Isolation and Identification of Alkaloids from Poisons of Fire Salamanders (Salamandra salamandra)

Article abstract of DOI:10.1021/acs.jnatprod.9b00065

Fire salamanders (Salamandra salamandra) are conspicuously colored amphibians secreting a skin poison that contains unique steroid alkaloids such as samandarine (1) and samadarone (2), exhibiting toxic as well as antimicrobial activities. Because of their antipredatory and anti-infectious functions, alkaloids from Salamandra poison are of interest with regard to the threat that the lethal fungus Batrachochytrium salamandrivorans (Bsal) poses to salamanders. Nevertheless, reliable data on the biological activity of Salamandra alkaloids are scarce, in part due to the difficulty to obtain and study those substances. Thus, isolation of pure salamander alkaloids is an important task that might directly contribute to the understanding of Bsal infections. Here we present a noninvasive isolation procedure for samandarine (1) and O-acetylsamandarine (3), as well as for two new alkaloids, O-3-hydroxybutanoylsamandarine (4) and samanone (6), using HPLC. For the first time, high-field NMR data are presented for these alkaloids. Analysis using GC/MS and ESI⁺-MS, provided important information on the structural variability of these salamander alkaloids.

Full text of DOI:10.1021/acs.jnatprod.9b00065

Article
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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Isolation and Identification of Alkaloids from Poisons of Fire
Salamanders (Salamandra salamandra)
†
‡,§
Janosch Knepper, Tim Luddecke, Kathleen Preißler,§ Miguel Vences,§ and Stefan Schulz*,†
̈
†
̈
Institute of Organic Chemistry, Technische Universitat Braunschweig, Hagenring 30, 38106 Braunschweig, Germany
‡Animal Venomics Research Group, Fraunhofer Institute for Molecular Biology and Applied Ecology, Winchesterstraße 2, 35394
Gießen, Germany
§
̈
Zoological Institute, Technische Universitat Braunschweig, Mendelssohnstraße 4, 38106 Braunschweig, Germany
S
* Supporting Information
ABSTRACT: Fire salamanders (Salamandra salamandra) are con-
spicuously colored amphibians secreting a skin poison that contains
unique steroid alkaloids such as samandarine (1) and samadarone
(2), exhibiting toxic as well as antimicrobial activities. Because of
their antipredatory and anti-infectious functions, alkaloids from
Salamandra poison are of interest with regard to the threat that the
lethal fungus Batrachochytrium salamandrivorans (Bsal) poses to
salamanders. Nevertheless, reliable data on the biological activity of
Salamandra alkaloids are scarce, in part due to the difficulty to obtain
and study those substances. Thus, isolation of pure salamander
alkaloids is an important task that might directly contribute to the
understanding of Bsal infections. Here we present a noninvasive
isolation procedure for samandarine (1) and O-acetylsamandarine (3), as well as for two new alkaloids, O-3-
hydroxybutanoylsamandarine (4) and samanone (6), using HPLC. For the first time, high-field NMR data are presented for
these alkaloids. Analysis using GC/MS and ESI+-MS, provided important information on the structural variability of these
salamander alkaloids.
uropean fire salamanders (Salamandra salamandra) are
Europe.19−22 In this regard an understanding of salamander
Ewell known for their distinct black and yellow color toxins, especially their involvement in pathogen defense, is
important, as discussed by us recently.23
pattern. This color pattern is assumed to act as an aposematic
Salamander alkaloids are not commercially available, as they
are difficult to obtain in large quantities from the protected
animals and even more difficult to synthesize. Earlier isolations
were performed by manual extraction and crystallization
methods, while no modern chromatographic isolation methods
for this substance class are available.24,25 However, for
determination of the alkaloids’ bioactivity and their potential
in Bsal inhibition, it is necessary to produce larger quantities of
purified alkaloids.
warning signal since the salamanders produce a defensive
poison that is secreted by skin glands. These glands are
distributed across the animal’s dorsum and occur in
concentrated form in a macroglandular system at the head,
known as parotoid glands.
The first chemical investigations of fire salamander poison
were performed by Zalesky in 1866, who isolated an
amorphous base that was identified as an alkaloid and named
samandarine.1 Later studies on salamander poison were
Here we developed a new isolation procedure based on
HPLC and reevaluated the poison composition with modern
methods. We isolated the four salamander alkaloids
samandarine (1), O-acetylsamandarine (3), O-(S)-3-hydrox-
ybutanoylsamandarine (4), and samanone (6), of which 4 and
6 have not been reported before as natural products.
̈
predominantly performed by Schopf and Habermehl. They
described the main constituents as steroid alkaloids and
elucidated their absolute configurations.2−11 Later the focus
shifted toward functional and applied aspects of these
compounds, such as biological properties, biosynthesis, and
total synthesis. In this context, two main biological functions of
the salamander alkaloids were discovered: First, they cause
convulsions followed by lethal respiratory paralysis in various
vertebrates.12−15 Second, they possess antifungal and anti-
microbial activity, but the mode of action of that phenomenon
remained unresolved.16−18 This second aspect is of special
interest due to the lethal salamander plague, caused by the
fungus Batrachochytrium salamandrivorans (Bsal), which led to
the extinction of fire salamander populations in several parts of
RESULTS AND DISCUSSION
■
The skin secretion of 100 wild-caught individuals of S.
salamandra from the Solling forest, Germany, was obtained
via a noninvasive milking approach. Gentle application of
Received: January 22, 2019
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.9b00065
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and H-3 of the ether bridge, while H-16 appeared at 4.31 ppm.
COSY correlations led to four spin systems, H-1−H-2, H-3−
H-4−H-5−H-6−H-7, H-9−H-11−H-12, and H-14−H-15−H-
16−H-17, as shown in Figure 2. No coupling to other
hydrogens was observed for the two methyl groups, implying
their localization at a quaternary carbon. Characteristic 13C
signals were observed for both oxa-carbons, C-1 and C-3, at
80.9 and 89.6 ppm. The signal of C-16 at 72.2 ppm indicated a
CH-OH group. HMBC correlations (Figure 2) completed and
confirmed the structure. Additional structural confirmation was
1
achieved by H−15N HMBC experiments showing couplings
from the amine hydrogen to neighboring carbons, C-1 and C-
4. The experimental NMR data were also highly similar to
simulated spectra using the ACD/NMR Predictors software
(ACD Laboratories). NOESY correlations confirmed the
reported relative configuration of the salamander alkaloids, as
most of the CH2 protons were differentiated. The relative and
absolute configuration of the main salamander alkaloids has
been determined earlier, including X-ray crystallography.26
Compound D could not be isolated in pure form due to
coeluting compounds. Although no EI-MS data were reported
in the literature, the close similarity with the EI mass spectrum
of 1, differing mostly in the 2 amu lower molecular ion at m/z
303 (see Supporting Information Figure S4), the occurrence of
ions characteristic for the N,O-acetal ring, m/z 56/57 and 85/
86, and the known co-occurrence with 1 suggested this
compound to be samandarone (2).27
pressure by the thumbs on the parotoid glands and the tail root
caused the release of the poison, which was poured into a glass
container. The secretions were analyzed by HPLC coupled to
heated electrospray ionization mass spectrometry (HESI-MS)
after addition of MeOH. Chromatographic separation of most
compounds was achieved as shown in Figure 1.
Eleven compounds (A−K) were detected by their [M + H]+
ions, of which the major constituents were A, D, H, and J.
Because the structures of only nine salamander alkaloids
(Supporting Information, Figure S1) have been reported so
far,23 unknown compounds must have been present. There-
fore, the compounds were isolated to clarify their structures
and to obtain material for bioactivity testing. Purification of
each compound was performed by scaling up the separation
method to semipreparative HPLC conditions, which was
complicated by lack of UV−vis activity of the target
compounds.
Compound J proved to be O-acetylsamandarine (3), already
known from fire salamander poisons,25 although again no
NMR data have so far been published. The molecular formula
was determined to be C21H34NO3 by HR-ESI-MS. The EI-
mass spectrum (Figure S5) was similar to that of 1 (Figure S3
Supporting Information) with a molecular ion at m/z 347.
1
The H and 13C signals (Table 1) were assigned as for
samandarine. Differences were found for C-16, which was
detected at 76.1 ppm, typical for an ester. The ester carbon C20
was verified by a shift of 172.9 ppm. Additionally, HMBC
couplings from C-20 to the hydrogens of the acetyl CH3 group
as well as to H-16 were detected. NOESY experiments revealed
the same relative configuration as for 1.
For compound H a molecular formula of C23H37NO4 was
determined by HR-ESI-MS. GC/EI-MS analysis revealed a
spectrum shown in Figure 3a with M+ at m/z 391, not
matching with any of the known salamander alkaloids.
By this method, 4 mg of compound A was isolated and
identified as samandarine (1), already known as a major
alkaloid of fire salamander poisons.1 The molecular formula
was determined as C19H31NO2 by HR-ESI-MS. GC/EI-MS
analysis showed a spectrum similar to the published one of 1
(Figure S3).10 As no high-field NMR data of 1 have been
reported so far, NMR data of the sample were obtained
1
confirming the structure. The H NMR spectrum (Table 1)
showed signals at 5.44 and 4.49 ppm that were assigned to H-1
Figure 1. Base-peak chromatogram of salamander skin poison analyzed by HPLC/HESI-MS. The masses of [M + H]+ ions are shown. Major
components are shown in bold letters. The separation was optimized using a gradient composed of CH3CN, H2O, and 2% formic acid in H2O. The
CH3CN content increased from 5% to 18.8% over 9 min, was constant for 3 min, and then increased again to 30% over 3 min, while a constant
0.1% formic acid concentration was used.. For details of the gradient, see Supporting Information Table S1.
B
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Table 1. 1H and 13C NMR Shifts of Samandarine (1), O-Acetylsamandarine (3), and O-3-Hydroxybutanoylsamandarine (4) in
a
CD3OD (1H 600 MHz, 13C 150 MHz)
1
3
4
position
δC, type
δH (J in Hz)
δC, type
δH(J in Hz)
δC, type
δH(J in Hz)
1
2
3
4
5
6
7
8
80.8, CH
44.2, CH2
89.6, CH
30.8, CH2
34.5, CH
26.2, CH2
27.4, CH2
36.7, CH
42.5, CH
39.5, C
21.6, CH2
40.2, CH2
41.1, C
54.7, CH
37.8, CH2
72.2, CH
51.8, CH2
19.4, CH3
17.6, CH3
4.49, d (6.74)
3.40, m; 3.16, m
5.44, s
2.17, m; 1.39, m
1.74, m
1.85, tt (4.60, 21.01); 1.32, m
1.51, m; 1.09, m
1.49, m
80.7, CH
44.4, CH2
89.6, CH
31.2, CH2
34.6, CH
26.2, CH2
27.4, CH2
36.7, CH
42.5, CH
39.5, C
21.6, CH2
39.7, CH2
41.0, C
54.3, CH
35.4, CH2
76.1, CH
49.0, CH2
18.8, CH3
17.6, CH3
172.9, C
4.80, m
3.34, m; 3.14, m
5.40, s
2.16, m; 1.37, m
1.75, m
1.85, m; 1.31, m
1.48, m; 1.11, m
1.48, m
80.6, CH
44.5, CH2
89.6, CH
31.3, CH2
34.6, CH
26.2, CH2
27.4, CH2
36.7, CH
42.5, CH
39.5, C
21.6, CH2
39.7, CH2
41.0, C
54.4, CH
35.5, CH2
76.1, CH
49.6, CH2
18.8, CH3
17.6, CH3
173.2, C
4.45, d (6.71)
3.33, m; 3.14, m
5.38, s
2.15, m; 1.35, m
1.76, m
1.85, m; 1.31, m
1.49, m; 1.12, m
1.49, m
9
1.70, m
1.72, m
1.73, m
10
11
12
13
14
15
16
17
18
19
20
21
22
23
1.60, m; 1.39, m
1.74, m; 1.18, dt (4.11, 12.85)
1.63, m; 1.39, m
1.76, m; 1.21, dt (3.84, 12.79)
1.62, m; 1.39, m
1.76, m; 1.21, dt (3.98, 13.04)
1.04, m
2.17, m; 1.27, m
4.31, m
1.56, m; 1.49, m
0.95, s
0.95, s
1.09, m
2.28, m; 1.35, m
5.11, m
1.63, m; 1.58, m
0.91, s
0.95, s
1.09, m
2.29, m; 1.37, m
5.15, m
1.62, m
0.92, s
0.94, s
21.2, CH3
1.99, s
45.1, CH2
65.7, CH
23.4, CH3
2.39, m
4.14, dq (1.30, 6.18)
1.20, d (6.28)
a
For atom numbering see structure block.
identified as the new salamander alkaloid O-(S)-3-
hydroxybutanoylsamandarine (4).
Compound I, coeluting with H (4), was assigned to the only
other mass in the poison extract not assignable to any known
molecular ion. The molecular formula was determined to be
C19H31NO by HR-ESI-MS. GC/EI-MS analysis indicated a
compound with m/z 289 and a retention index (RI) of 2574,
whose spectrum is shown in Figure 3b. While the previous
mass spectra were similar especially in the intensity differences
from high to low amu, the spectrum of I was dominated by two
intense signals, m/z 289 [M]+ and 274 [M − CH3]+. No loss
of H2O was observed in the ESI-MS spectra, different from the
samandarine-type compounds. MS2 experiments with different
ionization energies only led to either no fragmentation or
complete degradation of the compound. Thus, two potential
structures were proposed, i.e., an oxidized form of samanine
(5) or a dehydroxylated form of samandarine (1). Given the
spectral similarity to the mass spectrum of 533 (Figure 3c), its
oxidation product samanone (6) seemed to be the most likely
structure. To verify this proposal, compound I was isolated
from the fraction H/I by an additional separation, yielding
about 1 mg of product (Table S2 and Figure S2 Supporting
Information).
The NMR data supported again a samandarine-type
skeleton, but lacking the oxygen bridge in ring A. COSY
correlations confirmed four spin systems, H-1−H-2, H-3−H-
4−H-5−H-6−H-7−H-8−H-9, H-11−H-12, and H-14−H-15
(Figure 4). Two methyl groups with no coupling to any other
hydrogen were observed, suggesting that they are localized at a
quaternary carbon. A keto group with a shift of 220.9 ppm was
located at C-15 by HMBC, while no other oxygen-substituted
carbons were present (Table 2). Further structure elucidation
was performed using HMBC correlations that connected the
Figure 2. COSY (thick bonds), selected HMBC (blue arrows), and
1H−15N HMBC (green arrows) correlations of samandarine (1), O-
acetylsamandarine (3), and O-3-hydroxybutanoylsamandarine (4).
Interestingly, the mass spectrum below m/z 288 was found
to be very similar to that of compound 3, revealing similarity in
the ring systems of both. Therefore, the additional atoms are
1
likely to be located outside the ring system. The H and 13C
signals (Table 1) from C/H-1 to C/H-19 were almost identical
to those of 3, suggesting a modified acyl side chain. COSY,
HSQC, and HMBC experiments revealed the presence of a 3-
hydroxybutanoyl group, including a value of 65.7 ppm for the
carbinol C-22 and an ester carbonyl at 173.2 ppm at C-20.
While the configuration of the ring system was proven to be
identical to those of 1 and 3, the configuration of the
hydroxybutanoyl group had to be elucidated. Therefore,
transesterification was performed with trimethylsulfonium
hydroxide (TMSH) in MeOH, resulting in the formation of
methyl 3-hydroxybutanoate (MHB).28 Chiral-phase GC using
a Lipodex G phase showed that MHB had an S-configuration
(Figure S6). The R-enantiomer of 3-hydroxybutyric acid
predominates in animals, as it is formed during fatty acid
biosynthesis, while fatty acid catabolism produces (S)-3-
hydroxybutyric acid.29−32 Compound H was therefore
C
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1
Table 2. H and 13C NMR Shifts for Samanone (6) in
a
CD3OD (1H 600 MHz, 13C 150 MHz)
position
δC, type
δH
1
2
3
4
5
6
7
8
38.9, CH2
41.9, CH2
48.9, CH2
31.1, CH2
45.7, CH
29.1, CH2
28.1, CH2
36.8, CH
49.9, CH
38.1, C
21.9, CH2
39.1, CH2
40.1, C
52.4, CH
40.1, CH2
220.9, C
1.92, m; 1.69, m
3.27, m; 3.15, m
1.29, m
1.94, m; 1.53, m
1.74, m
2.14, m; 1.54, m
1.47, m; 1.05, m
1.53, m
9
3.35, m
10
11
12
13
14
15
16
17
18
19
1.53, m
1.87, m; 1.50, m
1.64, m
2.18, m; 1.96, m
56.7, CH2
18.3, CH3
22.1, CH3
2.04, m
0.91, s
1.08, s
a
For carbon numbering see structure block.
compounds facilitating a delivery system.38 Therefore, it is
important to determine the exact composition of the alkaloid
cocktail and other compounds produced by salamanders. Our
work suggests that the inventory of steroid alkaloids in
salamanders is not yet complete, and it remains to be
experimentally determined which biological function the
individual compounds exhibit alone or in combination with
others.
By the experimental approach described here, relevant
quantities of pure salamander alkaloids were obtained relying
on a collection method less harmful and invasive for the
animals than in previous protocols.39,40 The isolated alkaloids
will be used for bioassays to test their biological properties
toward the pathogenic fungi Batrachochytrium dendrobatidis
and B. salamandrivorans, as well as beneficial and pathogenic
bacteria colonizing the skin.41,42 A comparison of assays using
these alkaloids alone and in biologically realistic mixtures of
the entire salamander secretion may contribute to the
understanding of the functional properties of the salamander
defensive system.
Figure 3. EI-mass spectra of (a) O-3-hydroxybutanoylsamandarine
(4), (b) samanone (6), and (c) samanine (5).
Figure 4. COSY (thick bonds) and characteristic HMBC (blue
arrows) correlations of samanone (6).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured using an MCP 150 polarimeter from Anton Paar. IR
spectra were recorded with a Dani Instruments DiscovIR IR detector
coupled to an Agilent Technologies 7890B gas chromatograph. NMR
spectra were acquired with an AV II-600 (Bruker Biospin) instrument,
operated at 600 MHz for 1H and 150 MHz for 13C. Tetramethylsilane
(TMS, δ = 0 ppm) was used as an internal standard. Spin
multiplicities of 13C signals were determined via DEPT-135
measurements. High-resolution mass spectra of purified compounds
were measured using an LTQ Orbitrap Velos (Thermo Scientific) via
direct injection (DI). DI measurements were performed using a
custom-made microspray device to allow flow rates of 1 μL·min−1
through a stainless-steel capillary (90 μm i.d.). Measurements were
performed with a scan range from 50 to 2000 amu with a resolution of
100 000 fwhm at m/z 400. Typical spray voltages ranged between 2.3
and 2.8 kV for positive and between 1.7 and 2.5 kV for negative mode
measurements. Additionally, the cation of tetradecyltrimethyl-
ammonium bromide (M = 256.29988 amu) was set as lock mass
structural parts of the individual H-spin systems. The NMR
data showed high similarity, except near the CO group, to
the published data of synthetic samanine.34 Therefore,
compound I was identified as samanone (6), another new
salamander alkaloid.
In the present study, we described a separation method that
provided access to various salamander alkaloids and led to the
identification of new structures occurring along with the
previously known compounds. In other alkaloid-containing
amphibians, the compound diversity of the alkaloid cocktail
has been interpreted as one factor influencing overall toxicity35
and thus one important component in the intertwined
evolution of toxicity and aposematism.36,37 Differently
structured alkaloids might confer different biological activities,
sometimes in complex interactions with other chemical
D
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and was used as internal mass reference. An HPLC system consisting
of an Accela photodiode array (PDA) Detector, Accela autosampler,
and Accela 1250 pump was coupled to an LTQ XL mass spectrometer
(from Thermo Scientific) for HPLC/HESI-MS analyses. Heated
electrospray ionization was used with an enhanced scan range of 120
to 2000 amu. Gradient HPLC solvent programs consisted of LCMS-
grade H2O, CH3CN, and 2% formic acid in H2O. An Agilent Zorbax
Eclipse Plus C18 (3.5 μm, 2.1 × 150 mm) column was used, which
was kept at 30 °C. The PDA detector was set to a scanning range
from 190 to 600 nm with 1 nm wavelength steps. Purification of
compounds was performed via semipreparative HPLC-DAD using a
Dionex Ultimate 3000 HPLC system consisting of a DAD,
autosampler, pump, and automated fraction collector (Thermo
Scientific). Gradient programs used HPLC-grade H2O, CH3CN,
and 2% formic acid in H2O with a flow rate of 4.73 mL·min−1. A
Nucleodur C18 HTec (5 μm, 10 × 250 mm) column was used.
Analyses via GC/EI-MS were performed with a 7890A GC system
coupled to a 5975 Series mass detector (Agilent Technologies)
equipped with an Agilent HP-5MS capillary column (0.25 μm film,
0.25 mm × 30 m) and helium as the carrier gas. Ionization was
performed via electron impact (EI) with 70 eV, and the following
temperature program was used: isothermal at 50 °C for 5 min,
followed by heating at 10 °C·min−1 up to 320 °C. Gas chromato-
graphic retention indices were calculated from a homologous series of
n-alkanes (C8−C38). Chiral-phase separations were performed with a
7890A GC-FID system (Agilent Technologies) using a Lipodex G
column (50 m × 0.25 mm) and hydrogen as the carrier gas with the
following temperature program: isothermal at 50 °C for 15 min,
followed by heating at 25 °C·min−1 up to 220 °C.
O-3-Hydroxybutanoylsamandarine (4): [α]20 +40 (c 0.4,
D
MeOH); GC-IR νmax 3335, 2969, 2935, 2856, 1730, 1452, 1379,
1303, 1179, 1115, 1086, 1039, 1022, 999, 952, 915, 858, 831 cm−1;
1H and 13C NMR data (CD3OD), Table 1; HR-ESI-MS m/z
392.27986 [M + H]+ (calcd for C23H38NO4, 392.28008); EI-MS
spectrum, Figure 3a.
Samanone (6): colorless semisolid; [α]20 −9.2 (c 0.1, MeOH);
D
GC-IR νmax 2960, 2931, 2875, 2860, 1726, 1601, 1582, 1465, 1381,
1283, 1263, 1126, 1076, 745 cm−1; 1H and 13C NMR data (CD3OD),
Table 2; HR-ESI-MS m/z 290.24826 [M + H]+ (calcd for C19H32NO,
290.24839); EI-MS spectrum, Figure 3b.
Transesterification of O-3-Hydroxybutanoylsamandarine
(4). The absolute configuration of the 3-hydroxybutanoyl side chain
was determined after transesterification of 4 with TMSH according to
Schomburg et al.28 A small amount of the compound was dissolved in
100 μL of CH2Cl2, and 100 μL of 0.25 M TMSH in MeOH was
added. After 1 h, the mixture was diluted with CH2Cl2 and analyzed
by GC/EI-MS.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.9b00065.
Overview of salamander alkaloids identified so far, GC/
EI-MS data for samandarine (1) samandarone (2), and
O-acetylsamandarine (3), NMR spectra of all com-
pounds, chromatograms of chiral-phase analysis, as well
as purification gradients for semipreparative HPLC
(PDF)
Extraction and Isolation. The glandular secretion (1 g) was
obtained by a noninvasive “milking” procedure applied to fire
salamanders from three sites in the Solling area (geographical
coordinates 51°53′25 N 9°36′17 E; 51°48′24 N 9°29′31 E; and
51°45′15 N 9°40′32 E). Each animal was taken into one hand with
the head directed toward the thumb. Then the thumb of the
controlling hand was placed on top of one of the parotoids and the
thumb of the other hand underneath. A glass container was placed
close to the parotoids, and by applying gentle pressure with both
thumbs the skin poison was collected. As an alternative, the
salamander’s tail may serve as another source of poison. By applying
gentle pressure on the root of the tail and then gliding up to the tail
tip, secretions can be released. This procedure however causes a wider
dispersion of poison droplets and therefore appears to be less
accurate. We refer to the herein described method as “milking” (as
suggested by Geßner and Craemer43) in analogy to the term often
used for venom sampling in snakes and other venomous animals.
This secretion was extracted with 15 mL of MeOH for 20 min with
regular mixing via soft shaking. The extract was filtered and diluted
1:10 in MeOH for method development and purification. Because
salamander alkaloids cannot be detected via PDA, development of the
method was performed via HPLC/HESI-MS, followed by scaling up
to semipreparative HPLC for purification. Pure fractions of
samandarine (1), O-acetylsamandarine (3), and O-3-hydroxybuta-
noylsamandarine (4) were obtained within the first purification step
and analyzed. The solvent system used is shown in the Supporting
Information Table S1. A second purification method resulted in the
separation of samanone (6). Solvent gradient information is shown in
the Supporting Information Table S2.
AUTHOR INFORMATION
Corresponding Author
*E-mail: stefan.schulz@tu-bs.de.
■
ORCID
Stefan Schulz: 0000-0002-4810-324X
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The research was funded by the German Ministry of Science
and Education in the framework of the program ANOBIN.
Fieldwork was performed in accordance with animal welfare
(33.19-42502-04-18/2792) and under the permission of the
administrative districts of Northeim (VI.3-NAT-1738/18) and
Holzminden (366 3245 30 44/2018). We are grateful to E.
Sanchez for valuable discussions on the subject and to S.
Gippner, M. Dinis, M. Wagler, F. Goudarzi, R. Schmidt, U.
Seidel, and S. Steinfartz for their much appreciated help during
the fieldwork.
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