compound C) and (8S,9R)-9-acetoxy-O-(2-methylbutyryl)-8,9dihydrooroselol (compound D) were obtained using heptane/ethyl acetate/methanol/water (2:1:2:1, v/v) in the less than 40 min. The method yielded 4.6 mg of a mixture of compounds B and C (11:89) and 3.7 mg of compound D. These amounts were obtained from the crude extract (0.5 g) in a single run. Although the compounds are known, their isolation by countercurrent chromatography and the analysis of their relative stereochemistry by two-dimensional NMR spectroscopy have been performed for the first time. Additionally, heptane/ethyl acetate/methanol/water (3:2:3:2, v/v) led to the isolation of oxypeucedanin (1.2 mg; compound A). This is the first time that angular dihydrofuranocoumarin was isolated from plant extract by counter-current chromatography.
1. Introduction CCC is a form of LLE in which either a centrifugal or gravitational force is used to retain one liquid phase in a coil or train of chambers, while a second, immiscible phase is passed through, making contact with the first phase [1]. The technique is an all-liquid method, which relies on the partition of a sample between two immiscible solvents to achieve separation [2]. In HPCCC, to obtain phase distribution and retention of the stationary phase, the specific planetary motion is used. Thanks to the centrifugal force, mixing and settling zones occur, where separation takes place [3]. The most important step in HPCCC is selecting the proper solvent system. Facility in scale up allows for the best separation without wasting of solvents and can provide satisfactory amounts of pure compounds [4]. Plant extracts are mixtures of a large number of active constituents with similar structures and widely different polarities, thus the separation and structural identification are particularly important for driving pharmaceutical development. CCC, having series of advantages mentioned above, find broad application in the isolation of natural products. Possibilities of implementation of different elution modes including stepwise elution, extrusion elution, reverse elution, recycling elution and its combination in two dimensions, allowing the establishment of routine methods and the enhancement of separation efficiency and let to separate both minor and major compounds with different polarities and high purities in single run [5–7].
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Plants belonging to genus Peucedanum (Apiaceae) have great value in traditional medicine. They were used in ancient times for the treatment of sore throat. They were also known as being effective in the treatment of epilepsy, joint pain, respiratory and gastrointestinal disorders [8–11]. In recent times, the antiviral, antidiabetic, and anticholesterol activities were confirmed [12–14]. These various bioactivities were the reasons for searching for the constituents responsible for the pharmacological action. Many bioactive compounds have been isolated so far from species belonging to the genus Peucedanum. The coumarin derivatives are the most important [15], among which also novel compounds (alsaticol and alsaticocoumarin) or very rare (notoptol, ledebouriellol, and divaricatol) were purified [16, 17]. All of these mentioned derivatives were obtained by the application of CCC. The aim of present study was to separate both the major and minor coumarin derivatives from a non-polar extract of the fruits of Peucedanum cervaria (L.) Lap., which could not be identified because of the lack of available standards. Four compounds were isolated and identified. According to the available literature, there is no report on the use of CCC for the separation and purification of dihydrooroselol derivatives from plant source. Additionally, a well-known furanocoumarin, oxypeucedanin, was isolated in a very short time. The optimal method was transferred easily from analytical to semi-preparative scale. Although isolated dihydrooroselol derivatives are known, they are very rare, and analysis of their relative stereochemistry by 2D NMR spectroscopy has been performed here for the first time. Tumor promotor-induced phenomena in vitro was described for dihydrooroselol derivatives [18], thus, to make quantities of those active molecules accessible for further extensive pharmacological study, it is important to find the efficient separation technique.
2.
Materials and methods
2.1
Apparatus A commercially available Spectrum high-performance counter-current chromatograph
(HPCCC) from Dynamic Extractions (Slough, UK) was used in this study. The apparatus was equipped with polytetrafluoroethylene multilayer coils for both analytical and semipreparative scale analysis. The analytical coil of 0.8 mm bore had a capacity of 22 mL, and a 1 mL sample loop was used. For semi-preparative purposes, a coil with a volume 136 mL and 1.6 mm bore, and a 6 mL sample loop was used. The chromatograph was connected to a This article is protected by copyright. All rights reserved.
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Sapphire UV detector and Alpha 10 pump (ECOM, Prague, Czech Republic). The apparatus was run at the maximum rotation speed 1600 rpm to obtain the maximum centrifugal force of 240 g field.
2.2 Reagents All organic solvents used to obtain the biphasic system and the dichloromethane used for the preparation of the extract from the plant material were of analytical grade and purchased from POCh Gliwice. Water was purified using a Millipore laboratory ultra pure water system Millipore, Simplicity (France). Methanol for HPLC was purchased from J.T. Baker, Germany. Oxypeucedanin standard was provided by ChromaDex (Irvine, USA).
2.3 Plant material Fruits of Peucedanum cervaria L. were collected from the Medicinal Plant Garden (Medical University of Lublin) in October 2010. Plant material was identified by specialist in botany, and the voucher specimens No. 14/4–5 are deposited with the Department of Pharmacognosy, Medical University, Lublin. The plant material was dried and immediately milled. The extract was obtained using the following procedure: prepared fruits P. cervaria (50 g) were placed in a round-bottomed flask with reflux condenser and extracted with dichloromethane (200 mL) by heating to boiling point of the solvent for 30 min. The procedure was repeated three times. Extracts were mixed and evaporated to obtain crude oily sample (5.92 g).
2.4 Selection of two phase system The composition of the two-phase solvent system was selected according to the partition coefficient (K) and peak resolution of the target compounds. Different ratios of heptane, ethyl acetate, methanol, and water (HEMW) were tested. To find a suitable volume ratio, the partition coefficient was determined by HPLC analysis. Four mixtures of HEMW solutions were made according to the method described by Garrard [1]. About 1 mg of crude sample was added to a test tube containing 2 mL of each phase of tested two-phase solvent systems. The tubes were shaken for 1 min. Then equal volumes (500 μL) of upper and lower phases were evaporated to dryness separately. The residues were dissolved in methanol (1 mL) before HPLC analysis.
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2.5 Solvent system preparation HEMW mixtures for the CCC purification were prepared in a separation funnel and thoroughly equilibrated at room temperature. The two phases were separated shortly before use. Upper and lower phases were collected in labeled bottles and sonicated for 30 min. The upper phase was used as a stationary phase and the lower phase as a mobile phase in a reversed-phase system.
2.6 Separation procedure Selection of optimal conditions for separation was carried over firstly in analytical, then in semi-preparative, scale. A constant temperature was established during the experiment equal 30°C. The multilayer coil was first filled with upper stationary phase. Then the rotation speed was set to 1600 rpm, and the mobile phase was introduced with flow speed 1 mL/min for analytical and 6 mL/min for semi-preparative scale. When there was no more stationary phase eluting from coil, the equilibrium was reached. The volume of displaced stationary phase allowed us to calculate the retention of the stationary phase on the coil. In this condition sample solution was injected. 80 mg of crude dichloromethane extract of the fruits of P. cervaria was dissolved in solvent system (1 mL, 0.5 mL upper and 0.5 mL lower phase—analytical purpose), whereas a sample (500 mg) were dissolved in biphasic system (6 mL) for semi-preparative purpose. The effluent from the tail end of the column was continuously monitored with a UV absorbance detector at 320 nm. 1 min fractions were collected, which were evaporated, dissolved in methanol, and analyzed with HPLC with diode array detection (DAD).
2.7 HPLC–DAD–ESI-TOF-MS, GC–MS and NMR analysis and identification of HPCCC peak fractions All analyses were performed using HPLC coupled with DAD and ESI-TOF-MS. A 1200 Agilent HPLC system consisting of a binary pump, membrane degasser, thermostatted column compartment, autosampler, DAD and 6210 MSD TOF mass spectrometer was used. An HPLC–DAD system was used for analysis of crude extract and all collected fractions, which were separated on Zorbax Eclipse XDB C18 stainless-steel column (250 mm × 4.6 mm) packed with 5 μm by use of a stepwise mobile phase gradient prepared from methanol (A) and water (B). The gradient was: 0 min, 50% A in B; 5 min, 60% A in B; 25 min, 80% A in
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B; 30–40 min, 100% A. The flow rate was 1 mL/min, the column temperature was 25°C, and the injection volume was 10 μL. Fractions containing pure compounds were subjected to HPLC–DAD–ESI-TOF-MS analysis, performed using the analytical column Zorbax Stable Bond RP-18 (150 mm x 2.1 mm, 3.5 m). The following gradient was applied: solvent A: 60% acetonitrile in water (+ 5 mM ammonium formate with 0.1% HCOOH), solvent B: 90% acetonitrile in water (+ 5 mM ammonium formate with 0.1% HCOOH); 0–8 min: isocratic 100% A, then 8–30 min: 0–80% B, 30–35 min: isocratic at 80% B, and 35–38 min: 80 to 0% of B. The total analysis time was 38 min with 12 min post-time; injection volume was 10 μL, DAD spectra were recorded from 200 to 400 nm; ESI-TOF-MS spectra were recorded using the following parameters: m/z 100–1000 amu, ESI source: (+)-positive mode, gas temp. 350°C, N2 flow rate 10 L/min, nebulizer pressure: 35 psig, skimmer 65 V, Octopole 1RFVpp 250 V, fragmentor: 215 and 260 V. GC/MS was performed with a Shimadzu GC-2010 Plus GC instrument coupled to a Shimadzu QP2010 Ultra mass spectrometer. Compounds were separated on a fused-silica capillary column ZB-5 MS (30 m, 0.25 mm) with a film thickness of 0.25 μm (Phenomenex). The following oven temperature program was initiated at 50°C, held for 3 min, then increased at the rate of 5°C/min to 250°C, and held for 15 min. The mass spectrometer was operated in the electron-impact (EI) mode, the scan range was 40–500 amu, the ionization energy was 70 eV, and the scan rate was 0.20 s per scan. Injector, interface and ion source were kept at 250, and 220°C, respectively. Split injection (1 μL) was conducted with a split ratio of 1:20, and helium was used as carrier gas at 1.0 mL/min flow rate. The retention indices were determined in relation to a homologous series of n-alkanes (C8–C24) under the same operating conditions. Compounds were identified using a computer-supported spectral library [19], mass spectra of reference compounds, as well as MS data from the literature [20,21]. A 600 MHz Bruker spectrometer was used for 1D NMR (1H, 175 MHz for 13C NMR spectra in CDCl3) and 2D NMR spectroscopy [correlation spectroscopy (COSY), heteronuclear
multiple-quantum
correlation
(HMQC),
heteronuclear
correlation (HMBC), nuclear Overhauser effect spectroscopy (NOESY)].
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multiple-bond
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3 Results and discussion 3.1 Selection of HPCCC conditions To achieve an efficient resolution of the target compounds, four different CCC solvent systems were examined. K values, expressed as the concentration of target compounds in the upper stationary phase divided by that in the lower mobile phase. A suitable solvent system is one in which the target components have a K value between approximately 0.2 and 5 [1]. Results, which have been published so far, indicated mixtures of HEMW as the most suitable and the most often used [16, 17, 22]. Nevertheless some authors successfully replaced alkanes with light petroleum ether [15, 23] and also mixtures of chloroform, methanol and water have been applied for purification of simple coumarins and some chromones derivatives [24]. Thus, to find suitable condition for separation of target compounds HEMW solvent systems with different volume ratios were tested for the HPCCC separation, as well as other mixtures published so far. The best separation conditions were achieved when different HEMW systems were applied. When the HEMW solvent system with the volume ratio of 5:2:5:2 was used, all target compounds were eluted before 20 min, which did not give satisfactory separation, as the target compounds had higher affinity to the mobile phase. Increasing the polarity from 5:2:5:2 v/v to 2:1:2:1 v/v led to an increase in K values from 0.85, 0.83 and 1.18 to 1.50, 1.43 and 1.87 for compounds B, C and D, respectively, and efficient separation was obtained in less than 37 min. Further increasing the polarity of the tested solvent system to 3:2:3:2 slightly changed the K values of compounds B, C and D, keeping the separation time almost the same, but the purities of the isolated compounds were significantly less. The main difference was that the more polar solvent system allowed for the isolation of compound A, which in previous experiments was eluted almost with the front of the mobile phase. Finally, a two-phase HEMW system (2:1:2:1 v/v) was used for the efficient isolation of compounds B, C, and D, while the system 3:2:3:2 v/v led to the isolation of the more polar compound A. Retention of the stationary phase was 80 and 81%, respectively. All experiments were performed first on analytical scale, and then readily transferred to a semipreparative scale. The results of increasing sample concentration were also tested on an analytical scale. As the sample concentration was increased from 15–80 mg/mL, resolution of target compounds was satisfactory. With a higher concentration, precipitation of the extract was
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observed, therefore 80 mg/mL was chosen as the maximum and optimum concentration for separation.
3.2 HPLC, MS and NMR analyses of fractions The HPLC chromatogram of the crude dichloromethane extract from the fruits of P. cervaria is presented in Fig. 1I. UV-DAD spectral analysis suggests that many of the constituents are coumarin-like. Fractions collected from the HPCCC run were analyzed (60 fractions each time) and the target compounds were detected as follows. When the HEMW (2:1:2:1 v/v) system was used, the mixture of compounds B and C (4.6 mg, with compound C being the major component present at 89% by HPLC peak area) was isolated between 26–28 min, while compound D was eluted between 35–36 min (3.7 mg). Those target compounds were detected also in fractions 25, 29–32 (compounds B + C) and 34 and 37 (compound D), but their purity was much lower than expected. To isolate compound A, more polar HEMW system (3:2:3:2 v/v) was applied which led to pure compound A between 19–20 min (1.2 mg). HPLC chromatograms of the isolated compounds, together with their UV-DAD spectra, are presented in Figs. 1 II–IV. The HPCCC chromatogram of crude extract with HEMW (2:1:2:1 v/v) is shown in Fig. 2. The HPCCC chromatogram with HEMW (3:2:3:2 v/v) is shown in Fig. S2’. The structures of the isolated compounds were confirmed by HPLC–DAD–ESI-TOFMS, GC–MS, and 1D and 2D NMR spectroscopy. In this way, various hyphenated chromatographic and mass spectrometric techniques together with 1D and 2D NMR spectroscopy were utilized to determine the structures of the isolated compounds. It has been done as it is described below, also including relative stereochemistry determination. Compound A, as a known furanocoumarin, was identified on the basis of comparison of DAD, ESI-TOF-MS and NMR spectra together with available standard and literature data [25–27]. The DAD spectrum of compound B showed prominent bands at 205 and 322 nm with a small bathochromic shift (in comparison with D), indicating a possible angular type of furanocoumarin [28]. The ESI-TOF-MS spectrum of compound B in the positive ion mode exhibited the sodium adduct of the molecular ion [C21H22NaO7]+ at m/z 409.1263 (theoretical mass 409.1258; 1.3 ppm error) and the major peak at m/z 227.0706 for C14H11O3+ (theoretical mass 227.0703; 1.3 ppm error). This indicated the possible presence of two esterified acid chains at C8 and C9. The structure and localization of these two acidic residues was This article is protected by copyright. All rights reserved.
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confirmed by 1D and 2D NMR spectroscopy, as well as by GC–MS. Using the GC–MS method, the presence of acetic and 2-(or 3)-methylcrotonic acids was confirmed by the presence of characteristic product ions at m/z 43 and 83, respectively A [18]. The complete structure was established by extensive 1D and 2D NMR spectroscopy. In the 1H NMR spectrum three pairs of doublets were present for protons 3–4, 5–6, and 8–9. They were the following: 3–4 (6.25 and 7.63 ppm, J = 9.6 Hz), 5–6 (7.43 and 6.87 ppm, J = 8.4 Hz), and 8– 9 (5.22 and 7.02 ppm, J = 6.9 Hz). Thus, the structure of an angular dihydrofuranocoumarin with a cis-orientation of the two esterified residues was confirmed. It was in agreement with previously published paper on this type of furanocoumarin [18, 29]. The COSY and HMQC spectra were used to confirm these findings, while HMBC and NOESY spectra allowed establishment of the localization of the acetic and 3-methylcrotonic acid residues, at C-8 and C-9, respectively. Surprisingly, the proton at C-9 gave a prominent cross-peak not with the C=O signal of the acetic acid ester (as in compound D), but with the C=O at =164.1 ppm of the 3-methylcrotonic acid ester in the HMBC spectrum. 3-Methylcrotonic acid ester (Osenecioyl-) was established by the presence of a broad singlet at 5.60 ppm, together with two methyl singlets at 2.02 and 1.92 ppm. The singlet (3H) of the acetyl moiety was at 2.26 ppm. In this way, compound B was determined as (8S,9R)-9-(3-methylbutenoyloxy)-Oacetoxy-8,9-dihydrooroselol, previously described in Ref. [18]. Using HMBC and NOESY techniques the different localization of the acetic acid residue in comparison with compound D was again demonstrated. HMBC correlations for compound B are presented in Fig. 3. The DAD spectrum of compound C showed a similar UV spectrum to compound B indicating a possible angular type of furanocoumarin [28]. The ESI-TOF-MS spectrum of compound C in the positive mode exhibited the same futures as for compound B, indicating an isomeric structure. It indicated also the possible presence of two esterified acid chains at C-8 and C-9. The structure and localization of these two acidic residues was confirmed by 1D and 2D NMR spectroscopy, as well as by the GC–MS method. Using GC–MS, the presence of acetic and 2-(or 3)-methylcrotonic acids were confirmed by the presence of characteristic product ions at m/z 43 and 83, respectively [18]. The complete structure was established by extensive 1D and 2D NMR spectroscopy. In the 1H NMR spectrum three pairs of doublets were also present, respectively for the protons 3–4, 5–6, and 8–9. They were the following: 3–4 (6.25 and 7.64 ppm, J = 9.6 Hz), 5–6 (7.44 and 6.88 ppm, J = 8.4 Hz), and 8–9 (5.31 and 7.10 ppm, J = 7.0 Hz). Thus, the structure of an angular dihydrofuranocoumarin with a cis This article is protected by copyright. All rights reserved.
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orientation of the two esterified residues was again confirmed. It was similar to previously published papers on this type of furanocoumarin [18, 29]. Also, the COSY and HMQC spectra were used to confirm these findings, while the HMBC and NOESY spectra allowed establishment of the region-placement of the acetic and 2-methylcrotonic acid residues, at C-8 and C-9, respectively. Surprisingly, the proton at C-9 gave a prominent crosspeak not with the C=O signal of the acetic acid ester (=170.53 ppm; like for compound D) but with C=O at 165.93 ppm of the 2-methylcrotonic acid ester in the HMBC spectrum. 2Methyl-Z-crotonic acid ester (O-angeloyl-) was proved by the presence of a quartet at 6.09 ppm (J = 7.2 Hz) coupled with the methyl doublet at 1.99 ppm with the same coupling constant. It was also apparent in the NOESY spectrum, as the 2-methyl signal at 1.85 ppm gave a cross-peak with an unsaturated proton at 6.09 ppm. The singlet (3H) of the acetyl moiety was at 2.04 ppm. In this way, compound C was determined to be (8S,9R)-9-(2methyl-Z-butenoyloxy)-O-acetoxy-8,9-dihydrooroselol, also known as edultin [18]. Using HMBC and NOESY techniques different localization of acetic acid residue in comparison with compound D was established. HMBC correlations for compound C are presented in Fig.4. The DAD spectrum of compound D showed prominent bands at 202 and 320 nm, indicating a possible angular type of furanocoumarin [28]. The ESI-TOF-MS spectrum of compound D in the positive ion mode exhibited the sodium adduct of the molecular ion [C21H24NaO7]+ at m/z 411.1412 (theoretical mass 411.1414; 0.55 ppm error) and the major peak at m/z 227.0706 for C14H11O3+ (theoretical mass 227.0703; 1.3 ppm error). The proposed structure of the major product ion at m/z 227.0706 is shown in Fig. 5. It indicated the possible presence of two esterified acid chains at C-8 and C-9. The structure and regio-placement of these two acidic residues was confirmed by 1D and 2D NMR spectroscopy and GC–MS. Using GC–MS, the presence of acetic and 2-methylbutyric acids was confirmed by the presence of characteristic product ions at m/z 43 and 85, respectively [18]. The complete structure was established by extensive 1D and 2D NMR spectroscopy. In the 1H NMR spectrum once again three pairs of doublets were present, respectively for the protons 3–4, 5–6, and 8–9. They were the following: 3–4 (6.25 and 7.65 ppm, J = 9.6 Hz), 5–6 (7.45 and 6.87 ppm, J = 8.4 Hz), and 8–9 (5.28 and 7.00 ppm, J = 6.8 Hz). Thus, the structure of the angular dihydrofuranocoumarin with a cis orientation of the two esterified residues was once more confirmed. It was also in agreement with the This article is protected by copyright. All rights reserved.
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previously published literature on this type of furanocoumarin [18, 29]. Also, the COSY and HMQC spectra were used to confirm these findings, while the HMBC and NOESY spectra allowed for the establishment of the regio-localization of the acetic and 2-methylbutyric acid residues, at C-9 and C-8, respectively. The proton at C-9 gave a prominent cross-peak with the C=O signal of the acetic acid ester (C-1’’, =170.58 ppm) in the HMBC spectrum, but not with the C=O at 174.72 ppm (C-1’, for 2-methylbutyric acid ester). The 2-methyl orientation of the acidic residue at -8 was confirmed using the NOESY technique for the 2methylbytyric residue. The singlet (3H) of the acetyl moiety was at 2.05 ppm. In this way, the compound
D
was
determined
as
(8S,9R)-9-acetoxy-O-(2-methylbutyryl)-8,9-
dihydrooroselol, previously described from Peucedanum oreoselinum (L.) Moench. [29]. Although, it is known compound, its complete relative stereochemistry together with 2D NMR spectra have now been performed for the first time. HMBC correlations for compound D are presented in Fig. 6. Before, to isolate dihydrooroselol derivatives from a non-polar extract from fruits of Angelica edulis, time-consuming conventional isolation over silica gel, followed by prepHPLC were applied [18]. Edulisin III was separated previously from fruits of Peucedanum oreoselinum Moench. in two steps, chromatography on silica gel and further recrystallization of obtained sediments [29]. Oxypeucedanin, a known and widely-distributed furanocoumarin, and has been purified previously by time-consuming (4–8 h) CCC [30, 31]. The currently proposed method decreased the separation time to 21 min.
3.3 LC–DAD–ESI-TOF-MS, GC–MS, and NMR data of identified compounds All data of identified compounds can be found in the Supporting Information.
4. Conclusions This is the first time that angular dihydrofuranocoumarin has been isolated from a plant extract by the application of CCC. (8S,9R)-9-(3-Methylbutenoyloxy)-O-acetyl-8,9dihydrooroselol
(compound
B),
(8S,9R)-9-(2-methyl-Z-butenoyloxy)-O-acetyl-8,9-
dihydrooroselol (edultin, compound C), and (8S, 9R)-9-acetoxy-O-(2-methylbutyryl)-8,9dihydrooroselol (compound D) were isolated, and a linear scale-up process from analytical to semi-preparative HPCCC has been successfully used. Although, the compounds are known,
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their relative stereochemistry has been analyzed by 2D NMR spectroscopy for the first time. The present study indicates that HPCCC is a powerful technique for the preparative separation and purification of bioactive components from plant materials and provides a significant advantage over the traditional separation techniques.
Acknowledgments This study was supported by grant no. N N405 617538 from the National Science Centre in Krakow, Poland. The authors have declared no conflict of interest.
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Fig 1. The HPLC chromatogram of the crude dichloromethane extract from Peucedanum cervaria fruits (I) together with isolated compounds with their UV-DAD spectra: oxypeucedanin (compound A, II), (8S,9R)-9-(3-methylbutenoyloxy)-O-acetyl-8,9dihydrooroselol (compound B, III), (8S,9R)-9-(2-methyl-Z-butenoyloxy)-O-acetyl-8,9dihydrooroselol (compound C, III) and (8S,9R)-9-acetoxy-O-(2-methylbutyryl)-8,9dihydrooroselol (compound D, IV). HPLC conditions: column: Zorbax Eclipse XDB C18 (250 mm x 4.6 mm, 5 μm); mobile phase: methanol/water (methanol: 0 min 50%; 5 min 60%; 25 min 80%; 30–40 min 100%); flow rate: 1 mL/min, temperature: 25°C, detection wavelength: 320 nm.
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Fig. 2. HPCCC chromatogram of the crude extract using HEMW (2:1:2:1). Other conditions are given in the text.
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Fig. 3. Chemical structure and HMBC correlations for (8S,9R)-9-(3-methylbutenoyloxy)-Oacetyl-8,9-dihydrooroselol (compound B);
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selected HMBC correlations.
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Fig. 4. Chemical structure and HMBC correlations for (8S,9R)-9-(2-methyl-Z-butenoyloxy)O-acetyl-8,9-dihydrooroselol (edultin, compound C);
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selected HMBC correlations.
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Fig. 5. The proposed structure of the major product ion m/z 227.0703 of compounds B–D observed in ESI-TOF-MS spectrum in positive mode.
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Fig. 6. Chemical structure and HMBC correlations for (8S,9R)-9-acetoxy-O-(2methylbutyryl)-8,9-dihydrooroselol (compound D);
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selected HMBC correlations.
