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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Three new triterpene saponins from roots of Eryngium planum a
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Mariusz Kowalczyk , Milena Masullo , Barbara Thiem , Sonia c
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Piacente , Anna Stochmal & Wiesław Oleszek a
Department of Biochemistry, Institute of Soil Science and Plant Cultivation, State Research Institute, Puławy, Poland b
Department of Pharmaceutical Botany and Plant Biotechnology, Karol Marcinkowski University of Medical Sciences, Poznań, Poland c
Dipartimento di Farmacia, Università degli Studi di Salerno, Via Giovanni Paolo II n. 132, 84084 Fisciano, Salerno, Italy Published online: 17 Mar 2014.
To cite this article: Mariusz Kowalczyk, Milena Masullo, Barbara Thiem, Sonia Piacente, Anna Stochmal & Wiesław Oleszek (2014) Three new triterpene saponins from roots of Eryngium planum, Natural Product Research: Formerly Natural Product Letters, 28:9, 653-660, DOI: 10.1080/14786419.2014.895722 To link to this article: http://dx.doi.org/10.1080/14786419.2014.895722
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Natural Product Research, 2014 Vol. 28, No. 9, 653–660, http://dx.doi.org/10.1080/14786419.2014.895722
Three new triterpene saponins from roots of Eryngium planum
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Mariusz Kowalczyka*, Milena Masulloc, Barbara Thiemb, Sonia Piacentec, Anna Stochmala and Wiesław Oleszeka a Department of Biochemistry, Institute of Soil Science and Plant Cultivation, State Research Institute, Puławy, Poland; bDepartment of Pharmaceutical Botany and Plant Biotechnology, Karol Marcinkowski University of Medical Sciences, Poznan´, Poland; cDipartimento di Farmacia, Universita` degli Studi di Salerno, Via Giovanni Paolo II n. 132, 84084 Fisciano, Salerno, Italy
(Received 19 September 2013; final version received 14 February 2014) Saponin composition of the roots of Eryngium planum L. was investigated. Triterpene saponins found in E. planum and also present in Eryngium maritimum were different from those described previously in Eryngium campestre L. Three primary saponins were isolated and their tentative identifications, based on the electrospray MS/MS fragmentation patterns, were subsequently confirmed by 1D and 2D NMR analyses. Their structures were established as 3-O-b-D -glucopyranosyl-(1 ! 2)-b-D -glucuronopyranosyl-21-O-acetyl22-O-angeloyl-R1-barrigenol (1) and 3-O-b-D -glucopyranosyl-(1 ! 2)-b-D -glucuronopyranosyl-22-O-angeloyl-A1-barrigenol (2) and 3-O-b-D -glucopyranosyl-(1 ! 2)-b-D glucuronopyranosyl-22-O-angeloyl-R1-barrigenol (3). Concentrations of the newly identified compounds in aerial parts and roots of both species were estimated using the liquid chromatography–mass spectrometry method. Keywords: triterpene saponins; barrigenol; Eryngium; liquid chromatography – mass spectrometry; nuclear magnetic resonance
1. Introduction Eryngium is the largest genus in the Apiaceae family. It comprises of over 300 species, many of which have a long tradition of use in folk medicine (Ku¨peli et al. 2006). Eryngium species are known to contain triterpene saponins, phenolic acids, flavonoids, coumarin derivatives, essential oils and acetylenes (Muckensturm et al. 2010; Wang et al. 2012). As revealed by taxonomic and molecular biology studies, relationships between Eryngium species are very complex (Calvin˜o et al. 2010). Thus, subgeneric taxa, such as sections and subsections, were recognised within the genus (Wolff 1913; Calvin˜o et al. 2008). According to Wolff’s classification, species recorded in Poland and investigated in this work – Eryngium campestre L., Eryngium maritimum L. and Eryngium planum L. – represent three different sections: Campestria, Halobia and Plana (Wolff 1913). Kartal et al. (2005, 2006) extensively investigated triterpene saponins from roots of E. campestre. Extending on this research, we compared saponin compositions of E. campestre, E. planum and E. maritimum roots. 2. Results and discussion Barrigenol derivatives described by Kartal et al. in the roots of E. campestre were readily identifiable on the liquid chromatography –mass spectrometry (LC –MS) chromatogram of the
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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methanolic extract from this material (Figure S1a and Table S1). Based on their fragmentation patterns in the negative ion electrospray, deprotonated ions at m/z 1071 and 1055 could represent compounds mentioned in the initial report by Kartal et al. (2005), while ions at m/z 893, 909 and 951 may correspond to those described in the follow-up publication (Kartal et al. 2006). Numerous other peaks presumed to represent other, yet unknown, saponins were also visible. However, saponins detected previously in E. campestre were completely absent in extracts from roots of E. maritimum (Figure S1b) and E. planum (Figure S1c). Chromatogram of E. maritimum root extract shows several peaks, which were recognised as saponins, based on their MS/MS fragmentation spectra (Table S1). Two of these, forming [M 2 H]2 ions at m/z 1041 and 1099, may be similar to eryngiosides H (or I) and J described in Eryngium yuccifolium (Zhang et al. 2008). Similarly, an ion at m/z 1119 on the chromatogram of E. planum root extract may represent eryngioside C (Zhang et al. 2008). Although different sets of saponins are observed in the roots of E. maritimum and E. planum, at least two compounds, presenting deprotonated ions at m/z 967 (Figure S1b and S1c, RT 26.6 min, compound 1) and 909 (Figure S1b and S1c, RT 30.5 min, compound 2) appear in both species. We attempted to isolate these compounds from the roots of E. planum and further characterise them. This was accomplished by a combination of reversed-phase flash chromatography in isocratic conditions followed by preparative HPLC on a reversed-phase C18 column with gradient elution. E. planum saponin producing [M 2 H]2 ion at m/z 925 (Figure S1c, RT 14.7 min, compound 3) was also isolated. Results of co-chromatography of isolated compounds with E. maritimum extracts (not shown) indicated that the latter plant contains the same compounds as E. planum. The sequence of monosaccharides in the carbohydrate side chain of compound 1 could be deduced from the negative ion electrospray MS/MS fragmentation spectrum of its precursor ion at m/z 967 (Figure S2). Low-intensity ion at m/z 805 indicated a loss of dehydrohexose, while ion at m/z 629 could be attributed to a neutral loss of dehydrohexuronosyl-hexose. The base peak at m/z 697 (corresponding to a loss of 270 mass units) is presumably a result of 1,4X cross-ring cleavage of the hexuronic acid moiety followed by a loss of water. Crucial for the structural elucidation of compound 1 is the loss of 82 (m/z 885) and 100 (m/z 867) mass units, which indicates acylation with angelic or tiglic acid moiety. Losses of 42 and 60 mass units (lowintensity ions at m/z 925 and 907, respectively) point to additional acylation with acetyl residue. Acidic hydrolysis of compound 1 followed by determination of absolute configurations of carbohydrates confirmed the presence of D -glucose and D -glucuronic acid. In HR-ESI-MS, quasi-molecular [M 2 H]2 ion of compound 1 exhibited a peak at m/z 967.4894, consistent with a molecular formula of C49H75O19. The 1H NMR spectrum of the aglycone part of 1 revealed signals for seven tertiary methyl groups at d 0.90 (6H), 1.02, 1.05, 1.09, 1.12 and 1.42, one olefinic proton at d 5.50 (t, J ¼ 3.5), five oxygen-bearing methine protons at d 3.25 (dd, J ¼ 11.5, 4.5 Hz), 3.78 (d, J ¼ 4.0 Hz), 3.80 (d, J ¼ 4.0 Hz), 5.85 (d, J ¼ 10 Hz) and 5.60 (d, J ¼ 10.0 Hz) and one primary alcoholic function at d 3.32 and 3.07 (each, d, J ¼ 9.3 Hz) (Table S2). The HMBC spectrum allowed us to assign the secondary alcoholic function on the triterpene skeleton. The HMBC correlations between the proton at d 3.25 with the methyl carbons at d 16.6 (C-24) and 28.3 (C-23) established its position at C-3, the HMBC correlations between the proton at d 5.85 with the methyl carbons at d 19.6 (C-30) and 29.5 (C-29) allowed to locate it at C-21, while the correlation between the proton at d 3.78 and the methyl carbon at d 21.0 (C-27) assigned the secondary alcoholic function at C-15. The remaining secondary alcoholic groups at d 3.80 and 5.60 were assigned at C-16 and C-22, respectively, on the basis of the COSY correlations between the proton at d 3.80 and the proton at d 3.78 (H-15) and between the proton at d 5.60 and the proton at d 5.85 (H-21). A detailed analysis of 2D NMR data suggested the structure of an aglycone of 1 as an R1-barrigenol. The configuration at C-3, C-15, C-16, C-21 and C-22 of an
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R1-barrigenol were confirmed by ROESY experiment and by the multiplicity and coupling constants of the proton signals. Indeed, in the ROESY spectrum cross-peaks between the proton at d 3.24 (H-3) and the protons at d 0.83 (H-5) and 1.12 (H-23) established the b-orientation of the hydroxyl group at C-3, while correlations between the protons at d 3.80 (H-16) and d 5.60 (H-22) with the protons at d 3.32 and 3.07 (H2-28) allowed to deduce the a-orientation at C-16 and C-22. Orientations of the alcoholic groups at C-15 and C-21 were established as a and b by, respectively, coupling constants of H-15 at d 3.78 (d, J ¼ 4.0 Hz) and H-21 at d 5.85 (d, J ¼ 10 Hz). These data were in agreement with those reported in the literature for R1-barrigenol (Errington et al. 1967; Matsushita et al. 2004; Tabopoda et al. 2012). The 1H and 13C NMR spectra revealed additional signals for methyl groups at d 1.91 (br s) and at 2.00 (br dd, J ¼ 7.1, 1.3 Hz) and an olefinic proton at d 6.15 (dq, J ¼ 7.1, 1.3 Hz) which are typical of an angeloyl moiety as well as a signal characteristic for acetyl group at d 1.97. The downfield shift of H-22 (d 5.60) and the HMBC correlations between the proton at d 5.60 (H-22) with the carbon resonance at d 169.4 indicate the occurrence of an angeloyl group at C-22, while the downfield shift of H-21 at d 5.85 and the HMBC correlation between the proton signals at d 5.85 and 1.97 with the carbon resonance at d 172.3 allowed us to locate the acetyl function at C-21. For the sugar portion, compound 1 displayed signals corresponding to two anomeric protons at d 4.55 (d, J ¼ 7.5 Hz) and 4.72 (d, J ¼ 7.8 Hz) (Table S3). On the basis of 2D NMR data, one b-glucuronopyranosyl unit (d 4.55) and one b-glucopyranosyl (d 4.72) unit were identified. The determination of the sequence and linkage sites was obtained from the HMBC correlations between the proton signal at 4.72 (H-1glc) with the carbon signal at d 80.2 (C-2glcA) and the signal at d 4.55 (H-1glcA) with the carbon resonance at d 91.4 (C-3). On the basis of these data, compound 1 was identified as 3-O-b-D -glucopyranosyl-(1 ! 2)-b-D -glucuronopyranosyl-21-Oacetyl-22-O-angeloyl-R1-barrigenol (Figure 1), a compound not reported previously in the literature. In the negative ion MS/MS spectrum of compound 2 obtained from a precursor ion at m/z 909 (Figure S3), ions at m/z 747 and 571 point to subsequent loss of dehydrohexosyl and dehydrohexuranosyl-hexosyl residues, indicating carbohydrate sequence identical to that of compound 1. Similarly, the base peak at m/z 639 is a result of the loss of 270 mass units that we attributed to a cross-ring cleavage of hexuronic acid residue and subsequent loss of water. Ions at m/z 827 and 809 suggest acylation with an angeloyl or a tigloyl group. Acidic hydrolysis of compound 2 yielded the same monosaccharides as for compound 1: D -glucose and D -glucuronic acid. In negative ion HR-ESI-MS, compound 2 revealed a deprotonated ion at m/z 909.4845, corresponding to a molecular formula of C47H73O17. The 1H NMR spectrum of the aglycone moiety of compound 2 revealed signals for seven tertiary methyl groups at d 0.90, 0.95, 1.02, 1.05, 1.08, 1.11 and 1.43, one olefinic proton at d 5.47 (t, J ¼ 3.5), three oxygen-bearing methine protons at d 3.25 (dd, J ¼ 11.5, 4.5 Hz), 3.95 (d, J ¼ 4.0 Hz), 3.81 (d, J ¼ 4.0 Hz) and 5.49 (dd, J ¼ 12.0, 6.4 Hz) and one primary alcoholic function at d 3.35 and 3.16 (each, d, J ¼ 9.3 Hz) (Table S2). In comparison with compound 1, compound 2 indicated the absence of the secondary alcoholic function at C-21. 1H and 13C NMR spectroscopic data were in agreement with those of A1-barrigenol (Konoshima & Lee 1986). Methyl signals at d 1.94 (br s) and 2.00 (br dd, J ¼ 7.1, 1.3 Hz) and the olefinic proton at d 6.15 (dq, J ¼ 7.1, 1.3 Hz), ascribable to an angeloyl group, were evident. Two anomeric protons at d 4.55 (d, J ¼ 7.5 Hz) and 4.72 (d, J ¼ 7.8 Hz) (Table S2) assigned to one b-glucuronopyranosyl unit (d 4.55) and one b-glucopyranosyl (d 4.72) unit were identified. The determination of the sequence and linkage sites obtained from the HMBC was the same as reported for compound 1. The structure of compound 2 was determined as previously unreported 3-O-b-D glucopyranosyl-(1 ! 2)-b-D -glucuronopyranosyl-22-O-angeloyl-A1-barrigenol.
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Figure 1. Saponins from roots of E. planum and E. maritimum.
Positive and negative ion fragmentation spectra of compound 3 were almost identical to the spectra of compound 1, except that the m/z values for the most prominent ions were 42 mass units lower (Figure S4). Also, ions resulting from the loss of acetyl group were not observed, indicating that compound 3 may be de-acetylated compound 1. An ion at m/z 925.4786, consistent with a molecular formula of C47H73O18, was observed for compound 3 in HR-ESI-MS. Except the absence of signals from C-21 acetyl group, 1H and 13C NMR spectra of compound 3 were superimposable on the analogous spectra of compound 1; therefore, 3 was identified as 3-O-b-D -glucopyranosyl-(1 ! 2)-b-D -glucuronopyranosyl-22-O-angeloyl-R1-barrigenol. Concurring with previous studies on Eryngium species, triterpene glycosides isolated and characterised in this work are derivatives of acylated R1- and A1-barrigenols. Extracts of E. planum roots collected from the natural habitats contained similarly high amounts of compounds 1 and 2, which in both cases exceeded 2 mg/mg of fresh weight (FW) (Table 1). In analogous samples from E. maritimum, these concentrations were much lower, at only 0.2 and 0.4 mg/mg FW for compounds 1 and 2, respectively. Only small amount, approximately 30 ng/
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Table 1. Estimated contents of isolated saponins (ng/g FW) in roots and rosette leaves of E. planum and E. maritimum.a E. planum
Roots Rosette leaves
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a
E. maritimum
1
2
3
1
2
3
2346.7 ^ 456.6 53.3 ^ 14.0
2616.0 ^ 334.0 132.0 ^ 27.7
176.0 ^ 32.0 6.7 ^ 2.3
442.0 ^ 83.5 20.0 ^ 6.9
198.7 ^ 26.6 49.3 ^ 12.9
24.0 ^ 8.0 n.d.
Mean ^ standard deviation (n ¼ 3), n.d. – not detected.
mg FW, of compound 3 was detected in the roots of E. maritimum, but roots of E. planum contained at least an order of magnitude more of this saponin. Aerial parts (rosette leaves) of E. maritimum contained only minute amounts of compound 1; higher concentration was detected in E. planum.
3. Experimental 3.1. Materials Roots of intact plants of E. planum L. and E. campestre L. were collected from natural habitats in Poland (Łukaszewo, Kuyavian-Pomeranian province) in August 2008. E. maritimum L. roots were collected from plants grown on an artificial dune plot in Poznan´ University Botanical Garden in September 2009. The taxonomic identification of species was made according to Flora Europea (Tutin et al. 1968). The voucher specimens (EP-2008-08-01K, EC-2008-08-01K and EM-2009-08-01K) belonging to Department of Pharmaceutical Botany and Plant Biotechnology, K. Marcinkowski, University of Medical Sciences in Poznan´ have been temporarily deposited in the Herbarium of Medicinal Plant Garden in Institute of Natural Fibers and Medicinal Plants in Poznan´. Collected roots were washed with tap water, blot-dried on a filter paper and lyophilised. HPLC gradient-grade solvents were from J.T. Baker (Phillipsburg, NJ, USA); all other chemicals were from Sigma-Aldrich (St. Louis, MO, USA). 3.2. Extraction, purification and LC –MS analyses Lyophilised plant material (roots of E. planum, approximately 100 g) was defatted with chloroform (2.5 L) and extracted three times with 80% MeOH (2.0 L each time) on an ultrasonic bath at room temperature. Combined extracts (15 g) were concentrated under reduced pressure and lyophilised, dissolved in 45% methanol and loaded onto a C18 flash chromatography cartridge (Grace Revelerisw RP C18 Flash Cartridge, 120 g) connected to a Reveleris flash chromatography system (Grace-Davison, Columbia, MD, USA) equipped with UV and ELS detectors. Following isocratic elution with 45% acetonitrile containing 0.1% acetic acid, 35 fractions containing main peaks detected by an evaporative light scattering device were collected and analysed using LC-MS as described later. Fractions containing compounds of interest (23 – 27 for compound 1, 28 – 31 for compound 2 and 15 –16 for compound 3), were combined and further chromatographed using a gradient of 35 –42% acetonitrile containing 0.5% of acetic acid elution on a semi-preparative Gilson HPLC (Gilson, Inc., Middleton, WI, USA) equipped with a PrepELS (Evaporative Light Scattering) detector and an RP-18 column (10 mm £ 250 mm; 5 mm; Kromasil C18) at flow rate 5.0 mL/min. These three separations yielded fractions containing compounds 1 (12 mg), 2 (15 mg) and 3 (4 mg). Collected peaks were dried down in a stream of nitrogen and stored in 2 208C.
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Semi-quantitative and qualitative LC-MS analyses were carried out as described earlier (Kowalczyk et al. 2011) except that in semi-qualitative analyses deprotonated quasi-molecular ions at m/z 925, 909 and 967 were observed and digoxin (final conc. 8 ng/ml) was used as an internal standard. HR-ESI-MS analyses were performed on a Waters Synapt G2-S (Waters Corp., Milford, MA, USA) mass spectrometer using direct sample infusion.
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3.3. Acid hydrolysis Samples of approximately 1 mg were dissolved in 0.5 mL of dioxane/water (1:1 v/v) mixture and then hydrolysed for 6 h with 2 mL of 2 M HCl in 808C. After hydrolysis, samples were neutralised with Amberlite IRA-400, decanted and partitioned three times against 6 mL of ethyl acetate. Resulting water phase was evaporated to dryness and re-dissolved in 50 mL of pyridine. Derivatisation and determination of absolute configurations were then performed as described by Tanaka et al. except that separations were performed on a Waters HPLC consisting of 616 pump 717 autosampler, 996 PDA detector and using Knauer Eurospher 250 £ 4.6 mm, 5 mm column (Knauer, Berlin, Germany). 3.4. NMR analyses NMR experiments were performed on a Bruker DRX-600 spectrometer (Bruker BioSpin GmBH, Rheinstetten, Germany) equipped with a Bruker 5 mm TCI CryoProbe at 300 K. All 2D NMR spectra were acquired in CD3OD (99.95%, Sigma-Aldrich) and standard pulse sequences and phase cycling were used for DQF-COSY, HSQC, HMBC and ROESY spectra. The NMR data were processed using UXNMR software. 3.5. Chemical data of compounds 1 –3 21 KBr Compound 1: Purity . 80% (HPLC-DAD), ½a25 D 2 12.7 (c 0.1 MeOH); IR nmax cm : 3430 (. OH), 2925 (. CH), 1680 (CvO), 1656 (CvC); negative ion ESI-MS m/z 967 [M 2 H]2, positive ion ESI-MS m/z 991 [M þ Na]þ, negative and positive ion ESI-MS/MS spectra are reported in Figure S2, negative ion HR-ESI-MS m/z 967.4894 [M 2 H]2 (calcd for C49H75O2 19 967.4903) aglycone. 1H NMR (CD3OD, 600 MHz) dH (ppm, J in Hz): 3.25 (1H, dd, J ¼ 11.5, 4.5, 3-CH), 5.50, (1H, t, J ¼ 3.5, 12-CH), 3.78 (1H, d, J ¼ 4.0, 15-CH), 3.80 (1H, d, J ¼ 4.0, 16CH), 5.85 (1H, d, J ¼ 10.0, 21-CH), 5.60 (1H, d, J ¼ 10.0, 22-CH), 1.12 (3H, s, 23-CH3), 0.90 (3H, s, 24-CH3), 1.02 (3H, s, 25-CH3), 1.05 (3H, s, 26-CH3), 1.42 (3H, s, 27-CH3), 3.32 (1H, d, J ¼ 9.3, 28-CH), 3.07 (1H, d, J ¼ 9.3 Hz, 28-CH), 0.90 (3H, s, 29-CH3), 1.09 (3H, s, 30-CH3), C-21 acetyl, 1.97 (3H, s, 2-CH3), C-22 angeloyl, 6.15 (1H, dq, J ¼ 7.1, 1.3, 3-CH), 2.00 (3H, br dd, J ¼ 7.1, 1.3, 4-CH3), 1.91 (3H, br s, 5-CH3) aglycone. 13C NMR (CD3OD, 150 MHz) dC (ppm): 40.0 (C-1), 26.7 (C-2), 91.4 (C-3), 39.5 (C-4), 56.4 (C-5), 19.3 (C-6), 36.8 (C-7), 40.8 (C-8), 48.0 (C-9), 37.3 (C-10), 24.5 (C-11), 126.7 (C-12), 143.0 (C-13), 48.4 (C-14), 68.0 (C-15), 74.5 (C-16), 48.0 (C-17), 41.8 (C-18), 46.9 (C-19), 36.0 (C-20), 80.2 (C-21), 73.0 (C-22), 28.3 (C-23), 16.6 (C-24), 16.7 (C-25), 17.6 (C-26), 21.0 (C-27), 63.5 (C-28), 29.5 (C-29), 19.6 (C-30); C-21 acetyl, 172.3 (C-1), 20.6 (C-2); C-22 angeloyl; 169.4 (C-1), 128.7 (C-2), 139.4 (C-3), 15.8 (C-4), 20.6 (C-5). 21 KBr Compound 2: Purity . 75% (HPLC-DAD), ½a25 D 2 15.0 (c 0.1 MeOH); IR nmax cm : 3420 (. OH), 2925 (. CH), 1675 (CvO), 1660 (CvC); negative ion ESI-MS m/z 909 [M 2 H]2, positive ion ESI-MS m/z 933 [M þ Na]þ, negative and positive ion ESI-MS/MS are shown in Figure S3; negative ion HR-ESI-MS m/z 909.4845 [M 2 H]2 (calcd for C47H73O2 17 909.4848) aglycone. 1H NMR (CD3OD, 600 MHz) dH (ppm, J in Hz): 3.25 (1H, dd, J ¼ 11.5, 4.5, 3-CH), 5.47, (1H, t, J ¼ 3.5, 12-CH), 3.81 (1H, d, J ¼ 4.0, 15-CH), 3.95 (1H, d, J ¼ 4.0, 16-CH), 2.25
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(1H, t, J ¼ 12.0, 21-CH), 5.49 (1H, dd, J ¼ 12.0, 6.4, 22-CH), 1.11 (3H, s, 23-CH3), 0.90 (3H, s, 24-CH3), 1.02 (3H, s, 25-CH3), 1.05 (3H, s, 26-CH3), 1.43 (3H, s, 27-CH3), 3.35 (1H, d, J ¼ 9.3, 28-CH), 3.16(1H, d, J ¼ 9.3, 28-CH), 0.95 (3H, s, 29-CH3), 1.08 (3H, s, 30-CH3), C-22 angeloyl, 6.15 (1H, dq, J ¼ 7.1, 1.3, 3-CH), 2.00 (3H, br dd, J ¼ 7.1, 1.3, 4-CH3), 1.94 (3H, br s, 5-CH3) aglycone. 13C NMR (CD3OD, 150 MHz) dC (ppm): 40.0 (C-1), 26.7 (C-2), 91.2 (C-3), 40.0 (C-4), 56.5 (C-5), 19.4 (C-6), 37.0 (C-7), 41.9 (C-8), 47.9 (C-9), 37.5 (C-10), 24.6 (C-11), 126.1 (C-12), 144.1 (C-13), 48.1 (C-14), 68.4 (C-15), 75.2 (C-16), 48.1 (C-17), 42.1 (C-18), 47.5 (C-19), 32.0 (C-20), 41.8 (C-21), 73.2 (C-22), 28.2 (C-23), 16.6 (C-24), 16.1 (C-25), 17.8 (C-26), 20.7 (C-27), 63.6 (C-28), 33.4 (C-29), 24.9 (C-30); C-22 angeloyl; 169.3 (C-1), 129.7 (C-2), 137.9 (C-3), 15.8 (C-4), 20.7 (C-5). 21 KBr Compound 3: Purity .85% (HPLC-DAD), ½a25 D 2 9.5 (c 0.1 MeOH); IR nmax cm : 3430 (.OH), 2930 (.CH), 1675 (CvO), 1656 (CvC); negative ion ESI-MS m/z 925 [M 2 H]2; positive ion ESI-MS m/z 949 [M þ Na]þ; negative and positive ion ESI-MS/MS spectra are reported in Figure S4; negative ion HR-ESI-MS m/z 925.4786 [M 2 H]2 (calcd for C47H73O2 18 925.4773) aglycone. 1H NMR (CD3OD, 600 MHz) dH (ppm, J in Hz): 3.24 (1H, dd, J ¼ 11.5, 4.5, 3CH), 5.49, (1H, t, J ¼ 3.5, 12-CH), 3.78 (1H, d, J ¼ 4.0, 15-CH), 3.80 (1H, d, J ¼ 4.0, 16-CH), 4.3 (1H, d, J ¼ 10.0, 21-CH), 5.36 (1H, d, J ¼ 10, 22-CH), 1.12 (3H, s, 23-CH3), 0.90 (3H, s, 24-CH3), 1.02 (3H, s, 25-CH3), 1.05 (3H, s, 26-CH3), 1.42 (3H, s, 27-CH3), 3.32 (1H, d, J ¼ 9.3, 28-CH), 3.05 (1H, d, J ¼ 9.3, 28-CH), 1.02 (3H, s, 29-CH3), 1.04 (3H, s, 30-CH3), C-22 angeloyl, 6.13 (1H, dq, J ¼ 7.1, 1.3, 3-CH), 2.03 (3H, br dd, J ¼ 7.1, 1.3, 4-CH3), 1.98 (3H, br s, 5-CH3) aglycone. 13C NMR (CD3OD, 150 MHz) dC (ppm): 39.8 (C-1), 26.7 (C-2), 91.4 (C-3), 40.1 (C-4), 56.0 (C-5), 19.3 (C-6), 37.0 (C-7), 41.7 (C-8), 47.9 (C-9), 37.0 (C-10), 24.4 (C-11), 126.6 (C-12), 143.7 (C-13), 48.1 (C-14), 68.3 (C-15), 74.5 (C-16), 48.0 (C-17), 41.3 (C-18), 47.7 (C-19), 36.0 (C-20), 77.5 (C-21), 76.6 (C-22), 28.4 (C-23), 16.6 (C-24), 16.0 (C-25), 17.6 (C-26), 21.0 (C-27), 63.8 (C-28), 29.7 (C29), 18.4 (C-30); C-22 angeloyl; 170.4 (C-1), 129.6(C-2), 138.4 (C-3), 15.7 (C-4), 20.6 (C-5). Carbohydrate parts of compounds 1 –3: 1H NMR (CD3OD, 600 MHz) b-D -GlcA (at C-3) dH (ppm, J in Hz); 4.55 (1H, d, J ¼ 7.5, H-1), 3.67 (1H, dd, J ¼ 7.5, 9.0, H-2), 3.62 (1H, dd, J ¼ 9.0, 9.0, H-3), 3.57 (1H, dd, J ¼ 9.0, 9.0, H-4), 3.81 (1H, d, J ¼ 9.0, H-5); b-D -Glc (at C2GlcA), 4.72 (1H, d, J ¼ 7.8, H-10 ), 3.26 (1H, dd, J ¼ 7.8, 9.0, H-20 ), 3.39 (1H, dd, J ¼ 9.0, 9.0, H-30 ), 3.24 (1H, dd, J ¼ 9.0, 9.0, H-40 ), 3.28 (1H, m, H-50 ), 3.86 (1H, dd, J ¼ 2.5, 12.0, H-60 ), 3.65 (1H, dd, J ¼ 4.5, 12.0, H-60 ). 13C NMR (CD3OD, 150 MHz) dC (ppm): b-D -GlcA (at C-3), 105.1 (C-1), 80.2 (C-2), 77.5 (C-3), 72.6 (C-4), 76.6 (C-5), 171.8 (C-6), b-D -Glc (at C-2glcA), 103.9 (C-10 ), 75.6 (C-20 ), 78.0 (C-30 ), 70.3 (C-40 ), 78.0 (C-50 ), 62.9 (C-60 ). 4. Conclusions Our results indicate a large diversity, both qualitative and quantitative, in saponin composition between the investigated species. Although aglycones of newly identified and isolated from E. planum and E. maritimum saponins are similar or the same as those reported from E. campestre, their oligosaccharides contain D -glucose instead of L -rhamnose as a terminal carbohydrate. Furthermore, saponins containing D -rhamnose were not detected in extracts from the two tested species. In addition to structural differences, plant materials collected from plants in their natural environment contained varied amounts of saponins. To a large extent, this was also species dependent, with E. maritimum containing relatively small amount of saponins. Although the subsection classification of Eryngium was developed mainly on the basis of morphological features, it appears to be reflected on a phytochemical level as well. Supplementary material Supplementary material relating to this article is available online, alongside Figures S1 –S7 and Tables S1 and S2.
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Acknowledgements Parts of this work were supported by the Ministry of Science and Education, Warsaw, Poland from educational sources of 2011– 2013 as grant no. NN405683340 and from the 7th Framework Program of European Community, PROFICIENCY (contract no. 245751).
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References Calvin˜o CI, Martı´nez SG, Downie SR. 2008. The evolutionary history of Eryngium (Apiaceae, Saniculoideae): rapid radiations, long distance dispersals, and hybridizations. Mol Phylogen Evol. 46:1129–1150. Calvin˜o CI, Martı´nez SG, Downie SR. 2010. Unraveling the taxonomic complexity of Eryngium L. (Apiaceae, Saniculoideae): phylogenetic analysis of 11 non-coding cpDNA loci corroborates rapid radiations. Plant Div Evol. 128:137–149. Errington SG, White DE, Fuller MW. 1967. The structures of A1-barrigenol and R1-barrigenol. Tetrahedron Lett. 8:1289–1294. Kartal M, Mitaine-Offer A-C, Abu-Asaker M, Miyamoto T, Calis I, Wagner H, Lacaille-Dubois M-A. 2005. Two new triterpene saponins from Eryngium campestre. Chem Pharm Bull. 53:1318–1320. Kartal M, Mitaine-Offer A-C, Paululat T, Abu-Asaker M, Wagner H, Mirjolet J-F, Guilbaud N, Lacaille-Dubois M-A. 2006. Triterpene saponins from Eryngium campestre. J Nat Prod. 69:1105–1108. Konoshima T, Lee K-H. 1986. Antitumor agents, 82. Cytotoxic sapogenols from Aesculus hippocastanum. J Nat Prod. 49:650–656. Kowalczyk M, Pecio Ł, Stochmal A, Oleszek W. 2011. Qualitative and quantitative analysis of steroidal saponins in crude extract and bark powder of Yucca schidigera Roezl. J Agr Food Chem. 59:8058–8064. Ku¨peli E, Kartal M, Aslan S, Yesilada E. 2006. Comparative evaluation of the anti-inflammatory and antinociceptive activity of Turkish Eryngium species. J Ethnopharmacol. 107:32– 37. Matsushita A, Sasaki Y, Warashina T, Miyase T, Noguchi H, Vander Velde D. 2004. Hydrocotylosides I –VII, new oleanane saponins from Hydrocotyle sibthorpioides. J Nat Prod. 67:384–388. Muckensturm B, Boulanger A, Farahi M, Reduron JP. 2010. Secondary metabolites from Eryngium species. Nat Prod Res. 24:391–397. Tabopda TK, Mitaine-Offer A-C, Miyamoto T, Tanaka C, Mirjolet JF, Duchamp O, Ngadjui BT, Lacaille-Dubois M-A. 2012. Triterpenoid saponins from Hydrocotyle bonariensis Lam. Phytochemistry. 73:142–147. Tutin TG, Heywood VH, Burges NA, Moore DM, Valentine DH, Walters SM, Webb DA. 1968. Flora Europea. Vol. 2. Cambridge: Cambridge University Press. Wang P, Su Z, Yuan W, Deng G, Li S. 2012. Phytochemical constituents and pharmacological activities of Eryngium L. (Apiaceae). Pharm Crops. 3:99–120. Wolff H. 1913. IV. 228: Umbelliferae-Saniculoideae. In: Engler A, editor. Das Pflanzenreich. Leipzig: Engelmann; p. 106–271. Zhang Z, Li S, Ownby S, Wang P, Yuan W, Zhang W, Scott Beasley R. 2008. Phenolic compounds and rare polyhydroxylated triterpenoid saponins from Eryngium yuccifolium. Phytochemistry. 69:2070–2080.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Three new triterpene saponins from roots of Eryngium planum a
c
b
Mariusz Kowalczyk , Milena Masullo , Barbara Thiem , Sonia c
a
a
Piacente , Anna Stochmal & Wiesław Oleszek a
Department of Biochemistry, Institute of Soil Science and Plant Cultivation, State Research Institute, Puławy, Poland b
Department of Pharmaceutical Botany and Plant Biotechnology, Karol Marcinkowski University of Medical Sciences, Poznań, Poland c
Dipartimento di Farmacia, Università degli Studi di Salerno, Via Giovanni Paolo II n. 132, 84084 Fisciano, Salerno, Italy Published online: 17 Mar 2014.
To cite this article: Mariusz Kowalczyk, Milena Masullo, Barbara Thiem, Sonia Piacente, Anna Stochmal & Wiesław Oleszek (2014) Three new triterpene saponins from roots of Eryngium planum, Natural Product Research: Formerly Natural Product Letters, 28:9, 653-660, DOI: 10.1080/14786419.2014.895722 To link to this article: http://dx.doi.org/10.1080/14786419.2014.895722
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Natural Product Research, 2014 Vol. 28, No. 9, 653–660, http://dx.doi.org/10.1080/14786419.2014.895722
Three new triterpene saponins from roots of Eryngium planum
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Mariusz Kowalczyka*, Milena Masulloc, Barbara Thiemb, Sonia Piacentec, Anna Stochmala and Wiesław Oleszeka a Department of Biochemistry, Institute of Soil Science and Plant Cultivation, State Research Institute, Puławy, Poland; bDepartment of Pharmaceutical Botany and Plant Biotechnology, Karol Marcinkowski University of Medical Sciences, Poznan´, Poland; cDipartimento di Farmacia, Universita` degli Studi di Salerno, Via Giovanni Paolo II n. 132, 84084 Fisciano, Salerno, Italy
(Received 19 September 2013; final version received 14 February 2014) Saponin composition of the roots of Eryngium planum L. was investigated. Triterpene saponins found in E. planum and also present in Eryngium maritimum were different from those described previously in Eryngium campestre L. Three primary saponins were isolated and their tentative identifications, based on the electrospray MS/MS fragmentation patterns, were subsequently confirmed by 1D and 2D NMR analyses. Their structures were established as 3-O-b-D -glucopyranosyl-(1 ! 2)-b-D -glucuronopyranosyl-21-O-acetyl22-O-angeloyl-R1-barrigenol (1) and 3-O-b-D -glucopyranosyl-(1 ! 2)-b-D -glucuronopyranosyl-22-O-angeloyl-A1-barrigenol (2) and 3-O-b-D -glucopyranosyl-(1 ! 2)-b-D glucuronopyranosyl-22-O-angeloyl-R1-barrigenol (3). Concentrations of the newly identified compounds in aerial parts and roots of both species were estimated using the liquid chromatography–mass spectrometry method. Keywords: triterpene saponins; barrigenol; Eryngium; liquid chromatography – mass spectrometry; nuclear magnetic resonance
1. Introduction Eryngium is the largest genus in the Apiaceae family. It comprises of over 300 species, many of which have a long tradition of use in folk medicine (Ku¨peli et al. 2006). Eryngium species are known to contain triterpene saponins, phenolic acids, flavonoids, coumarin derivatives, essential oils and acetylenes (Muckensturm et al. 2010; Wang et al. 2012). As revealed by taxonomic and molecular biology studies, relationships between Eryngium species are very complex (Calvin˜o et al. 2010). Thus, subgeneric taxa, such as sections and subsections, were recognised within the genus (Wolff 1913; Calvin˜o et al. 2008). According to Wolff’s classification, species recorded in Poland and investigated in this work – Eryngium campestre L., Eryngium maritimum L. and Eryngium planum L. – represent three different sections: Campestria, Halobia and Plana (Wolff 1913). Kartal et al. (2005, 2006) extensively investigated triterpene saponins from roots of E. campestre. Extending on this research, we compared saponin compositions of E. campestre, E. planum and E. maritimum roots. 2. Results and discussion Barrigenol derivatives described by Kartal et al. in the roots of E. campestre were readily identifiable on the liquid chromatography –mass spectrometry (LC –MS) chromatogram of the
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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methanolic extract from this material (Figure S1a and Table S1). Based on their fragmentation patterns in the negative ion electrospray, deprotonated ions at m/z 1071 and 1055 could represent compounds mentioned in the initial report by Kartal et al. (2005), while ions at m/z 893, 909 and 951 may correspond to those described in the follow-up publication (Kartal et al. 2006). Numerous other peaks presumed to represent other, yet unknown, saponins were also visible. However, saponins detected previously in E. campestre were completely absent in extracts from roots of E. maritimum (Figure S1b) and E. planum (Figure S1c). Chromatogram of E. maritimum root extract shows several peaks, which were recognised as saponins, based on their MS/MS fragmentation spectra (Table S1). Two of these, forming [M 2 H]2 ions at m/z 1041 and 1099, may be similar to eryngiosides H (or I) and J described in Eryngium yuccifolium (Zhang et al. 2008). Similarly, an ion at m/z 1119 on the chromatogram of E. planum root extract may represent eryngioside C (Zhang et al. 2008). Although different sets of saponins are observed in the roots of E. maritimum and E. planum, at least two compounds, presenting deprotonated ions at m/z 967 (Figure S1b and S1c, RT 26.6 min, compound 1) and 909 (Figure S1b and S1c, RT 30.5 min, compound 2) appear in both species. We attempted to isolate these compounds from the roots of E. planum and further characterise them. This was accomplished by a combination of reversed-phase flash chromatography in isocratic conditions followed by preparative HPLC on a reversed-phase C18 column with gradient elution. E. planum saponin producing [M 2 H]2 ion at m/z 925 (Figure S1c, RT 14.7 min, compound 3) was also isolated. Results of co-chromatography of isolated compounds with E. maritimum extracts (not shown) indicated that the latter plant contains the same compounds as E. planum. The sequence of monosaccharides in the carbohydrate side chain of compound 1 could be deduced from the negative ion electrospray MS/MS fragmentation spectrum of its precursor ion at m/z 967 (Figure S2). Low-intensity ion at m/z 805 indicated a loss of dehydrohexose, while ion at m/z 629 could be attributed to a neutral loss of dehydrohexuronosyl-hexose. The base peak at m/z 697 (corresponding to a loss of 270 mass units) is presumably a result of 1,4X cross-ring cleavage of the hexuronic acid moiety followed by a loss of water. Crucial for the structural elucidation of compound 1 is the loss of 82 (m/z 885) and 100 (m/z 867) mass units, which indicates acylation with angelic or tiglic acid moiety. Losses of 42 and 60 mass units (lowintensity ions at m/z 925 and 907, respectively) point to additional acylation with acetyl residue. Acidic hydrolysis of compound 1 followed by determination of absolute configurations of carbohydrates confirmed the presence of D -glucose and D -glucuronic acid. In HR-ESI-MS, quasi-molecular [M 2 H]2 ion of compound 1 exhibited a peak at m/z 967.4894, consistent with a molecular formula of C49H75O19. The 1H NMR spectrum of the aglycone part of 1 revealed signals for seven tertiary methyl groups at d 0.90 (6H), 1.02, 1.05, 1.09, 1.12 and 1.42, one olefinic proton at d 5.50 (t, J ¼ 3.5), five oxygen-bearing methine protons at d 3.25 (dd, J ¼ 11.5, 4.5 Hz), 3.78 (d, J ¼ 4.0 Hz), 3.80 (d, J ¼ 4.0 Hz), 5.85 (d, J ¼ 10 Hz) and 5.60 (d, J ¼ 10.0 Hz) and one primary alcoholic function at d 3.32 and 3.07 (each, d, J ¼ 9.3 Hz) (Table S2). The HMBC spectrum allowed us to assign the secondary alcoholic function on the triterpene skeleton. The HMBC correlations between the proton at d 3.25 with the methyl carbons at d 16.6 (C-24) and 28.3 (C-23) established its position at C-3, the HMBC correlations between the proton at d 5.85 with the methyl carbons at d 19.6 (C-30) and 29.5 (C-29) allowed to locate it at C-21, while the correlation between the proton at d 3.78 and the methyl carbon at d 21.0 (C-27) assigned the secondary alcoholic function at C-15. The remaining secondary alcoholic groups at d 3.80 and 5.60 were assigned at C-16 and C-22, respectively, on the basis of the COSY correlations between the proton at d 3.80 and the proton at d 3.78 (H-15) and between the proton at d 5.60 and the proton at d 5.85 (H-21). A detailed analysis of 2D NMR data suggested the structure of an aglycone of 1 as an R1-barrigenol. The configuration at C-3, C-15, C-16, C-21 and C-22 of an
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R1-barrigenol were confirmed by ROESY experiment and by the multiplicity and coupling constants of the proton signals. Indeed, in the ROESY spectrum cross-peaks between the proton at d 3.24 (H-3) and the protons at d 0.83 (H-5) and 1.12 (H-23) established the b-orientation of the hydroxyl group at C-3, while correlations between the protons at d 3.80 (H-16) and d 5.60 (H-22) with the protons at d 3.32 and 3.07 (H2-28) allowed to deduce the a-orientation at C-16 and C-22. Orientations of the alcoholic groups at C-15 and C-21 were established as a and b by, respectively, coupling constants of H-15 at d 3.78 (d, J ¼ 4.0 Hz) and H-21 at d 5.85 (d, J ¼ 10 Hz). These data were in agreement with those reported in the literature for R1-barrigenol (Errington et al. 1967; Matsushita et al. 2004; Tabopoda et al. 2012). The 1H and 13C NMR spectra revealed additional signals for methyl groups at d 1.91 (br s) and at 2.00 (br dd, J ¼ 7.1, 1.3 Hz) and an olefinic proton at d 6.15 (dq, J ¼ 7.1, 1.3 Hz) which are typical of an angeloyl moiety as well as a signal characteristic for acetyl group at d 1.97. The downfield shift of H-22 (d 5.60) and the HMBC correlations between the proton at d 5.60 (H-22) with the carbon resonance at d 169.4 indicate the occurrence of an angeloyl group at C-22, while the downfield shift of H-21 at d 5.85 and the HMBC correlation between the proton signals at d 5.85 and 1.97 with the carbon resonance at d 172.3 allowed us to locate the acetyl function at C-21. For the sugar portion, compound 1 displayed signals corresponding to two anomeric protons at d 4.55 (d, J ¼ 7.5 Hz) and 4.72 (d, J ¼ 7.8 Hz) (Table S3). On the basis of 2D NMR data, one b-glucuronopyranosyl unit (d 4.55) and one b-glucopyranosyl (d 4.72) unit were identified. The determination of the sequence and linkage sites was obtained from the HMBC correlations between the proton signal at 4.72 (H-1glc) with the carbon signal at d 80.2 (C-2glcA) and the signal at d 4.55 (H-1glcA) with the carbon resonance at d 91.4 (C-3). On the basis of these data, compound 1 was identified as 3-O-b-D -glucopyranosyl-(1 ! 2)-b-D -glucuronopyranosyl-21-Oacetyl-22-O-angeloyl-R1-barrigenol (Figure 1), a compound not reported previously in the literature. In the negative ion MS/MS spectrum of compound 2 obtained from a precursor ion at m/z 909 (Figure S3), ions at m/z 747 and 571 point to subsequent loss of dehydrohexosyl and dehydrohexuranosyl-hexosyl residues, indicating carbohydrate sequence identical to that of compound 1. Similarly, the base peak at m/z 639 is a result of the loss of 270 mass units that we attributed to a cross-ring cleavage of hexuronic acid residue and subsequent loss of water. Ions at m/z 827 and 809 suggest acylation with an angeloyl or a tigloyl group. Acidic hydrolysis of compound 2 yielded the same monosaccharides as for compound 1: D -glucose and D -glucuronic acid. In negative ion HR-ESI-MS, compound 2 revealed a deprotonated ion at m/z 909.4845, corresponding to a molecular formula of C47H73O17. The 1H NMR spectrum of the aglycone moiety of compound 2 revealed signals for seven tertiary methyl groups at d 0.90, 0.95, 1.02, 1.05, 1.08, 1.11 and 1.43, one olefinic proton at d 5.47 (t, J ¼ 3.5), three oxygen-bearing methine protons at d 3.25 (dd, J ¼ 11.5, 4.5 Hz), 3.95 (d, J ¼ 4.0 Hz), 3.81 (d, J ¼ 4.0 Hz) and 5.49 (dd, J ¼ 12.0, 6.4 Hz) and one primary alcoholic function at d 3.35 and 3.16 (each, d, J ¼ 9.3 Hz) (Table S2). In comparison with compound 1, compound 2 indicated the absence of the secondary alcoholic function at C-21. 1H and 13C NMR spectroscopic data were in agreement with those of A1-barrigenol (Konoshima & Lee 1986). Methyl signals at d 1.94 (br s) and 2.00 (br dd, J ¼ 7.1, 1.3 Hz) and the olefinic proton at d 6.15 (dq, J ¼ 7.1, 1.3 Hz), ascribable to an angeloyl group, were evident. Two anomeric protons at d 4.55 (d, J ¼ 7.5 Hz) and 4.72 (d, J ¼ 7.8 Hz) (Table S2) assigned to one b-glucuronopyranosyl unit (d 4.55) and one b-glucopyranosyl (d 4.72) unit were identified. The determination of the sequence and linkage sites obtained from the HMBC was the same as reported for compound 1. The structure of compound 2 was determined as previously unreported 3-O-b-D glucopyranosyl-(1 ! 2)-b-D -glucuronopyranosyl-22-O-angeloyl-A1-barrigenol.
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Figure 1. Saponins from roots of E. planum and E. maritimum.
Positive and negative ion fragmentation spectra of compound 3 were almost identical to the spectra of compound 1, except that the m/z values for the most prominent ions were 42 mass units lower (Figure S4). Also, ions resulting from the loss of acetyl group were not observed, indicating that compound 3 may be de-acetylated compound 1. An ion at m/z 925.4786, consistent with a molecular formula of C47H73O18, was observed for compound 3 in HR-ESI-MS. Except the absence of signals from C-21 acetyl group, 1H and 13C NMR spectra of compound 3 were superimposable on the analogous spectra of compound 1; therefore, 3 was identified as 3-O-b-D -glucopyranosyl-(1 ! 2)-b-D -glucuronopyranosyl-22-O-angeloyl-R1-barrigenol. Concurring with previous studies on Eryngium species, triterpene glycosides isolated and characterised in this work are derivatives of acylated R1- and A1-barrigenols. Extracts of E. planum roots collected from the natural habitats contained similarly high amounts of compounds 1 and 2, which in both cases exceeded 2 mg/mg of fresh weight (FW) (Table 1). In analogous samples from E. maritimum, these concentrations were much lower, at only 0.2 and 0.4 mg/mg FW for compounds 1 and 2, respectively. Only small amount, approximately 30 ng/
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Table 1. Estimated contents of isolated saponins (ng/g FW) in roots and rosette leaves of E. planum and E. maritimum.a E. planum
Roots Rosette leaves
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a
E. maritimum
1
2
3
1
2
3
2346.7 ^ 456.6 53.3 ^ 14.0
2616.0 ^ 334.0 132.0 ^ 27.7
176.0 ^ 32.0 6.7 ^ 2.3
442.0 ^ 83.5 20.0 ^ 6.9
198.7 ^ 26.6 49.3 ^ 12.9
24.0 ^ 8.0 n.d.
Mean ^ standard deviation (n ¼ 3), n.d. – not detected.
mg FW, of compound 3 was detected in the roots of E. maritimum, but roots of E. planum contained at least an order of magnitude more of this saponin. Aerial parts (rosette leaves) of E. maritimum contained only minute amounts of compound 1; higher concentration was detected in E. planum.
3. Experimental 3.1. Materials Roots of intact plants of E. planum L. and E. campestre L. were collected from natural habitats in Poland (Łukaszewo, Kuyavian-Pomeranian province) in August 2008. E. maritimum L. roots were collected from plants grown on an artificial dune plot in Poznan´ University Botanical Garden in September 2009. The taxonomic identification of species was made according to Flora Europea (Tutin et al. 1968). The voucher specimens (EP-2008-08-01K, EC-2008-08-01K and EM-2009-08-01K) belonging to Department of Pharmaceutical Botany and Plant Biotechnology, K. Marcinkowski, University of Medical Sciences in Poznan´ have been temporarily deposited in the Herbarium of Medicinal Plant Garden in Institute of Natural Fibers and Medicinal Plants in Poznan´. Collected roots were washed with tap water, blot-dried on a filter paper and lyophilised. HPLC gradient-grade solvents were from J.T. Baker (Phillipsburg, NJ, USA); all other chemicals were from Sigma-Aldrich (St. Louis, MO, USA). 3.2. Extraction, purification and LC –MS analyses Lyophilised plant material (roots of E. planum, approximately 100 g) was defatted with chloroform (2.5 L) and extracted three times with 80% MeOH (2.0 L each time) on an ultrasonic bath at room temperature. Combined extracts (15 g) were concentrated under reduced pressure and lyophilised, dissolved in 45% methanol and loaded onto a C18 flash chromatography cartridge (Grace Revelerisw RP C18 Flash Cartridge, 120 g) connected to a Reveleris flash chromatography system (Grace-Davison, Columbia, MD, USA) equipped with UV and ELS detectors. Following isocratic elution with 45% acetonitrile containing 0.1% acetic acid, 35 fractions containing main peaks detected by an evaporative light scattering device were collected and analysed using LC-MS as described later. Fractions containing compounds of interest (23 – 27 for compound 1, 28 – 31 for compound 2 and 15 –16 for compound 3), were combined and further chromatographed using a gradient of 35 –42% acetonitrile containing 0.5% of acetic acid elution on a semi-preparative Gilson HPLC (Gilson, Inc., Middleton, WI, USA) equipped with a PrepELS (Evaporative Light Scattering) detector and an RP-18 column (10 mm £ 250 mm; 5 mm; Kromasil C18) at flow rate 5.0 mL/min. These three separations yielded fractions containing compounds 1 (12 mg), 2 (15 mg) and 3 (4 mg). Collected peaks were dried down in a stream of nitrogen and stored in 2 208C.
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Semi-quantitative and qualitative LC-MS analyses were carried out as described earlier (Kowalczyk et al. 2011) except that in semi-qualitative analyses deprotonated quasi-molecular ions at m/z 925, 909 and 967 were observed and digoxin (final conc. 8 ng/ml) was used as an internal standard. HR-ESI-MS analyses were performed on a Waters Synapt G2-S (Waters Corp., Milford, MA, USA) mass spectrometer using direct sample infusion.
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3.3. Acid hydrolysis Samples of approximately 1 mg were dissolved in 0.5 mL of dioxane/water (1:1 v/v) mixture and then hydrolysed for 6 h with 2 mL of 2 M HCl in 808C. After hydrolysis, samples were neutralised with Amberlite IRA-400, decanted and partitioned three times against 6 mL of ethyl acetate. Resulting water phase was evaporated to dryness and re-dissolved in 50 mL of pyridine. Derivatisation and determination of absolute configurations were then performed as described by Tanaka et al. except that separations were performed on a Waters HPLC consisting of 616 pump 717 autosampler, 996 PDA detector and using Knauer Eurospher 250 £ 4.6 mm, 5 mm column (Knauer, Berlin, Germany). 3.4. NMR analyses NMR experiments were performed on a Bruker DRX-600 spectrometer (Bruker BioSpin GmBH, Rheinstetten, Germany) equipped with a Bruker 5 mm TCI CryoProbe at 300 K. All 2D NMR spectra were acquired in CD3OD (99.95%, Sigma-Aldrich) and standard pulse sequences and phase cycling were used for DQF-COSY, HSQC, HMBC and ROESY spectra. The NMR data were processed using UXNMR software. 3.5. Chemical data of compounds 1 –3 21 KBr Compound 1: Purity . 80% (HPLC-DAD), ½a25 D 2 12.7 (c 0.1 MeOH); IR nmax cm : 3430 (. OH), 2925 (. CH), 1680 (CvO), 1656 (CvC); negative ion ESI-MS m/z 967 [M 2 H]2, positive ion ESI-MS m/z 991 [M þ Na]þ, negative and positive ion ESI-MS/MS spectra are reported in Figure S2, negative ion HR-ESI-MS m/z 967.4894 [M 2 H]2 (calcd for C49H75O2 19 967.4903) aglycone. 1H NMR (CD3OD, 600 MHz) dH (ppm, J in Hz): 3.25 (1H, dd, J ¼ 11.5, 4.5, 3-CH), 5.50, (1H, t, J ¼ 3.5, 12-CH), 3.78 (1H, d, J ¼ 4.0, 15-CH), 3.80 (1H, d, J ¼ 4.0, 16CH), 5.85 (1H, d, J ¼ 10.0, 21-CH), 5.60 (1H, d, J ¼ 10.0, 22-CH), 1.12 (3H, s, 23-CH3), 0.90 (3H, s, 24-CH3), 1.02 (3H, s, 25-CH3), 1.05 (3H, s, 26-CH3), 1.42 (3H, s, 27-CH3), 3.32 (1H, d, J ¼ 9.3, 28-CH), 3.07 (1H, d, J ¼ 9.3 Hz, 28-CH), 0.90 (3H, s, 29-CH3), 1.09 (3H, s, 30-CH3), C-21 acetyl, 1.97 (3H, s, 2-CH3), C-22 angeloyl, 6.15 (1H, dq, J ¼ 7.1, 1.3, 3-CH), 2.00 (3H, br dd, J ¼ 7.1, 1.3, 4-CH3), 1.91 (3H, br s, 5-CH3) aglycone. 13C NMR (CD3OD, 150 MHz) dC (ppm): 40.0 (C-1), 26.7 (C-2), 91.4 (C-3), 39.5 (C-4), 56.4 (C-5), 19.3 (C-6), 36.8 (C-7), 40.8 (C-8), 48.0 (C-9), 37.3 (C-10), 24.5 (C-11), 126.7 (C-12), 143.0 (C-13), 48.4 (C-14), 68.0 (C-15), 74.5 (C-16), 48.0 (C-17), 41.8 (C-18), 46.9 (C-19), 36.0 (C-20), 80.2 (C-21), 73.0 (C-22), 28.3 (C-23), 16.6 (C-24), 16.7 (C-25), 17.6 (C-26), 21.0 (C-27), 63.5 (C-28), 29.5 (C-29), 19.6 (C-30); C-21 acetyl, 172.3 (C-1), 20.6 (C-2); C-22 angeloyl; 169.4 (C-1), 128.7 (C-2), 139.4 (C-3), 15.8 (C-4), 20.6 (C-5). 21 KBr Compound 2: Purity . 75% (HPLC-DAD), ½a25 D 2 15.0 (c 0.1 MeOH); IR nmax cm : 3420 (. OH), 2925 (. CH), 1675 (CvO), 1660 (CvC); negative ion ESI-MS m/z 909 [M 2 H]2, positive ion ESI-MS m/z 933 [M þ Na]þ, negative and positive ion ESI-MS/MS are shown in Figure S3; negative ion HR-ESI-MS m/z 909.4845 [M 2 H]2 (calcd for C47H73O2 17 909.4848) aglycone. 1H NMR (CD3OD, 600 MHz) dH (ppm, J in Hz): 3.25 (1H, dd, J ¼ 11.5, 4.5, 3-CH), 5.47, (1H, t, J ¼ 3.5, 12-CH), 3.81 (1H, d, J ¼ 4.0, 15-CH), 3.95 (1H, d, J ¼ 4.0, 16-CH), 2.25
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Natural Product Research
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(1H, t, J ¼ 12.0, 21-CH), 5.49 (1H, dd, J ¼ 12.0, 6.4, 22-CH), 1.11 (3H, s, 23-CH3), 0.90 (3H, s, 24-CH3), 1.02 (3H, s, 25-CH3), 1.05 (3H, s, 26-CH3), 1.43 (3H, s, 27-CH3), 3.35 (1H, d, J ¼ 9.3, 28-CH), 3.16(1H, d, J ¼ 9.3, 28-CH), 0.95 (3H, s, 29-CH3), 1.08 (3H, s, 30-CH3), C-22 angeloyl, 6.15 (1H, dq, J ¼ 7.1, 1.3, 3-CH), 2.00 (3H, br dd, J ¼ 7.1, 1.3, 4-CH3), 1.94 (3H, br s, 5-CH3) aglycone. 13C NMR (CD3OD, 150 MHz) dC (ppm): 40.0 (C-1), 26.7 (C-2), 91.2 (C-3), 40.0 (C-4), 56.5 (C-5), 19.4 (C-6), 37.0 (C-7), 41.9 (C-8), 47.9 (C-9), 37.5 (C-10), 24.6 (C-11), 126.1 (C-12), 144.1 (C-13), 48.1 (C-14), 68.4 (C-15), 75.2 (C-16), 48.1 (C-17), 42.1 (C-18), 47.5 (C-19), 32.0 (C-20), 41.8 (C-21), 73.2 (C-22), 28.2 (C-23), 16.6 (C-24), 16.1 (C-25), 17.8 (C-26), 20.7 (C-27), 63.6 (C-28), 33.4 (C-29), 24.9 (C-30); C-22 angeloyl; 169.3 (C-1), 129.7 (C-2), 137.9 (C-3), 15.8 (C-4), 20.7 (C-5). 21 KBr Compound 3: Purity .85% (HPLC-DAD), ½a25 D 2 9.5 (c 0.1 MeOH); IR nmax cm : 3430 (.OH), 2930 (.CH), 1675 (CvO), 1656 (CvC); negative ion ESI-MS m/z 925 [M 2 H]2; positive ion ESI-MS m/z 949 [M þ Na]þ; negative and positive ion ESI-MS/MS spectra are reported in Figure S4; negative ion HR-ESI-MS m/z 925.4786 [M 2 H]2 (calcd for C47H73O2 18 925.4773) aglycone. 1H NMR (CD3OD, 600 MHz) dH (ppm, J in Hz): 3.24 (1H, dd, J ¼ 11.5, 4.5, 3CH), 5.49, (1H, t, J ¼ 3.5, 12-CH), 3.78 (1H, d, J ¼ 4.0, 15-CH), 3.80 (1H, d, J ¼ 4.0, 16-CH), 4.3 (1H, d, J ¼ 10.0, 21-CH), 5.36 (1H, d, J ¼ 10, 22-CH), 1.12 (3H, s, 23-CH3), 0.90 (3H, s, 24-CH3), 1.02 (3H, s, 25-CH3), 1.05 (3H, s, 26-CH3), 1.42 (3H, s, 27-CH3), 3.32 (1H, d, J ¼ 9.3, 28-CH), 3.05 (1H, d, J ¼ 9.3, 28-CH), 1.02 (3H, s, 29-CH3), 1.04 (3H, s, 30-CH3), C-22 angeloyl, 6.13 (1H, dq, J ¼ 7.1, 1.3, 3-CH), 2.03 (3H, br dd, J ¼ 7.1, 1.3, 4-CH3), 1.98 (3H, br s, 5-CH3) aglycone. 13C NMR (CD3OD, 150 MHz) dC (ppm): 39.8 (C-1), 26.7 (C-2), 91.4 (C-3), 40.1 (C-4), 56.0 (C-5), 19.3 (C-6), 37.0 (C-7), 41.7 (C-8), 47.9 (C-9), 37.0 (C-10), 24.4 (C-11), 126.6 (C-12), 143.7 (C-13), 48.1 (C-14), 68.3 (C-15), 74.5 (C-16), 48.0 (C-17), 41.3 (C-18), 47.7 (C-19), 36.0 (C-20), 77.5 (C-21), 76.6 (C-22), 28.4 (C-23), 16.6 (C-24), 16.0 (C-25), 17.6 (C-26), 21.0 (C-27), 63.8 (C-28), 29.7 (C29), 18.4 (C-30); C-22 angeloyl; 170.4 (C-1), 129.6(C-2), 138.4 (C-3), 15.7 (C-4), 20.6 (C-5). Carbohydrate parts of compounds 1 –3: 1H NMR (CD3OD, 600 MHz) b-D -GlcA (at C-3) dH (ppm, J in Hz); 4.55 (1H, d, J ¼ 7.5, H-1), 3.67 (1H, dd, J ¼ 7.5, 9.0, H-2), 3.62 (1H, dd, J ¼ 9.0, 9.0, H-3), 3.57 (1H, dd, J ¼ 9.0, 9.0, H-4), 3.81 (1H, d, J ¼ 9.0, H-5); b-D -Glc (at C2GlcA), 4.72 (1H, d, J ¼ 7.8, H-10 ), 3.26 (1H, dd, J ¼ 7.8, 9.0, H-20 ), 3.39 (1H, dd, J ¼ 9.0, 9.0, H-30 ), 3.24 (1H, dd, J ¼ 9.0, 9.0, H-40 ), 3.28 (1H, m, H-50 ), 3.86 (1H, dd, J ¼ 2.5, 12.0, H-60 ), 3.65 (1H, dd, J ¼ 4.5, 12.0, H-60 ). 13C NMR (CD3OD, 150 MHz) dC (ppm): b-D -GlcA (at C-3), 105.1 (C-1), 80.2 (C-2), 77.5 (C-3), 72.6 (C-4), 76.6 (C-5), 171.8 (C-6), b-D -Glc (at C-2glcA), 103.9 (C-10 ), 75.6 (C-20 ), 78.0 (C-30 ), 70.3 (C-40 ), 78.0 (C-50 ), 62.9 (C-60 ). 4. Conclusions Our results indicate a large diversity, both qualitative and quantitative, in saponin composition between the investigated species. Although aglycones of newly identified and isolated from E. planum and E. maritimum saponins are similar or the same as those reported from E. campestre, their oligosaccharides contain D -glucose instead of L -rhamnose as a terminal carbohydrate. Furthermore, saponins containing D -rhamnose were not detected in extracts from the two tested species. In addition to structural differences, plant materials collected from plants in their natural environment contained varied amounts of saponins. To a large extent, this was also species dependent, with E. maritimum containing relatively small amount of saponins. Although the subsection classification of Eryngium was developed mainly on the basis of morphological features, it appears to be reflected on a phytochemical level as well. Supplementary material Supplementary material relating to this article is available online, alongside Figures S1 –S7 and Tables S1 and S2.
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Acknowledgements Parts of this work were supported by the Ministry of Science and Education, Warsaw, Poland from educational sources of 2011– 2013 as grant no. NN405683340 and from the 7th Framework Program of European Community, PROFICIENCY (contract no. 245751).
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