Anti-inflammatory Potential of Flavonoids from the Aerial Parts of Corispermum marschallii

Article abstract of DOI:10.1021/acs.jnatprod.8b00152

The isolation of phenolics from aerial parts of Corispermum marschallii yielded a total of 13 compounds including nine previously undescribed patuletin and spinacetin glycosides. These were identified as patuletin 3-O-β-d-galactopyranosyl-7-O-β-d-glucopyranoside (1), spinacetin 3-O-β-d-galactopyranosyl-7-O-β-d-glucopyranoside (2), patuletin 3-O-(6″-O-β-d-glucopyranosyl)-β-d-galactopyranoside (3), patuletin 3-O-(6″-O-α-l-arabinopyranosyl)-β-d-galactopyranoside (4), patuletin 3-O-(2″-O-(5-O-α-l-arabinopyranosyl)-β-d-apiofuranosyl)-β-d-galactopyranoside (5), patuletin 3-O-(2″-O-β-d-apiofuranosyl)-β-d-galactopyranoside (6), spinacetin 3-O-β-d-galactopyranoside (7), patuletin 3-O-β-d-galactopyranosyl-7-O-(6-O-feruloyl)-β-d-glucopyranoside (8), and spinacetin 3-O-β-d-galactopyranosyl-7-O-(6-O-feruloyl)-β-d-glucopyranoside (9). Structure elucidation was based on UV-visible, multistage MS, and 1D and 2D NMR spectroscopy and chemical derivatization, which allowed the identification on the glycosides with two different hexose moieties occurring at different positions of the aglycones. Most of the compounds tested inhibited the production of pro-inflammatory factors such as ROS, IL-8, and TNF-α in stimulated neutrophils.

Full text of DOI:10.1021/acs.jnatprod.8b00152

Article
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Anti-inflammatory Potential of Flavonoids from the Aerial Parts of
Corispermum marschallii
†
†
Karolina Pawłowska, Monika E. Czerwinska, Marcin Wilczek,‡ Jakub Strawa,§ Michał Tomczyk,§
́
and Sebastian Granica*,†
†Department of Pharmacognosy and Molecular Basis of Phytotherapy, Faculty of Pharmacy, Medical University of Warsaw,
Banacha 1, 02-097 Warsaw, Poland
‡Laboratory of NMR Spectroscopy, Faculty of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
§Department of Pharmacognosy, Faculty of Pharmacy, Medical University of Białystok, Mickiewicza 2A, 15-230 Białystok, Poland
S
* Supporting Information
ABSTRACT: The isolation of phenolics from aerial parts of
Corispermum marschallii yielded a total of 13 compounds
including nine previously undescribed patuletin and spinacetin
glycosides. These were identified as patuletin 3-O-β-
D-galactopyranosyl-7-O-β-D-glucopyranoside (1), spinacetin
3-O-β-D-galactopyranosyl-7-O-β-D-glucopyranoside (2), patu-
letin 3-O-(6″-O-β-D-glucopyranosyl)-β-D-galactopyranoside (3),
patuletin 3-O-(6″-O-α-L-arabinopyranosyl)-β-D-galactopyrano-
side (4), patuletin 3-O-(2″-O-(5‴-O-α-L-arabinopyranosyl)-
β-D-apiofuranosyl)-β-D-galactopyranoside (5), patuletin 3-O-
(2″-O-β-D-apiofuranosyl)-β-D-galactopyranoside (6), spinace-
tin 3-O-β-D-galactopyranoside (7), patuletin 3-O-β-D-galacto-
pyranosyl-7-O-(6‴-O-feruloyl)-β-D-glucopyranoside (8), and spinacetin 3-O-β-D-galactopyranosyl-7-O-(6‴-O-feruloyl)-β-
D-glucopyranoside (9). Structure elucidation was based on UV−visible, multistage MS, and 1D and 2D NMR spectroscopy
and chemical derivatization, which allowed the identification on the glycosides with two different hexose moieties occurring at
different positions of the aglycones. Most of the compounds tested inhibited the production of pro-inflammatory factors such as
ROS, IL-8, and TNF-α in stimulated neutrophils.
Corispermum marshallii Steven (syn. C. borysthenicum Andrz.1),
commonly known as Marschall’s bugseed or gray bugseed
(from the German, Marschall Wanzensame or Grauer
Wanzensame), belongs to the family Amaranthaceae. It is an
annual herbaceous plant that grows 20−50 cm high with an
arborescent stem and oblong-lanceolate leaves and spikes. The
plant is found mainly in Central and Eastern Europe in gravelly
or sand habitats.2
Available research on the phytochemical composition of
Corispermum species has shown that species belonging to this
genus contain isoquinoline alkaloids such as salsoline and
salsolidine. In particular, the presence of salsolidine in aerial
parts of C. marschallii was confirmed.3 The phytochemical anal-
ysis of C. leptopyrum4 revealed another unknown basic com-
pound as well. Other research has revealed that seeds of
Corispermum species contain small amounts of ecdysteroids.5
Also, a previous study has indicated the presence of vitamins
and flavonoid glycosides in C. papillosum collected in a desert
region of Central Asia,6 revealing that studying the phenolic
profiles of other Corispermum species would be a worthwhile
endeavor.
were used in traditional Chinese medicines for treating a wide
variety of maladies,7−10 none of their effects can be attributed
to the activity of alkaloids. These activities may suggest pos-
sible anti-inflammatory effects; thus, further exploration of the
content of phenols and their antioxidant and anti-inflammatory
activities may be justified.
The aim of the present study was to develop a UHPLC-
DAD-MS method to analyze the phenolic profile of the
extracts from aerial parts of C. marschallii and to isolate and
identify the major flavonoids present in this plant material.
Additionally, the bioactivities of the isolated compounds were
evaluated using a human neutrophil model.
RESULTS AND DISCUSSION
■
The structures of 13 compounds isolated from C. marschallii
were determined based on UV−vis spectroscopy, multistage
MS (Table S1, Supporting Information), and NMR spectros-
copy (Figure 1), and a chromatographic method was
developed to identify the monosaccharide units present and
determine their configurations on hydrolysis. 1H NMR spectra
From previous studies, it is unclear whether or not Corispermum
species might be useful in pharmacotherapy. Although there
are some reports that various Asian species of Corispermum
Received: February 15, 2018
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.8b00152
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Article
Figure 1. Relevant HMBC correlations occurring in spectra of compounds isolated from C. marschallii.
were recorded for each isolated compound and compared with
the available literature. For structure elucidation of the major-
ity of the new isolated compounds, further NMR experiments,
such as 13C NMR, HSQC, HMBC, and COSY, were performed.
The aglycone in compound 1 was identified by analysis of
the chemical shifts in the 13C NMR spectrum and the HMBC
correlations proving the linkage of the methoxy substituent
(represented by a high-intensity singlet at δH 3.76 ppm) to C-6
carbon (δC 132.2 ppm). After comparison with previously pub-
lished data,11 the aglycone was identified unequivocally as
patuletin. The chemical shifts of the glycosyl signals were
assigned based on the 13C NMR and HSQC experiments and
previously published data on D-galactopyranose12,13 and
D-glucopyranose moieties.14 The glycosylation site of the
D-galactosyl unit was determined based on an HMBC exper-
iment, which showed a cross-peak between the H-1″ doublet
(δH 5.41, J = 7.7 Hz) and the C-3 carbon (δC 133.4 ppm).
Moreover, the value of the coupling constant indicated that the
glycoside was the β-anomer. The D-glucosyl moiety was linked
to the aglycone at the C-7 position based on an HMBC exper-
iment, which showed a cross-peak between the H-1‴ doublet
(δH 5.14, J = 7.5 Hz) and the C-7 carbon (δC 156.4 ppm).
Thus, compound 1 was determined as patuletin 3-O-β-
D-galactopyranosyl-7-O-β-D-glucopyranoside, a new compound.
Earlier reports of patuletin glycosides identified in plants from
the Amaranthaceae family (particularly those formerly
classified as in the family Chenopodiaceae) have concerned
only 3-O-glucosides, and not 3-O-galactosides.15,16
The NMR data of compound 2 were found to be similar to
those of compound 1. The chemical shifts and correlations in
the 2D spectra were mostly comparable; the exception was the
presence of an additional singlet in the 1H NMR spectrum (δH
3.85 ppm) and a 13C NMR signal (δC 56.0 ppm) caused by the
presence of a second methoxy group. The position of this
substituent was determined from the HMBC experiment in
which the singlet was correlated with the C-3′ carbon in the
B ring of flavonol (δC 147.0 ppm). The NMR data of the agly-
cone were in agreement with previous reports on spinacetin
3-O-glycoside.11 Compound 2 was found to be a dihexoside
based on its MS, which showed neutral losses characteristic of
the cleavage of a hexoside (m/z 162 amu). The NMR data
allowed the assignment of 2 as spinacetin 3-O-β-D-galactopyr-
anosyl-7-O-β-D-glucopyranoside with the chemical shifts and
B
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Chart 1
coupling constants of the spectra recorded in DMSO-d6 being
analogous to those in the same spectra of compound 1.
Compound 3 gave the same aglycone and glycosyl moieties
as were observed for compound 1. Changes in the chemical
shift of the H-1‴ doublet (δ = 4.06 ppm) and the shifts of
H-5″ and H-6″ along with the changes in the HMBC spectrum
(i.e., the absence of the H-1‴/C-7 cross-peak and the appear-
ance of two new correlations at H-1‴/C-6″ and vice versa) led
to the conclusion that compound 3 is patuletin 3-O-(6″-O-β-
D-glucopyranosyl)-β-D-galactopyranoside. Compound 3 has
not been reported previously.
both a hexose and two pentose moieties. The results of the
hydrolysis experiments indicated the presence of D-galactose,
L-arabinose, and D-apiose moieties. The galactopyranose struc-
ture was confirmed as described above for compound 1, and
the signals from the arabinopyranose atoms were compared
with the spectra of arabinopyranosides recorded in CD3OD.19,20
An apiofuranoside unit was identified based on the DEPT-
edited HSQC data, in which inverted signals were observed for
two groups of cross-peaks, revealing the presence of two CH2
units. Additionally, the 13C NMR signal corresponding to the
C-3‴ carbon (δ = 80.0 ppm) did not correlate to any 1H NMR
signals in the HSQC spectrum, meaning it was tertiary.
Chemical shifts were assigned based on the HMBC and COSY
spectra and comparison with available data,21,22 which enabled
the elucidation of an apiofuranose unit. The coupling constant
of the anomeric proton doublet (δH 5.48 ppm, J = 0.8 Hz)
indicated a trans relationship between the H-1‴ and H-2‴
protons, which in the case of the apiofuranose unit corre-
sponded to a β-configuration.21,22 The HMBC cross-peaks
between the H-1‴ doublet of the arabinofuranosyl moiety and
the C-2″ carbon of the galactopyranosyl unit (and, inversely,
the H-2″/C-1‴ cross-peak) indicated the glycosylation posi-
tion of the first pentoside, a β-apiofuranoside moiety, whereas
analysis of the cross-peak between the H-1″″ doublet of
The presence of a pentosyl unit in the structure of com-
pound 4 was suspected based on the MS fragmentation analysis
and was confirmed by the 13C NMR data. Both the 1H and 13
C
NMR chemical shifts and the coupling constant of the anomeric
proton doublet (δ = 3.97 ppm, J = 6.7 Hz, 1H) were consistent
with the published spectra of α-L-arabinopyranosides recorded
in DMSO-d6.17,18 The HMBC correlation between the H-1‴
doublet and C-6″ was indicative of linear glycosylation and a
structure analogous to that of compound 3. Compound 4 was
thus established as patuletin 3-O-(6″-O-α-L-arabinopyranosyl)-
β-D-galactopyranoside, a new natural compound.
The MS fragmentation pattern and the NMR spectra of
compound 5 were more complex and showed signals from
C
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Journal of Natural Products
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the arabinopyranosyl moiety and the signal from the C-5‴
carbon (as well as the H-5‴/C-1″″ cross-peak) indicated the
second residue to be a pentoside. Thus, the structure of the
new compound 5 was determined as patuletin 3-O-(2″-O-(5‴-
O-α-L-arabinopyranosyl)-β-D-apiofuranosyl)-β-D-galactopyra-
noside.
Compound 6, a further new compound, was identified by
analogy to 5. The structure of 6 lacked only the arabinopyrano-
side moiety, which resulted in slightly different chemical shifts
for the H-5‴ and C-5‴ signals. Thus, the structure of 6 was
elucidated as patuletin 3-O-(2″-O-β-D-apiofuranosyl)-β-D-gal-
actopyranoside.
Compound 7 was found to be a monohexoside based on its
MS data. The NMR spectra of compound 7 were found to be
similar to those of compound 2. The chemical shifts and corre-
lations in the 2D spectra were comparable in the case of the
aglycone, with the difference concerning the lack of the glucosyl
moiety that was present in the structure of compound 2.
The galactosyl unit in compound 7 was identified by analogy
to compound 1, which confirmed that 7 is spinacetin 3-O-β-
D-galactopyranoside, a new substance.
Compounds 8 and 9 were determined to be derivatives of
compounds 1 and 2, respectively, based on their NMR spectra,
which showed comparable chemical shifts and the same 2D
correlations between the analogous signals. The resonances in
the NMR spectra of compounds 8 and 9 that were noted in the
spectra of 1 and 2 were signals for an additional moiety includ-
ing two doublets and a doublet of doublets (with chemical
shifts that indicated an additional aromatic ring with an ABX
coupling pattern) and doublets with large coupling constants
(J ≈ 15.9 Hz) that corresponded to olefinic protons and
another high-intensity singlet from a methoxy group. The latter
showed an HMBC correlation with the C-3″″ carbon in the
aromatic system described. The 13C NMR data included a sec-
ond signal from a carbonyl group (δC 166.7 ppm) and signals
that showed an HMBC cross-peak with the H-6‴ glucopyr-
anosyl proton. These data allowed the identification of a
feruloyl moiety, and thus 8 and 9 were assigned as patuletin
3-O-β-D-galactopyranosyl-7-O-(6‴-O-feruloyl)-β-D-glucopyra-
noside and spinacetin 3-O-β-D-galactopyranosyl-7-O-(6‴-O-
feruloyl)-β-D-glucopyranoside, respectively. Similar compounds
have been isolated from Atriplex littoralis (Amaranthaceae)15
and Chenopodium bonus-henricus (Amaranthaceae).23 Never-
theless, both 8 and 9 are new.
The potential anti-inflammatory activities of the isolated
compounds were evaluated using a human neutrophil model.
None of the compounds were cytotoxic to the cells used up to
50 μM based on cytometric experiments (data not shown).
The ability of each test compound to decrease the production
or release of pro-inflammatory factors such as reactive oxygen
species (ROS), interleukin 8 (IL-8), and tumor necrosis factor-
α (TNF-α) was evaluated. As expected from previous reports,24−27
all constituents were able to decrease the production of ROS
from f-MLP (N-formylmethionyl-leucyl-phenylalanine)-stimu-
lated neutrophils (Figure S77, Supporting Information).
Compounds were tested in the concentration range of 5 to
20 μM. The activities of most of the tested compounds were
concentration-dependent, and their IC50 values are shown in
Table 3. The statistically significant differences between the
obtained values were analyzed. The most potent activities
[comparable to the positive control, quercetin (IC50 4.4
0.3 μM)] were observed for compounds 3−5, 9, and patuletin
3-O-β-D-galactopyranoside.
Each of the isolated compounds was tested for the ability to
decrease the production of IL-8 and TNF-α from lipopoly-
saccharide (LPS)-stimulated neutrophils at the concentration
range 10 to 50 μM. Patuletin 3-O-β-D-galactopyranoside was
confirmed to be active toward the inhibition of TNF-α
production. The most potent activities were observed for com-
pounds 3, 5, 6, patuletin 3-O-β-D-galactopyranoside, and
6-methoxykaempferol 3-O-β-D-galactopyranoside, and the IC50
values ranged from 7.7
1.5 μM for patuletin 3-O-β-D-
galactopyranoside to 12.8 4.5 μM for compound 6 (Table 3).
No obvious relationship between the chemical structure of the
tested flavonoid and their potential anti-inflammatory activities
could be established based on the results obtained. It was con-
firmed that all the investigated compounds can be considered,
to some extent, potential anti-inflammatory agents. Whenever
the activity of natural products is considered, the question
about their bioavailability must be addressed. In general flavo-
noids exhibit a very low bioavailability after oral intake.28 How-
ever, several studies have shown that the uptake of flavonoids
aglycones and/or glycosides can occur at different levels of the
gastrointestinal tract, leading to their presence in the circu-
lation in unchanged form.29 Additionally, it was proven that
administration of formulations containing multiple plant com-
ponents (similar to those in which other Corispermum species
can be found in TCM) may increase the absorption of flavo-
noids after oral intake.30
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a PerkinElmer polarimeter model 241 using a 1 cm
microcell. NMR spectra were acquired using Varian VNMRS Oxford
300 MHz (for compounds 2−7 and all known compounds) and
Bruker AVANCE II HD 500 MHz spectrometers (for compounds 1,
8, and 9). High-resolution mass spectra were acquired using a time-of-
flight high-resolution mass 6230 mass spectrometer (Agilent). The
parameters for the ESI source were as follows: nebulizer pressure
45 psi; dry gas flow 12 L/min; dry temperature 300 °C; and capillary
voltage 4.5 kV. Analysis was carried out using scans from m/z 100 to
1100. Mass spectra were recorded in the positive- and negative-ion
modes. UHPLC-DAD-MS analysis was conducted using a Dionex
Ultimate 3000RS system coupled with an Amazon SL ion trap mass
spectrometer (Bruker Daltonics, Bremen, Germany). The ion trap
AmazonSL mass spectrometer was equipped with an ESI interface.
The eluate was introduced into the ESI interface of the mass spectro-
meter without splitting. The parameters for the ESI source were as
follows: nebulizer pressure 40 psi; dry gas flow 9 L/min; dry tem-
perature 300 °C; and capillary voltage 4.5 kV. Analysis was carried out
using scanning from m/z 70 to 2200. Mass spectra were recorded in
the positive- and negative-ion modes. A sample of the crude plant extract
dissolved in methanol (20 mg/mL) was filtered through a 0.45 μm
syringe filter and subjected to UHPLC-DAD-MS analysis. The sepa-
ration was carried out on a Kinetex XB-C18 (150 mm × 3.0 mm ×
2.6 μm, Phenomenex, Torrance, CA, USA) column maintained at
25 °C. The mobile phases were 0.1% HCOOH in water (A) and 0.1%
HCOOH in acetonitrile (B), and elution was conducted with the
following gradient: 0 min, 0% B; 60 min, 26% B; and 70 min, 95% B.
The flow rate was 0.4 mL/min, and the injection volume was 2 μL of
the prepared extract. The UV−vis spectra of the detected compounds
were recorded over the 190−450 nm range. The chromatogram was
recorded at 254 nm. Compounds were characterized based on the
maxima observed in their UV−vis spectra and on their MS spectra.
Acetonitrile, methanol, and formic acid for UHPLC were purchased
from Merck (Darmstadt, Germany). Water for UHPLC was purified
with a Millipore Simplicity System (Bedford, MA, USA). Solvents for
the extraction of plant material and isolation were of analytical grade.
Column chromatography was carried out on Sephadex LH-20 (POCh,
Gliwice, Poland), Diaion HP-20 (Sigma−Aldrich, Steinheim, Germany),
D
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Table 1. NMR Spectroscopic Data for Patuletin (1, 3, 8) and Spinacetin Glycosides (2, 7, 9)
a
b
a
a
a
a
1
2
3
7
8
9
δH (J in
Hz)
δH (J in
Hz)
δH (J in
Hz)
position
δC, type δH (J in Hz) δC, type
δC, type
δC, type δH (J in Hz) δC, type δH (J in Hz) δC, type
flavonol moiety
2
3
4
5
6
7
157.0, C
159.3, C
135.3, C
156.5, C
133.2, C
177.7, C
151.6, C
131.3, C
157.5, C
156.3, C
156.9, C
156.9, C
133.1, C
177.8, C
151.3, C
132.4, C
156.4, C
94.0, CH
152.2, C
106.2, C
60.4, CH3
120.9, C
133.4, C
132.8, C
133.5, C
177.8, C
179.8, C
177.6, C
177.8, C
d
151.2, C
153.7,
C
152.4, C
151.2, C
132.2, C
133.8, C
157.8, C
131.3, C
132.3, C
156.4, C
157.7, C
156.4, C
8
6.93, s
93.9, CH 6.88, s
152.1, C
95.5, CH 6.50, s
93.9, CH 6.55, s
152.4, C
94.0, CH 6.98, s
151.6, C
93.9, CH 7.01, s
152.2, C
d
9
153.3, C
10
106.0, C
108.0, C
104.3, C
104.3, C
106.1, C
H3CO-6 3.76, s
60.2, CH3 3.91, s
121.1, C
61.5, CH3 3.75, s
122.8, C
60.0, CH3 3.75, s
121.1, C
60.0, CH3 3.76, s
121.1, C
60.4, CH3 3.76, s
121.0, C
1′
2′
7.54, d
(2.2)
116.2, CH 8.05, d (1.8) 114.6, CH 7.52, d
(2.1)
116.0, CH 8.02, d
(2.0)
113.6, CH 7.58, d (2.2) 116.2, CH 8.03, d (2.1) 113.6, CH
3′
4′
5′
144.9, C
148.6, C
148.4, C
151.0, C
144.8, C
148.5, C
147.0, C
149.4, C
144.9, C
148.7, C
147.0, C
149.3, C
6.83, d
(8.5)
115.1, CH 6.92, d (8.4) 115.9, CH 6.81, d
(8.5)
115.2, CH 6.92, d
(8.5)
115.2, CH 6.81, d (8.5) 115.1, CH 6.88, d (8.4) 115.1, CH
6′
7.66, dd
(8.5, 2.2)
122.0, CH 7.62, dd
(8.4, 1.8)
3.96, s
123.8, CH 7.66, dd
(8.5, 2.1)
122.0, CH 7.49, dd
121.9, CH 7.67, dd
122.0, CH 7.53, dd
(8.4, 2.1)
3.85, s
121.9, CH
56.0, CH3
(8.4, 2.0) (8.5, 2.2)
H3CO-3′
57.0, CH3
3.85, s
56.0, CH3
galactopyranose moiety
1″
5.41, d
(7.7)
101.5, CH 5.42, d (7.8) 104.1, CH 5.34, d
(7.6)
101.8, CH 5.51, d
(7.7)
71.3, CH 5.52, d (7.7) 101.5, CH 5.52, d (7.7) 101.5, CH
c
c
c
c
c
2″
3.57, t
(8.6)
71.2, CH 3.83
73.1, CH 3.53
71.1, CH 3.57
73.1, CH 3.58
71.3, CH 3.58
71.3, CH
c
c
c
c
c
c
3″
4″
3.35
73.2, CH 3.58
68.0, CH 3.86
74.9, CH 2.84, m
73.3, CH 3.41
68.0, CH 3.38−3.24
73.1, CH 3.38−3.24
73.1, CH
c
3.65, d
(3.1)
70.0, CH 3.68
67.9, CH 3.68, d
(2.2)
76.0, CH 3.68, d (3.0) 67.9, CH 3.68, d (3.0) 67.9, CH
c
c
c
c
c
c
5″
6″
3.33
75.9, CH 3.48
77.3, CH 2.93
76.6, CH 3.37
60.4, CH2 3.37
75.9, CH 3.37
75.9, CH
60.2, CH2
c
c
c
c
c
c
c
c
c
c
3.31
60.2, CH2 3.40−3.60
62.2, CH2 3.74, 3.40 67.1, CH2 3.50, 3.35 156.3, C
3.35, 3.48
60.2, CH2 3.35, 3.48
glucopyranose moiety
1‴
5.14, d
(7.5)
100.0, CH 5.12, d (6.9) 101.9, CH 4.06, d
(7.7)
102.9, CH
5.24, d (7.3) 100.1, CH 5.24, d (7.3) 100.1, CH
c
c
c
c
c
c
c
c
c
2‴
3‴
4‴
3.35
73.2, CH 3.58
76.7, CH 3.43
69.6, CH 3.41
74.8, CH 3.36
77.9, CH 2.98
71.3, CH 2.97
73.0, CH
76.6, CH
69.9, CH
3.35−3.24
73.1, CH 3.35−3.24
73.1, CH
76.3, CH
69.3, CH
c
c
c
3.32
3.36
76.3, CH 3.36
c
c
c
3.18 , t
3.34
69.3, CH 3.34
(8.8)
c
c
c
c
c
c
5‴
6‴
3.45 , m
77.3, CH 3.53
78.5, CH 3.55
73.8, CH
3.79
73.9, CH 3.79
73.9, CH
63.0, CH2
c
c
c
3.45 , m
60.6, CH2 3.40−3.60
62.5, CH2 3.55 , 3.36 60.9, CH2
4.39, dd
63.0, CH2 4.39, dd
(10.2, 1.7)
(10.2, 1.7)
4.24, dd
(12.2, 5.1)
4.24, dd
(12.2, 5.1)
feruloyl moiety
CO
166.7, C
166.7, C
α
6.40, d
(15.9)
114.0, CH 6.40, d
(15.9)
114.0, CH
β
7.48, d
(15.9)
145.4, CH 7.48, d
(15.9)
145.4, CH
125.4, C
1⁗
2⁗
3⁗
4⁗
5⁗
6⁗
125.4, C
7.22, d (1.9) 111.0, CH 7.22, d (1.9) 111.0, CH
147.9, C
149.6, C
147.9, C
149.6, C
6.69, d (8.1) 115.5, CH 6.69, d (8.1) 115.5, CH
6.91, dd
(8.3, 1.9)
123.1, CH 6.91, dd
(8.3, 1.9)
55.6, CH3 3.74, s
123.1, CH
H3CO
3.74, s
55.6, CH3
a
b
c
d
Recorded in DMSO-d6. Recorded in CD3OD. Signals overlapped, chemical shifts were derived from HSQC spectrum. Interchangeable.
and Toyopearl HW-40F (Tosoh, Shunan, Japan). Thin-layer chromatog-
raphy (TLC) was performed on Merck silica gel 60 (0.25 mm) plates.
Plant Material. The aerial parts of C. marschallii (700 g) were
collected on the banks of the Vistula River (Masovian District,
Poland; GPS: N 52°17′36.7″, E 20°58′19.4″) in September 2014, and
the species was authenticated by S. Granica and C. Zidorn. A voucher
specimen has been deposited in the plant collection of the Depart-
ment of Pharmacognosy and Molecular Basis of Phytotherapy
Medical University of Warsaw, Poland. A scan of the voucher is
available in Figure S3 (Supporting Information).
E
DOI: 10.1021/acs.jnatprod.8b00152
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Journal of Natural Products
Article
Table 2. NMR Spectroscopic Data for Patuletin Glycosides (4, 5, 6)
a
b
b
4
5
6
position
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
patuletin moiety
2
3
4
5
6
7
8
9
156.5, C
133.1, C
177.7, C
152.4, C
131.2, C
157.4, C
93.8, CH
151.5, C
104.4, C
60.0, CH3
121.1, C
115.9, CH
144.9, C
148.5, C
115.2, CH
122.0, CH
158.5, C
134.7, C
179.7, C
153.7, C
132.5, C
158.4, C
94.8, CH
153.8, C
106.4, C
61.0, CH3
123.3, C
117.3, CH
145.9, C
149.7, C
116.1, CH
123.3, CH
158.3, C
134.6, C
179.8, C
153.8, C
132.5, C
158.4, C
94.7, CH
153.6, C
106.3, C
60.9, CH3
123.2, C
117.3, CH
145.9, C
149.7, C
116.2, CH
123.2, CH
6.51, s
6.51, s
6.47, s
10
H3CO-6
3.75, s
3.87, s
3.87, s
1′
2′
3′
4′
5′
6′
7.52, d (1.9)
7.74, d (2.0)
7.71, d (2.1)
6.81, d (8.5)
7.67, dd (8.6, 2.0)
6.89, d (8.5)
7.66, dd (8.5, 2.1)
6.87, d (8.5)
7.63, dd (8.5, 2.1)
galactopyranose moiety
1″
2″
3″
4″
5″
6″
5.32, d (7.6)
101.7, CH
70.4, CH
73.0, CH
68.3, CH
74.4, CH
66.5, CH2
5.58, d (7.7)
3.99, t (8.1)
101.3, CH
76.4, CH
75.4, CH
70.7, CH
77.1, CH
62.1, CH2
5.50, d (7.7).
101.3, CH
76.7, CH
75.3, CH
70.6, CH
77.0, CH
62.0, CH2
c
c
c
c
c
3.17
3.37
3.64
3.55
3.97
3.73
3.85
c
c
3.70
3.83
3.46
3.47, 3.60
c
c
c
3.47, q (6.1)
c
c
c
c
c
c
3.67, 3.37
3.62, 3.50
apiopyranose moiety
1‴
2‴
3‴
4‴
5.48, d (0.8)
4.06, s
110.4, CH
78.6, CH
80.1, C
75.7, CH2
74.1, CH2
5.46, d (1.2)
4.06
110.7, CH
78.0, CH
80.8, C
75.5, CH2
66.2, CH2
c
c
c
c
c
3.68, 4.10, d (9.8)
3.70, 4.04
c
c
5‴
3.63, 4.10, d (9.8)
3.62, 3.75
arabinofuranose moiety
1⁗
2⁗
3⁗
4⁗
5⁗
3.97, d (6.7)
102.6, CH
71.0, CH
72.5, CH
67.4, CH
4.15, d (7.0)
105.2, CH
72.4, CH
74.1, CH
69.6, CH
66.9, CH2
c
c
3.56
3.51
3.30
3.73
3.85, 3.48
c
c
3.04
3.45
c
c
c
c
c
c
3.04, 3.53
65.0, CH2
a
b
c
Recorded in DMSO-d6. Recorded in CD3OD. Signals overlapped, chemical shifts were derived from HSQC spectrum.
Extraction and Isolation. Air-dried aerial parts of C. marschallii
subjected to a Sephadex LH-20 column (79 cm × 3.2 cm) eluted
with MeOH (isocratic flow). Altogether, 112 fractions were obtained,
and they were grouped into 10 main fractions, B1−B10, based on TLC
analysis (conditions as above). Fraction A4B6 was dissolved in DMSO
for preparative HPLC (P1 method: Shimadzu LC-20AP instrument
with a UV−vis detector, Kinetex SB-C18 (21.2 mm × 150 mm × 5 μm)
column; flow rate 22 mL/min, mobile phase A: 0.1% HCOOH in
H2O, B: 0.1% HCOOH in CH3CN; programmed gradient: 0−45 min
6−21% B, detection at 254 and 350 nm). This separation provided
three compounds: patuletin 3-O-β-D-galactopyranoside (13.7 mg, tR =
32.15−33.20 min), 6-methoxykaempferol 3-O-β-D-galactopyranoside
(3.6 mg, tR = 37.00−37.65 min), and 7 (23.6 mg, tR = 38.20−
40.05 min). Fraction A5 was separated according to the same protocol
as was used for fraction A4, and the resulting 150 fractions were com-
bined into main fractions C1−C6 based on TLC analysis. Fraction
A5C3 was further separated by preparative HPLC (P1 method), which
were powdered and extracted using an ultrasonic bath at room
temperature with an acetone−methanol−water (3:1:1) mixture (7 ×
4 L). After concentration of the combined extracts at 45 °C, a sample
was collected and freeze-dried for UHPLC analysis. The rest of the
aqueous residue was fractionated by liquid−liquid extraction with
chloroform (3 × 500 mL), diethyl ether (6 × 500 mL), ethyl acetate
(6 × 500 mL), and finally n-butanol (7 × 400 mL). The combined
layers were evaporated to dryness to yield 3.22 g of chloroform
residue, 6.38 g of diethyl ether residue, 7.58 g of ethyl acetate residue,
25.17 g of n-butanol residue, and 16.27 g of aqueous residue.
Afterward, to isolate the phenolics, the ethyl acetate and n-butanol
residues were purified.
The EtOAc residue (7.50 g) was dissolved in a mixture of ethyl
acetate and methanol, adsorbed onto silica gel (10.0 g), and subjected
to chromatography on a silica gel column (150 mm × 50 mm) using
an Interchim PuriFlash 430evo chromatograph. Elution was per-
formed with a mixture of chloroform−methanol (from 100:0 to 0:100
in 12 steps; 0%, 5%, 7%, 10%, 15%, 27%, 35%, 45%, 55%, 75%, and
100% methanol, 300 mL per step). The flow rate was 15 mL/min.
In total, 176 fractions were obtained and pooled into seven main
fractions, A1−A7, based on TLC analysis (silica gel, ethyl acetate−
formic acid−acetic acid−water, 100:11:11:26). Fraction A4 was
afforded pure naringenin 8-C-β-glucopyranoside (28.6 mg, tR
26.50−27.70 min).
=
The n-BuOH residue (25.0 g) was dissolved in water and separated
on a Diaion HP-20 column (20 cm × 5 cm) using an Interchim
PuriFlash 430evo device eluting with a gradient of H2O−MeOH (from
100:0 to 0:100 in 10 steps; 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,
50%, 75%, to 100%, 400 mL per step), and the flow rate was 15 mL/min.
F
DOI: 10.1021/acs.jnatprod.8b00152
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Journal of Natural Products
Article
13C NMR (DMSO-d6), Table 1; HRESIMS m/z 655.1504 [M − H]−
(calcd for C28H31O18−, 655.1510).
a
Table 3. IC50 Values Calculated for Isolated Compounds
ROS
TNF-α
secretion
Spinacetin 3-O-β-D-galactopyranosyl-7-O-β-D-glucopyranoside
(2): pale yellow, amorphous solid; [α]20 −56 (c 0.12, MeOH); UV
production IL-8 secretion
D
(MeOH) λmax (log ε) 211 (4.33), 257 (4.17), 354 (4.09) nm; 1H and
13C NMR (CD3OD and DMSO-d6), Table 1; HRESIMS m/z
669.1664 [M − H]− (calcd for C29H33O18−, 669.1667).
compound
IC50 SEM IC50 SEM IC50 SEM
1
2
3
4
5
6
7
8
9
26.9 2.9
31.5 7.4
5.7 1.1
5.7 0.7
5.4 0.9
7.1 1.4
38.5 5.6
36.6 3.3
6.0 1.3
66.0 12.1
25.7 4.8
28.9 12.1
25.5 13.2
24.5 5.3
7.2 1.2
16.3 5.5
9.3 3.1
Patuletin 3-O-(6″-O-β-D-glucopyranosyl)-β-D-galactopyranoside
(3): pale yellow, amorphous solid; [α]20 −20 (c 0.12, MeOH); UV
D
(MeOH) λmax (log ε) 213 (4.38), 259 (4.19), 359 (4.19) nm; 1H and
13C NMR (DMSO-d6), Table 1; HRESIMS m/z 655.1510 [M − H]−
(calcd for C28H31O18−, 655.1510).
9.7 1.0
12.8 4.5
16.3 10.8
23.2 13.1
20.7 5.6
11.7 3.9
51.9 4.7
46.7 12.3
Patuletin 3-O-(6″-O-α-L-arabinopyranosyl)-β-D-galactopyrano-
side (4): pale yellow, amorphous solid; [α]20 −7 (c 0.13, MeOH);
D
UV (MeOH) λmax (log ε) 209 (3.41), 261 (3.09), 361 (3.01) nm; 1H
and 13C NMR (DMSO-d6), Table 2; HRESIMS m/z 625.1395
[M − H]− (calcd for C27H29O17−, 625.1405).
6-methoxykaempferol 3-O-β- 15.4 3.2
18.1 5.5
11.4 2.3
8.0 0.7
D-galactopyranoside
Patuletin 3-O-(2″-O-(5‴-O-α-L-arabinopyranosyl)-β-D-apiofura-
nosyl)-β-D-galactopyranoside (5): pale yellow, amorphous solid;
naringenin 8-C-β-
13.2 3.5
3.4 0.6
4.4 0.3
23.7 6.6
7.7 1.5
20.4 1.9
glucopyranoside
[α]20 −72 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 213 (4.52),
patuletin 3-O-β-D-
D
1
260 (4.31), 355 (4.29) nm; H and 13C NMR (CD3OD), Table 2;
galactopyranoside
quercetin (positive control)
33.0 11.6
HRESIMS m/z 757.1826 [M − H]− (calcd for C32H37O21−, 757.1827).
Patuletin 3-O-(2″-O-β-D-apiofuranosyl)-β-D-galactopyranoside
a
Values given as μM.
(6): pale yellow, amorphous solid; [α]20 −128 (c 0.1, MeOH); UV
D
(MeOH) λmax (log ε) 211 (4.34), 259 (4.04), 346 (3.98) nm; 1H and
13C NMR (CD3OD), Table 2; HRESIMS m/z 625.1402 [M − H]−
(calcd for C27H29O17, 625.1405).
In total, 28 fractions were obtained and combined into main fractions
D1−D6, based on TLC analysis. Fraction D3 was subjected to
Toyopearl HW-40F column chromatography (55 cm × 2.2 cm) with
an isocratic mobile phase (70% MeOH). In total, 48 fractions were
collected, and they were combined into seven main fractions, E1−E7,
based on TLC analysis. Fraction D3E3 was dissolved in DMSO and
then subjected to preparative HPLC (P2 method: Shimadzu LC10vp
chromatographic system (Kyoto, Japan) equipped with a UV−vis
detector, Zorbax SB-C18 (21.2 × 150 mm × 5 μm) column, eluted
with A: 0.1% HCOOH in H2O, B: 0.1% HCOOH in CH3CN at
5 mL/min using the following gradient: 0−60 min 3−26% B; 60−
65 min 26−35% B, detection at 254 and 350 nm). This separation
provided compounds 1 (42.2 mg, tR = 24.30−30.70 min) and 2
(11.8 mg, tR = 34.20−35.25 min). Fraction D4 was separated using the
same conditions as used for fraction D3, with 60 fractions being
obtained, which were pooled into five main fractions, F1−F5, based on
TLC analysis. Four of the fractions were subjected to preparative
HPLC: D4F2, D4F3, D4F4, and D4F5 were combined with other
fractions featuring the same compounds. Dissolving fraction D4F2 in
DMSO and subjecting it to the P2 method provided two pure
compounds: 1 (40.0 mg, tR = 29.85−31.00 min) and 2 (86.0 mg, tR =
33.30−35.00 min). Preparative HPLC of fraction D4F3 dissolved in
DMSO using the P1 method gave pure 3 (24.6 mg, tR = 26.80−
28.00 min), 4 (4.4 mg, tR = 28.40−28.95 min), and 6 (17.6 mg, tR =
29.90−31.20 min). Fraction D4F4 was combined with fraction D3E6,
dissolved in DMSO, and separated by P2 to give pure patuletin 3-O-β-
D-galactopyranoside (18.7 mg, tR = 47.75−48.75 min). Combining
D4F5 and D3E7, dissolving them in DMSO, and purifying the mixture
by method P2 provided hyperoside (35.2 mg, tR = 45.60−46.35 min).
Fraction D5 was separated on a Toyopearl HW-40F column (79 cm ×
3.2 cm) eluted with 60% MeOH (isocratic flow). In total, 162 frac-
tions were collected and pooled into seven main fractions, G1−G7,
based on TLC analysis. Fraction D5G3 was dissolved in DMSO and
separated using preparative HPLC according to method P1. As a result,
four compounds were obtained: 2 (18.8 mg, tR = 20.25−20.85 min),
4 (1.6 mg, tR = 28.90−29.10 min), 5 (8.6 mg, tR = 29.45−30.00 min),
and 9 (2.1 mg, tR = 43.60−43.90 min). Fraction D5G4 was purified by
the same conditions used for D5G3 and gave five pure compounds:
3 (19.0 mg, tR = 27.60−28.55 min), 5 (17.6 mg, tR = 29.65−30.15 min),
6 (13.4 mg, tR = 30.75−31.25 min), 8 (6.5 mg, tR = 41.20−41.55 min),
and 9 (7.0 mg, tR = 43.65−43.90 min). A scheme of the isolation
procedure is provided in Figure S2 (Supporting Information).
Spinacetin 3-O-β-D-galactopyranoside (7): pale yellow, amor-
phous solid; [α]20 −58 (c 0.06, MeOH); UV (MeOH) λmax (log ε)
D
214 (3.95), 257 (3.71), 271 (3.70), 332 (3.68) nm; 1H and 13C NMR
(DMSO-d6), Table 1; HRESIMS m/z 506.0985 [M − H]− (calcd for
C23H23O13−, 507.1139).
Patuletin 3-O-β-D-galactopyranosyl-7-O-(6‴-O-feruloyl)-β-D-glu-
copyranoside (8): pale yellow, amorphous solid; [α]20D −104 (c 0.09,
MeOH); UV (MeOH) λmax (log ε) 210 (4.46), 260 (4.14), 273
(4.13), 330 (4.17) nm; 1H and 13C NMR (DMSO-d6), Table 1;
HRESIMS m/z 831.1975 [M − H]− (calcd for C38H39O21−, 831.1984).
Spinacetin 3-O-β-D-galactopyranosyl-7-O-(6‴-O-feruloyl)-β-D-
glucopyranoside (9): pale yellow, amorphous solid; [α]20 −116
D
(c 0.12, MeOH); UV (MeOH) λmax (log ε) 211 (4.43), 255 (4.16),
1
274 (4.11), 332 (4.20) nm; H and 13C NMR (DMSO-d6), Table 1;
HRESIMS m/z 845.2141 [M − H]− (calcd for C39H41O21−, 845.2140).
Hydrolysis of the Glycosides and Determination of the
Configurations of the Monosaccharides. Hydrolysis was
performed as previously described31 with some modification. Samples
of the isolated glycosides were refluxed under acidic conditions only,
for 2−12 h instead of 1 h. No additional purification (other than
evaporation of water and HCl under reduced pressure) was perfor-
med. The configurations of the monosaccharides were determined as
described in the previous report without changes.31 Dry CHCl3
extracts were dissolved in a CH3CN−H2O (20:80) mixture and
filtered through a PTFE syringe filter. The derivatives obtained were
analyzed by UHPLC-DAD-MS. Separations were performed on a
Kinetex XB-C18 (150 mm × 3.0 mm × 2.6 μm) column eluted with A:
0.1% HCOOH in H2O, B: 0.1% HCOOH in CH3CN at a flow rate of
0.4 mL/min, isocratic flow 35% B, duration of the analysis 25 min.
Samples were analyzed using extracted ion chromatograms that were
obtained from the following ions: 537 amu for hexoses, 465 amu for
pentoses, and 479 amu for rhamnose derivatives. Derivatives of the
standards had retention times as follows: L-arabinose, tR = 11.6 min;
L-xylose, tR = 12.0 min; D-arabinose, tR = 12.2 min; D-apiose, tR
=
12.4 min; D-xylose, tR = 12.5 min; D-galactose, tR = 14.5 min; L-glucose,
tR = 16.4 min; D-glucose, tR = 17.9 min: and L-rhamnose, tR = 20.0 min.
Comparing the ion masses and retention times of the derivatives of
standards and derivatives of the hydrolyzed samples allowed for the
identification of the saccharide moieties in the isolated glycosides. Based
on the experiments described, D-glucose was identified in 1, 2, 3, 8,
and 9; D-galactose in 1−9, patuletin 3-O-β-D-galactopyranoside, and
6-methoxykaempferol 3-O-β-D-galactopyranoside; L-arabinose in 4 and
5; and D-apiose in 5 and 6.
Patuletin 3-O-β-D-galactopyranosyl-7-O-β-D-glucopyranoside
(1): pale yellow, amorphous solid; [α]20 −62 (c 0.11, MeOH); UV
D
(MeOH) λmax (log ε) 212 (4.41), 260 (4.23), 359 (4.14) nm; 1H and
G
DOI: 10.1021/acs.jnatprod.8b00152
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Journal of Natural Products
Article
Biological Activity Assays. Compounds 1−9, patuletin 3-O-β-D-
galactopyranoside, and 6-methoxykaempferol 3-O-β-D-galactopyrano-
side were dissolved in DMSO to prepare 10 mM stock solutions.
Between experiments, the stock solutions were stored at −20 °C.
Stock solutions were diluted in the appropriate solvents (HBSS or
RPMI 1640) to obtain final concentrations of 5, 10, and 20 μM in the
wells for the ROS-inhibition assays and 10, 20, and 50 μM for other
assays. All isolated compounds used in the biological activity assays
were >95% pure by HPLC. A positive control, quercetin, was purchased
from Carl Roth (Karlsruhe, Germany) and was >99% pure by HPLC.
Isolation of Neutrophils and Determination of Their
Viability. The buffy coat for isolation of human neutrophils was pre-
pared as previously reported.32 The viability of the neutrophils was
determined by staining with propidium iodide (PI) using a previously
reported protocol with few alterations.32 Neutrophils (1 × 106/mL)
were cultured in a 96-well plate in RPMI 1640 medium in the absence
or presence of the investigated compounds at final concentrations of
10, 20, and 50 μM added 1 h before stimulation with LPS (100 ng/mL).
Triton X-100 (0.1%) was used as a positive control.
ROS Production. The ROS production by f-MLP-stimulated
neutrophils was determined using luminol-dependent chemilumines-
cence. The compounds were tested at concentrations of 5, 10, and 20 μM.
Following isolation, the cells were suspended in HBSS. The cell suspen-
sion (2.0 × 105/mL) was incubated with 50 μL of the samples containing
the tested compounds according to the previously reported protocol.33
IL-8 and TNF-α Production. Neutrophils (1.0 × 106/mL) for
evaluation of IL-8 and TNF-α production were cultured in 96-well
plates in RPMI 1640 medium with 10% fetal bovine serum, 10 mM
HEPES, and 2 mM L-glutamine in the absence or presence of LPS
(100 ng/mL) for 24 h at 37 °C with 5% CO2. The cells were treated
with the test compounds at final concentrations of 10, 20, and 50 μM
in 1 mL of cell suspension (96-well plates, 1 mL per well) 1 h before
stimulation. After 18 h, the solutions were centrifuged (2000 rpm;
10 min; 4 °C), and the supernatants were collected. The release of
cytokines by stimulated neutrophils was evaluated by ELISA following
the manufacturer’s instructions (BD Biosciences, San Jose, CA, USA,
or R&D Systems, Minneapolis, MN, USA). Quercetin (10, 20, and
50 μM) was used as a positive control.
́
R. Rudecki, and S. Doktor (all of the Medical University of
Warsaw) for their technical assistance during experiments. This
study was conducted in part with the use of equipment
purchased by the Medical University of Białystok as part of OP
DEP 2007-2013, Priority Axis I.3, Contact No. POPW.01.03.00-
20-001/12. This project also used CePT (Medical University of
Warsaw) infrastructure financed by the European Regional Devel-
opment Fund within the Operational Programme “Innovative
Economy” for 2007−2013. The research was financially
supported by the Medical University of Warsaw (students’
grant FW25/NM1/2017).
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Statistical Analysis. The results are expressed as the mean
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00152.
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Chromatogram of UHPLC-DAD-MS analysis, table with
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pounds (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: +48-22-5720942. E-mail: sgranica@wum.edu.pl.
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Simeonova, R.; Tzankova, V.; Aluani, D. Fitoterapia 2017, 118, 13−
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Monika E. Czerwinska: 0000-0002-8159-0174
Sebastian Granica: 0000-0002-1876-1321
Notes
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ACKNOWLEDGMENTS
The authors would like to thank Prof. C. Zidorn for help with
authentication of the plant material and M. Marciniak,
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