Three new triterpene saponins from roots of Eryngium planum

Article abstract of DOI:10.1080/14786419.2014.895722

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 isol

Full text of DOI:10.1080/14786419.2014.895722

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Three new triterpene saponins from
roots of Eryngium planum
Mariusz Kowalczyka, Milena Masulloc, Barbara Thiemb, Sonia
Piacentec, Anna Stochmala & Wiesław Oleszeka
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
Mariusz Kowalczyka*, Milena Masulloc, Barbara Thiemb, Sonia Piacentec, Anna Stochmala and
Wiesław Oleszeka
aDepartment of Biochemistry, Institute of Soil Science and Plant Cultivation, State Research Institute,
Puławy, Poland; Department of Pharmaceutical Botany and Plant Biotechnology, Karol Marcinkowski
b
c
University of Medical Sciences, Poznan, Poland; Dipartimento 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
isolatedandtheirtentativeidentifications,basedontheelectrosprayMS/MSfragmentation
patterns, weresubsequently confirmedby 1D and 2DNMR analyses. Their structures were
established as 3-O-b-D-glucopyranosyl-(1 ! 2)-b-D-glucuronopyranosyl-21-O-acetyl-
22-O-angeloyl-R1-barrigenol (1) and 3-O-b-D-glucopyranosyl-(1 ! 2)-b-D-glucurono-
pyranosyl-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: mkowalczyk@iung.pulawy.pl
q 2014 Taylor & Francis

654
M. Kowalczyk et al.
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 (low-
intensity 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-O-
acetyl-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.
1
The H 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.

656
M. Kowalczyk et al.
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-bar-
rigenol.
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
E. maritimum
1
2
3
1
2
3
Roots
Rosette
leaves
2346.7 ^ 456.6 2616.0 ^ 334.0 176.0 ^ 32.0 442.0 ^ 83.5 198.7 ^ 26.6 24.0 ^ 8.0
53.3 ^ 14.0 132.0 ^ 27.7 6.7 ^ 2.3 20.0 ^ 6.9 49.3 ^ 12.9 n.d.
a 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 2208C.

658
M. Kowalczyk et al.
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.
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
Compound 1: Purity .80% (HPLC-DAD), ½aꢀD 2 12.7 (c 0.1 MeOH); IR nKmBaxr cm21: 3430
25
(.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 C49H75O129
1
967.4903) aglycone. H 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, 16-
CH), 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).
Compound 2: Purity .75% (HPLC-DAD), ½aꢀD 2 15.0 (c 0.1 MeOH); IR nKmBaxr cm21: 3420
25
(.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 C47H73O127 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).
25
KBr
max
Compound 3: Purity .85% (HPLC-DAD), ½aꢀD 2 9.5 (c 0.1 MeOH); IR n
cm21: 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 C47H73O128
925.4773) aglycone. 1H NMR (CD3OD, 600 MHz)dH (ppm, J in Hz):3.24(1H, dd, J ¼ 11.5, 4.5, 3-
CH), 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. 13
C
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 (C-
29), 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 C-
2
GlcA), 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.

660
M. Kowalczyk et al.
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).
References
´
Calvin˜o CI, Martınez 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, Martınez 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.