Phytochemical Characterization of Low Molecular Weight Constituents from Marshmallow Roots (Althaea officinalis) and Inhibiting Effects of the Aqueous Extract on Human Hyaluronidase-1

Article abstract of DOI:10.1021/acs.jnatprod.6b00670

Extract RE was obtained from the roots of Althaea officinalis in a yield of 8.1%, related to the dried plant material, by extraction with MeOH-H₂O (1:1), followed by precipitation with EtOH to remove high molecular weight constituents. Phytochemical investigation of RE revealed the presence of N-phenylpropenoyl-l-amino acid amides 1-5, 8% glycine betaine 6, about 9% total amino acids with proline as the main compound, and about 61% mono- and oligomeric carbohydrates with sucrose as the main compound. Further fractionation revealed the presence of a hypolaetin diglycoside (12) and four hypolaetin glycosides (7-9 and 11) with O-sulfocarbohydrate moieties; additionally, 4′-O-methylisoscutellarein-8-O-β-d-(3-O-sulfo)glucuronopyranoside (10) and the diglycosylated coumarin haploperoside D (13) were identified. The hypolaetin-O-sulfoglycosides 7-10 are new natural products. RE inhibited the enzymatic activity of surface-displayed human hyaluronidase-1 on Escherichia coli F470 cells with an IC₅₀ of 7.7 mg/mL. RE downregulated mRNA expression of hyal-1 in HaCaT keratinocytes at 125 and 250 μg/mL, respectively. These data contribute to a deeper phytochemical understanding of marshmallow root extracts and to the positive influence of extracts used for therapy of irritated and inflamed buccal tissue and cough.

Full text of DOI:10.1021/acs.jnatprod.6b00670

Article
pubs.acs.org/jnp
Phytochemical Characterization of Low Molecular Weight
Constituents from Marshmallow Roots (Althaea officinalis) and
Inhibiting Effects of the Aqueous Extract on Human Hyaluronidase‑1
Jandirk Sendker,*,† Ines Boker,† Isabelle Lengers,‡ Simone Brandt,† Joachim Jose,‡ Timo Stark,§
̈
Thomas Hofmann,§ Careen Fink,⊥ Heba Abdel-Aziz,⊥ and Andreas Hensel†
‡
†Institute of Pharmaceutical Biology and Phytochemistry and Institute of Pharmaceutical and Medicinal Chemistry, University of
Munster, Correnstrasse 48, D-48149 Munster, Germany
̈
̈
§Chair of Food Chemistry and Molecular Sensory Science, Technical University of Munich, Lise-Meitner-Strasse 34, D-85354
Freising, Germany
⊥Steigerwald Arzneimittelwerk GmbH, Bayer Consumer Health, Havelstrasse 5, D-64295 Darmstadt, Germany
S
* Supporting Information
ABSTRACT: Extract RE was obtained from the roots of Althaea officinalis in
a yield of 8.1%, related to the dried plant material, by extraction with
MeOH−H2O (1:1), followed by precipitation with EtOH to remove high
molecular weight constituents. Phytochemical investigation of RE revealed
the presence of N-phenylpropenoyl-L-amino acid amides 1−5, 8% glycine
betaine 6, about 9% total amino acids with proline as the main compound,
and about 61% mono- and oligomeric carbohydrates with sucrose as the main
compound. Further fractionation revealed the presence of a hypolaetin
diglycoside (12) and four hypolaetin glycosides (7−9 and 11) with O-
sulfocarbohydrate moieties; additionally, 4′-O-methylisoscutellarein-8-O-β-D-
(3″-O-sulfo)glucuronopyranoside (10) and the diglycosylated coumarin
haploperoside D (13) were identified. The hypolaetin-O-sulfoglycosides 7−10 are new natural products. RE inhibited the
enzymatic activity of surface-displayed human hyaluronidase-1 on Escherichia coli F470 cells with an IC50 of 7.7 mg/mL. RE
downregulated mRNA expression of hyal-1 in HaCaT keratinocytes at 125 and 250 μg/mL, respectively. These data contribute
to a deeper phytochemical understanding of marshmallow root extracts and to the positive influence of extracts used for therapy
of irritated and inflamed buccal tissue and cough.
immune-activating effects of aqueous marshmallow root extract
and isolated polysaccharides.10
he roots of Althaea officinalis L. (Malvaceae), also known
Tas marshmallow roots, are used for the symptomatic
treatment of oral or pharyngeal irritation associated with dry
cough and for the symptomatic relief of mild gastrointestinal
discomfort.1 The herbal material contains 5−11% water-soluble
polysaccharides, mainly galacturorhamnans, arabinans, glucans,
and arabinogalactans.2 Other constituents are about 0.2%
flavone glycosides, phenolic acids, the coumarin scopoletin,
starch, pectin, and tannins.3,4 The traditional oral use of
aqueous extracts against dry cough caused by the irritation of
oral, pharyngeal, or gastric mucosa is related to the bioadhesive
effect of the polysaccharides to the epithelial mucosa, which
protects the cells from local irritations.5 Phytochemical
investigations, clinical particulars, and pharmacological proper-
ties provide evidence for a justifiable use of marshmallow
extract, and the ESCOP monograph gives therapeutic
indications for gastric and duodenal ulcers and for dry
cough.6 Additionally, an antitussive effect of marshmallow
root extract was found in an animal study using cats.7,8 Extract
applied topically to UV-irritated rabbit ears had an anti-
inflammatory effect.9 Other animal experiments indicated slight
The mucilaginous and bioadhesive effects of the poly-
saccharides from aqueous marshmallow root extract are usually
seen as the major pharmacodynamic effect, leading to the
formation of a protecting and shielding polysaccharide layer on
inflamed or destructed epithelial tissue. The formation of such
bioadhesive layers has been shown in detail by fluorescence
histology for pectin-like polygalacturonides with structural and
physicochemical patterns similar to marshmallow polysacchar-
ides.11 Additionally, strong adsorption of marshmallow
polysaccharides to porcine buccal mucosal tissue has been
shown.12 The polysaccharides from marshmallow roots also
exert stimulating effects on the cell viability and cellular
proliferation of epithelial KB cells.13
These findings lead to the hypothesis that the polysacchar-
ides are forming a bioadhesive layer on the epithelial tissue that
should lead to a better protection and rehydration of the tissue
and an improved shielding of the mucosal barrier against
Received: July 19, 2016
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Figure 1. Structural features of compounds obtained from Althaea officinalis.
physical or microbial stress noxes. Yet, reducing the clinical
RESULTS AND DISCUSSION
■
particulars of marshmallow extract only to the mucilaginous
effects of high molecular weight polysaccharides might be too
simplistic. In the present phytochemical investigation the low
molecular weight compounds from marshmallow extract were
also investigated, indicating the presence of unusual flavonoid-
O-sulfoglycosides.
Dried and pulverized roots from A. officinalis were extracted
twice with 50% aqueous MeOH. High molecular weight
material was removed by precipitation with EtOH, resulting in
a raw extract, hereafter referred to as RE (yield: 8.1%, related to
the dried plant material). Since preliminary TLC studies of RE
indicated the presence of carbohydrates, amino acids, and
highly polar caffeic acid derivatives, a direct investigation of RE
with specific screening methods was applied to obtain a proper
insight into the composition of RE prior to systematic
chromatographic fractionation.
N-Phenylpropenoyl-L-Amino Acid Amides (1−5).
Using a stable isotope dilution assay19 five N-phenyl-
propenoyl-L-amino acid amides were identified (Figure 1): N-
(E)-caffeoyl-L-dopa (1), N-(E)-caffeoyl-L-tyrosine (2), N-(E)-
coumaroyl-L-dopa (3), N-(E)-coumaroyl-L-tyrosine (4), and N-
(E)-cinnamoyl-L-aspartate (5). Absolute quantification indi-
cated a content of 60 μg/g dried plant material for 1, whereas a
content of 0.3 μg/g was determined for each of 2−4 and of 0.1
μg/g for 5.
Mono- and Oligosaccharides. Using carbohydrate-
specific IEC-HPLC with pulsed amperometric detection
(PAD),20,21 the following mono- and oligomeric carbohydrates
were identified from RE and quantified against the respective
reference substances: sucrose (59%, related to RE dry mass),
glucose (1.4%), fructose (1.0%), raffinose (0.4%), xylose
(0.1%), galactose (0.1%), and rhamnose (<0.1%). The extract
is therefore dominated by sucrose, which accounts for 4.7% of
the dry mass of the herbal material. The separation method
cannot discriminate enantiomers.
Amino Acids. Amino acids from RE were separated from
the carbohydrates by solid-phase extraction on a strong cationic
exchanger and identified and quantified after chromatographic
separation on AminoPac stationary phase using IEC-HPLC-
PAD.21 The following amino acids were identified in RE and
quantified against the respective reference substances: aspar-
agine (3.0%, related to RE dry mass), arginine (2.4%), proline
(1.0%), glutamine (0.7%), alanine (0.6%), tryptophan (0.3%),
As it is known that O-sulfopolysaccharides are strongly
involved in the formation and regulation of the extracellular
matrix (ECM) in the dermis and epidermis of the skin and the
mucosal tissue, a connection between these natural products
and the ECM is feasible. A special position in the regulation of
ECM, especially in wound healing and inflammation, is
attributed to hyaluronic acid (HA). High molecular weight
HA (>20 kDa) exerts a multitude of physicochemical effects on
the tissue, such as water-binding, regulation of osmotic
pressure, and also anti-inflammatory effects by influencing the
migration of leucocytes and macrophages. Induction of growth
factors for epithelial cells, cell proliferation, and differentiation
are also described for this polymer.14 In this regard, high
molecular weight HA seems to be relevant for wound healing
and tissue regeneration. Low molecular weight HA (<20 kDa)
promotes angiogenesis, inhibits apoptotic processes, stimulates
inflammation, and exerts reduced water-binding capacity.15−17
Low molecular weight HA is formed by hyaluronidases
(HYAL), of which HYAL-1 and HYAL-2 are the main enzymes
involved in the HA metabolism of human tissue. Whereas
HYAL-1 cleaves the substrate to form tetramers or larger
oligosaccharides, HYAL-2 produces only polymers of a
molecular weight of up to about 20 kDa. The inhibition of
HYAL with specific inhibitors might be a promising target for
improved wound healing and tissue regeneration. Advanced
test assays for inhibitor screening are available since HYAL-1
can be expressed on the surface of E. coli using an autodisplay
technique.18 Therefore, it seems interesting to investigate the
potential influence of marshmallow extract on the activity of
HYAL-1 for a better understanding of its mode of action during
the treatment of irritated mucosal membranes.
B
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Figure 2. Extraction and fractionation scheme of extract RE of Althaea officinalis roots. Sephadex LH-20 fractions A to N yielded compounds 7−13
after subsequent chromatographic separations. Sephadex LH-20 subfractions, which were tested for HYAL-1 inhibition, are supplemented by their
inhibitory effect (HYAL-1 surface-displayed on Escherichia coli F470 pAK009, pretreatment of cells, 70 mg/mL, positive control glycyrrhicinic acid, 1
mM).
aspartate (0.2%), histidine (0.2%), threonine (0.1%), valine
(0.1%), serine (0.1%), isoleucine (0.1%), leucine (0.1%), and
tyrosine (0.1%). Glycine, lysine, methionine, glutamate, and
cysteine were not detected. Thus, the total amino acid content
of RE is 9.0%, which accounts for 0.74% of the dry mass of the
herbal material. The separation method cannot discriminate
enantiomers.
Glycine Betaine (6). As the presence of 6 (Figure 1) in
marshmallow has been reported,22 a specific LC-MS method
was applied for the direct quantification of this highly polar
compound in RE. The chromatographic separation turned out
to be difficult because of the strong hydrophilicity of 6. Use of a
Monochrom stationary phase, with pentafluorophenyl groups
attached via an alkyl spacer to the silica matrix, provided
sufficient separation results: as the fluorination induces a
polarization of the aromatic system, strong interaction of this
stationary phase under HILIC conditions occurs with electron-
rich bases and cationic systems as present in 6. The LC-MS
method was validated according to ICH guidelines23 and
indicated a content of 8.2% of 6 in RE, which accounts for
0.66% of the dry mass of the herbal material.
Flavonoids (7−12). RE was fractionated using Sephadex
LH-20 as stationary phase and, successively, EtOH, MeOH, and
acetone−H2O (7:3) as mobile phase in a step-gradient elution.
This procedure resulted in 14 fractions, A−N (Figure 2).
Whereas the 12 fractions C to N constituted only a minor
fraction of the total dry mass of RE, fractions A (61% of RE)
and B (2% of RE) dominated the extract. TLC analysis
indicated a strong accumulation of carbohydrates and amino
acids in both fractions A and B.
TLC analyses of Sephadex LH-20 fractions H, I, and J
showed an intense spot for each, which was assumed to
originate from a flavonoid because of its detection behavior.
Using two different preparative MPLC systems on RP18 or
MCI gel and preparative RP18 HPLC, 7.9 mg of 7 was
obtained from 52 mg of the three combined Sephadex LH-20
fractions. The UV spectrum with λmax = 256, 271, and 353 nm
indicated a flavonoid structure. HRESIMS showed the [M +
H]+ ion at m/z 545.0599, corresponding to C21H20O15S and
fragmenting into m/z 465.0959 and 303.0487. The neutral loss
of 79.9640 amu is characteristic for the loss of SO3, indicating 7
to be an O-sulfo derivative. The mass difference of 162.0472
amu between the fragment ions is congruent with the
elimination of an O-linked hexose residue. 1H NMR and
COSY spectra showed the typical AMX spin system of a
dihydroxylated flavonoid B-ring (δ 7.65, d, H-2′; 7.33, dd, H-6′;
and 6.73, d, H-5′). A one-proton singlet at δ 6.42 did not show
any COSY correlations, but HMBC cross-peaks were detected
to C-2, C-4, C-10, and C-1′, which is indicative for a flavone.
The HMBC correlations of the A-ring signal at δ 6.08 did not
differentiate between C-6 and C-8. Also, the anomeric proton
doublet of the anomeric carbon C-1″ of the hexose moiety
correlated in the HMBC with signals that could not
differentiate between C-6 and C-8. To establish the A-ring
location of the hexosyl moiety, 13C NMR data were recorded
and the signals of C-5, C-6, C-8, and C-9 compared to
published data of different A-ring-substituted flavonoids. No
similarities were found with 5,6,7-trisubstituted hispidulin24 or
5,6,7-trisubstituted baicalein-6-O-β-glucuronide,25 whereas a
nearly exact correlation was found with data published for the
5,7,8-trisubstituted wogonin26 and 5,7,8-trisubstituted isoscu-
tellarein-8-O-β-D-glucuronopyranoside.27 8-Hydroxylation of 7
was established on the basis of these data and on the HRESIMS
fragment at m/z 303.0487. This finding was also proven by a
second method: glycosylated 8-hydroxyluteolin has been
described to isomerize to the more stable 6-hydroxyluteolin
during acid-catalyzed hydrolysis, whereas 6-hydroxyluteolin is
stable under these conditions.28 Treatment of 7 with strong
acid, followed by UHPLC analysis of the hydrolysate, resulted
in two different flavone peaks (data not shown). The UV
C
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spectra of the two peaks correlated well with UV data published
for 5,6,8- and 5,6,7-trisubstituted flavonoids, respectively; the
two spectra differ in the λmax of band II by 4−7 nm.29 This
difference was also found in the spectra of the two peaks of the
anomeric proton. The 3-O-sulfo group of the uronic acid
moiety was deduced from the typical downfield shift of the 13
C
and 1H NMR signals in comparison to an underivatized uronic
acid group. Pyridine/DMSO-catalyzed desulfonylation and
hydrolysis of the pyranosyloxy linkage followed by CZE proved
the presence of D-glucuronic acid as the moiety of 9. On the
basis of these data, the structure of 9 was defined as the new 4′-
O-methylhypolaetin-8-O-β-D-(3″-O-sulfo)glucuronopyranoside
(Figure 1).
hydrolysate of 7 (λmax = 281 nm for the main peak and λmax
=
277 nm for the other peak). The structure of 5,6,8,3′,4′-
pentahydroxyflavone (8-hydroxyluteolin, hypolaetin) was de-
duced from these data.
1
Using H NMR and COSY, the carbohydrate part of the
glycoside was identified as a glucopyranoside with a β-oriented
anomeric group. The strong downfield shifts observed for H-2″
(δ 4.34) and C-2″ (δ 80.04) and upfield shifts for C-1″ (+0.5
ppm) and C-3″ (+1.5 ppm) are typical for O-sulfocarbohy-
drates.30−32 Considering the SO3 loss observed in the
HRESIMS fragmentation of 7, a 2-O-sulfoglucosyl moiety was
deduced. For determination of its absolute configuration, 7 was
desulfonylated with pyridine/DMSO33,34 and hydrolyzed using
trifluoroacetic acid, and the carbohydrate identified as D-glucose
by capillary zone electrophoresis (CZE) of the derivatized
hydrolysate.35 On the basis of these data, the structure of 7 was
defined as the new compound hypolaetin-8-O-β-D-(2″-O-
sulfo)glucopyranoside (Figure 1), a compound that to the
best of our knowledge has not been described previously.
Compound 8 resembled 7 in TLC analysis and was isolated
from Sephadex LH-20 fractions F and G by MPLC on MCI
stationary phase and subsequent preparative HPLC on RP-18.
This procedure yielded 10.2 mg of a yellow substance with UV
characteristics similar to those of 7. The typical UV spectrum
with λmax = 273, 330 nm indicated a flavonoid-type structure.
HRESIMS showed the [M + H]+ ion at m/z 559.0765,
corresponding to C22H22O15S and fragmenting to m/z
479.1180 [M + H − SO3]+, 317.0656 [M + H − SO3 −
hexose]+, and 302.0430 [M + H − SO3 − hexose − CH3]+. 1H
NMR data showed the AMX spin system of a dihydroxylated
flavonoid B-ring (δ 8.10, d, H-6′; 7.86, s, H-2′; 7.26, d, H-5′). A
three-proton singlet (δ 3.98, s, −OCH3) could be assigned to
the 4′-O-methyl group via HMBC data. All other analytical data
obtained by NMR, HPLC, or CZE after desulfonylation and
hydrolysis were identical to those described for 7. On the basis
of these data, the structure of 8 was defined as the new 4′-O-
methylhypolaetin-8-O-β-D-(2″-O-sulfo)glucopyranoside (Fig-
ure 1).
Compound 12 (4.4 mg) was isolated from the Sephadex LH-
20 fraction E by MPLC on MCI stationary phase and
subsequent preparative HPLC on RP-18. The TLC behavior
of 12 was indicative for a flavonoid glycoside; this compound
showed a lower Rf value than the flavone monoglycosides 7, 8,
and 9, suggesting a higher glycosylation level. The UV
spectrum of 12 with λmax = 256, 270, and 350 nm also
indicated a flavonoid structure. HRESIMS showed the [M +
H]+ ion at m/z 641.1393, corresponding to C27H28O18 and
fragmenting to m/z 303.0505 [M + H − hexose]+ and
479.0756 [M + H − hexose − hexuronic acid]+. These data are
in accordance with a pentahydroxylated flavone, glycosylated
with a hexose and a hexuronic acid unit. 1H NMR data showed
the AMX spin system of a 3′,4′-dihydroxylated flavonoid B-ring
(δ 7.24, d, H-2′; 7.19, d, H-6′; 6.77, d, H-5′). Proton singlets at
δ 6.25 and 6.09 were assigned to C-3 and C-6, respectively. All
other analytical data obtained for the aglycone of 12 by NMR
and hydrolysis experiments coupled with HPLC data were
identical to those described for compound 7, 8, and 11. Thus,
the aglycone was again established as hypolaetin, with a
glycosidic moiety at C-8. The proton signals of hexose and
uronic acid moieties were assigned by COSY and TOCSY
1
experiments. H NMR signals at δ 4.16−3.76 were assigned to
the protons of the uronic acid group, the β-orientation and
pyranose form of the uronic acid being deduced from the
coupling constant of the anomeric proton (δ 4.85, J1,2 = 7.9
Hz). The other coupling constants of the uronic acid ranged
between 7.6 and 9.9 Hz and thus indicated the relative
configuration of glucuronic acid. CZE after hydrolysis and
derivatization confirmed the presence of D-glucuronic acid. The
hexose moiety was identified as glucose by its coupling
constants, and CZE after hydrolysis and derivatization
confirmed the presence of D-glucose. HMBC data showed a
cross-peak between H-4″ of the glucuronic acid and C-1‴ of
the glucose part, thus indicating a 1‴→4″ linkage, which was
confirmed additionally by a NOESY experiment. On the basis
of these data, the structure of 12 was established as the new
hypolaetin-8-O-β-D-glucopyranosyl-(1‴→4″)-β-D-glucurono-
pyranoside (Figure 1). A similar compound from the leaves of
A. officinalis, hypolaetin-8-O-gentiobioside, has been de-
scribed.36
Compound 9 resembled 7 in TLC analysis and was isolated
from Sephadex LH-20 fraction K by MPLC on MCI stationary
phase and subsequent preparative HPLC on RP18. This
procedure yielded 3.6 mg of a yellow substance with UV
characteristics similar to those of 7. The UV spectrum with λmax
= 253, 271, and 347 nm indicated a flavonoid-type structure.
The HRESIMS data showed the [M + H]+ ion at m/z
573.0573, corresponding to C22H20O16S and fragmenting to
m/z 493.0999 [M + H − SO3]+, 317.0668 [M + H − SO3 −
hexuronic acid]+, and 302.0417 [M + H − SO3 − hexuronic
acid − CH3]+. 1H NMR data showed an AMX spin system of a
dihydroxylated flavonoid B-ring (δ 7.38, d, H-6′; 7.28, s, H-2′;
6.93, d, H-5′). A three-proton singlet (δ 3.91, s, −OCH3) could
be assigned to a 4′-O-methyl group via HMBC data. All other
analytical data obtained for the aglycone part of 9 by NMR and
hydrolysis experiments in connection with HPLC were
identical to those described for 7, indicating the aglycone to
Compound 10 (2.5 mg) was isolated from the Sephadex LH-
20 fraction K by MPLC on MCI stationary phase and
subsequent preparative HPLC on RP18. TLC analysis showed
higher Rf values when compared with the aforementioned
hypolaetin glycosides, indicating a less hydrophilic compound.
The UV spectrum with λmax = 271 and 328 nm indicated a
flavonoid structure. HRESIMS showed the [M + H]+ ion at
m/z 557.1395, corresponding to C22H21O15S and fragmenting
to m/z 477.1046 [M + H − SO3]+, 301.0727 [M + H − SO3 −
hexuronic acid]+, and 286.0462 [M + H − SO3 − hexuronic
1
be a 4′-O-methylhypolaetin unit. H NMR signals at δ 5.01−
1
3.90 were assigned to the protons of a uronic acid group. The
β-orientation of the anomeric group and its pyranose form were
deduced from the coupling constants (J1,2 = 8.0 Hz) of the
acid − CH3]+. H NMR data showed two proton signals at δ
7.92, d (H-2′ and H-6′) and 6.99, d (H-3′ and H-5′). Using
HMBC data, a three-proton singlet (δ 3.90, s, −OCH3) could
D
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be assigned to the 4′-O-methyl group of a monohydroxylated
B-ring. By comparing the NMR data with published data,37 the
aglycone of 10 was established as 4′-O-methylisoscutellarein.
Additionally, the 5,7,8-trihydroxylation of the A-ring was again
shown after acid-catalyzed hydrolysis of 10, followed by
UHPLC, which resulted in the detection of two distinct
Influence of RE and Sephadex LH-20 Fractions on
Human Hyaluronidase-1 Enzyme Activity and Gene
Expression. RE was tested on potential inhibition of HYAL-1
as described recently.18 E. coli F470 cells with the pAK009
constitutive expression system for HYAL-1 were incubated for
5 min with variable concentrations of RE or its Sephadex LH-20
fractions; glycyrrhicinic acid (1 mM) served as reference
inhibitor and control. For quality control reasons E. coli F470
host cells without the HYAL-1 expression system were used in
parallel to exclude unspecific cell activating or inhibiting effects.
HYAL-1 activity was monitored by its degrading properties
against added hyaluronic acid over a period of 5 min.
Undegraded and thus still high molecular weight polysaccharide
was quantified by “stains-all reagent”, a carbocyanin dye
interacting with high molecular weight hyaluronic acid. This
interaction leads to a spectroscopic shift that can be monitored
specifically at λ = 650 nm. RE inhibited HYAL-1 activity with
an IC50 of 7.7 mg/mL (Figure 3). In a further experiment, the
1
peaks as expected for 7,8-dihydroxylated flavonoids. H NMR
signals between δ 4.99 and 3.90 were assigned to the protons of
the uronic acid group. The β-orientation of the anomeric group
and pyranose form were deduced from the coupling constant of
the anomeric proton doublet at δ 4.99 (J1,2 7.99 Hz). The 3-O-
sulfo group of the uronic acid was deduced from the typical
downfield shift of the 13C and 1H NMR signals when compared
with an underivatized uronic acid.27 Pyridine/DMSO-catalyzed
desulfonylation, hydrolysis, and derivatization of the O-
glycoside followed by CZE proved the presence of a D-
glucuronic acid moiety of 10. On the basis of these data, the
structure of 10 was defined as the new 4′-O-methylisoscutellar-
ein-8-O-β-D-(3″-O-sulfo)glucuronopyranoside (Figure 1).
Compound 11, which resembled 8 in TLC analysis, was
isolated from the Sephadex LH-20 fraction L by MPLC on
MCI stationary phase and subsequent preparative HPLC on
RP18. This procedure yielded 6.3 mg of a yellow substance
with UV, HRESIMS, and NMR characteristics similar to those
of 7. Differences were observed concerning the nature of the
carbohydrate moiety, which turned out to be a 3-O-
sulfoglucuronopyranoside, connected via a β-O-glucosidic
linkage to C-8 of hypolaetin. The structure of 8 was thus
d e fi n e d a s h y p o l a e t i n - 8 - O - β - D - ( 3 ″ - O - s u l f o ) -
glucuronopyranoside. This compound was described as
theograndin II from Theobroma grandiflorum; its analytical
data correspond well to those of 8.38
Coumarins. The Sephadex LH-20 fraction A was further
fractionated by MPLC on an RP18 stationary phase into eight
subfractions (A1−A8), from which A1−A6 contained mainly
carbohydrates and amino acids. Further fractionation of A7 by
preparative TLC yielded 6.8 mg of a yellow compound (13).
HRESIMS data showed the [M + H]+ ion at m/z 501.1550,
congruent with a molecular formula of C22H28O13. Fragments
at m/z 355.0974 and 193.0479 indicated the loss of one
deoxyhexose followed by the loss of one hexose moiety. The
aglycone was established as scopoletin by its UV spectrum and
by spiking experiments of the acid-catalyzed hydrolysate of 13
with authentic scopoletin using UHPLC. CZE of the
derivatized hydrolysate indicated the presence of D-glucose
and L-rhamnose as carbohydrate moieties of 13. 1H NMR data
confirmed the presence of scopoletin and showed further
signals indicative for a carbohydrate moiety, which could be
linked only to C-7 of the aglycone moiety. TOCSY data
permitted the assignment of two distinct spin systems to a
hexose moiety, which was recognized as a β-glucosyl unit by its
1H coupling constants and NOESY correlations, and a
deoxyhexose moiety with a J1,2 value of about 2 Hz. A 1→6
interglycosidic linkage was established by a 7 ppm downfield
shift of C-6 of the β-glucosyl unit relative to the respective
signal of scopolin (scopoletin-7-O-β-D-glucopyranoside).39 On
the basis of these data, the structure of 13 was defined as
scopoletin-7-O-α-L-rhamnopyranosyl-(1″→6′)-β-D-glucopyra-
noside (syn. scopolin-6′-O-α-L-rhamnopyranoside) (Figure 1).
Figure 3. IC50 value determination of aqueous extract RE from Althaea
officinalis for the determination of HYAL-1 inhibiting activity:
Inhibition of HYAL-1, surface-displayed on E. coli F470, was
determined after cell incubation with variable concentrations of RE
and subsequent whole cell stains-all assay. IC50 values were calculated
using GraphPadPrism5 software; IC50 values were determined by
interpolation (50% inhibition of the HYAL-1 activity of the plot (n =
3, error bars SD)).
Sephadex LH-20 fractions A, E−I, and K (Figure 2) were
investigated concerning potential inhibitory effects on HYAL-1
(due to limited amounts, not all fractions could be tested). No
activity was found for fractions H, I, and K, whereas fraction A
showed the highest enzyme inhibition (84%). Fractions E
(73%), F (73%), and G (83%) were found to also exert
remarkable anti-hHyal-1 activity (Figure 2).
To investigate if RE also influences the gene expression of
hyal-1 in epithelial cells, its expression was analyzed by
quantitative real-time PCR in HaCaT keratinocytes, a human
immortalized, nontumorigenic cell line, which represents skin
and mucosal epithelial cells.13,41 In a preliminary experiment,
HaCaT keratinocytes showed expression of mRNA of this
protein. Treatment of the cells with 125 and 250 μg/mL of RE
for 24 h reduced the formation of this mRNA in comparison
with untreated cells, as shown by the analysis of hyal-1
expression via qPCR (Figure 4).
These results permit the conclusion that on one hand RE
inhibits the enzymatic activity of HYAL-1 (and therefore
probably its destructive action on the extracellular matrix) and
on the other hand it changes the cell physiology of
This compound has been reported as haploperoside A with 13
C
NMR data matching those acquired from HSQC and HMBC
experiments of 13.40
E
DOI: 10.1021/acs.jnatprod.6b00670
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Journal of Natural Products
Article
Capillary Zone Electrophoresis after Desulfonylation.34,35
Desulfonylation: 1 mg of the flavonoid-(O-sulfo)glycoside was
dissolved in a solution of pyridine in H2O (0.1 mol/L, pH 4.8) and
lyophilized. The remaining residue was dissolved in 0.5 mL of DMSO
and heated for 3 h at 80 °C. DMSO was removed by lyophilization.
Hydrolysis of the desulfonylated flavonoid-O-glycosides was per-
formed by dissolving the remaining residue in 0.5 mL of TFA (23%),
heating for 1 h at 120 °C, and subsequent lyophilization. The
carbohydrates for CZE electrophoresis were derivatized as described
by Noe and Freissmuth.35 CZE: Beckman Coulter P/ACE MDQ
(Beckman Coulter, Brea, USA); fused silica capillary, 70/77 cm × 50
μm i.d.; running buffer, 50 mM Na2B2O7 pH 10.3, MeCN 4.4 mol/L
added; injection, 5−10 s at 0.5 psi; voltage, 30 kV; detection, λ = 200
nm; software, 32 Karat software version 5.0 (Beckman Coulter).
LC-MS Quantification of Glycine Betaine. UHPLC: Dionex
Ultimate 3000 RS (solvent rack SRD-3600, pump DGP-3600,
autosampler WPS 3000 TSL, oven TCC-3000 RS, detector DAD-
3000 RS) (Dionex, Sunnyvale, USA) connected to a Bruker Daltonics
micrOTOF-QII time-of-flight MS, Bruker Apollo ESI source (Bruker,
Billerica, MA, USA). Stationary phase: Monochrom MS, 5 μm, 100 ×
2 mm (Varian, Darmstadt, Germany); mobile phase, (A) NH4OAc 5
mM in H2O, containing 0.1% HOAc; (B) MeCN; t0 min 7% A, t7.0 min
7% A, t7.5 min 100% A, t12.5 min 100% A, t13 min 7% A, t19 min 7% A; flow
rate, 0.4 mL/min; injection volume, 2 μL; oven temperature, 40 °C.
Detection MS: ionization mode positive polarity, 2 Hz, mass range
50−1000 m/z; nebulizer gas, N2 3.0 bar; dry gas, N2 5.0 L/min, 200
°C; voltage, 4500 V; end plate offset, −500 V; ion energy, 50 eV;
isolation mass, 118 m/z; collision gas, N2; collision energy, 10 eV;
collision RF, 140 Vpp; transfer time, 70 μs; PrePulseStorage, 5 μs;
internal calibration, 10 mM sodium formate in 2-propanol−water, 1:1
(v/v).
Figure 4. Influence of aqueous extract RE (125 and 250 μg/mL) from
Althaea officinalis roots on the expression of hyal-1 in HaCaT
keratinocytes after 24 h of incubation. UC: untreated control; bars
represent the mean outcome of two experiments.
keratinocytes toward a reduced expression of hyal-1 mRNA
expression.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Unless stated otherwise,
solvents, reagents, and consumables were obtained from VWR
International (Darmstadt, Germany). All solvents and reagents were
of analytical quality. Water was produced by a Millipore Simplicity 185
system (Schwalbach, Germany). Dried root material of A. officinalis,
batch no WE101781, was obtained from Alpinamed AG (Freidorf,
Switzerland). The material was identified by coauthor A.H. A voucher
specimen of the material is retained in the archives of the Institute of
Hypolaetin-8-O-β-D-(2″-O-sulfo)glucopyranoside (7): yellow,
amorphous solid; UV (MeCN, H2O) λmax 256, 271, 353; ECD
(H2O, λ [nm] (Δϵ), c 0.079) 232 (36.8), 350 (−44.7), 350 (10.4); 1H
NMR (600 MHz, methanol-d4) δ 7.65 (d, J = 2.3 Hz, 1H, H-2′), 7.33
(dd, J = 2.3, 8.5 Hz, 1H, H-6′), 6.73 (d, J = 8.6 Hz, 1H, H-5′), 6.42 (s,
1H, H-3), 6.08 (s, 1H, H-6), 4.82 (d, J = 7.6 Hz, 1H, H-1″), 4.34 (dd, J
= 7.6, 8.9 Hz, 1H, H-2″), 3.54 (dd, J = 2.3, 12.0 Hz, 1H, H-6″a), 3.52
(t, J = 9.0 Hz, 1H, H-3″), 3.49 (dd, J = 4.4, 11.8 Hz, 1H, H-6″b), 3.44
(dd, J = 9.0, 9.8 Hz, 1H, H-4″), 3.07 (ddd, J = 2.5, 4.4, 9.8 Hz, 1H, H-
5″); 13C NMR (150 MHz, methanol-d4) δ 182.48 (C-4), 164.99 (C-
2), 156.71 (C-5), 157.68 (C-7), 149.61 (C-4′), 149.55 (C-9), 145.42
(C-3′), 123.86 (C-8), 121.93 (C-1′), 119.48 (C-6′), 115.64 (C-5′),
113.29 (C-2′), 103.98 (C-10), 102.06 (C-3), 102.06 (C-1″), 98.90 (C-
6), 80.04 (C-2″), 76.35 (C-5″), 75.70 (C-3″), 69.44 (C-4″), 60.58 (C-
6″); ESITOFMS m/z 545.0599 (calcd for C21H21O15S, 545.0596).
4′-O-Methylhypolaetin-8-O-β-D-(2″-O-sulfo)glucopyranoside (8):
yellow, amorphous solid; UV (MeCN, H2O) λmax 273, 330; ECD
(H2O, λ [nm] (Δϵ), c 0.102) 217 (6.4), 232 (−2.2), 246 (4.0), 276
Pharmaceutical Biology and Phytochemistry, Munster, Germany,
̈
under the designation IPBP 286. ECD spectra were recorded using
a Jasco-J-815 spectrometer (Jasco, Groß-Umstadt, Germany).
Extract RE Preparation. Pulverized roots (1 kg) of A. officinalis
were extracted with 10.0 L of a MeOH−H2O (1:1 v/v) mixture under
ultrasonic treatment at 40 °C for 15 min. The suspension was
centrifuged (18000g at 18 °C for 14 min). The resulting pellet was
again extracted with 5 L of a MeOH−H2O (1:1 v/v) mixture under
the same conditions as described above. The combined extracts were
concentrated at a maximum of 40 °C to 2 L using a rotary evaporator.
High molecular weight compounds were precipitated by adding the
extract dropwise into a 4-fold volume of ice cold EtOH until a final
EtOH concentration of 65−70% was reached. The suspension was
kept at 8 °C for 24 h, followed by centrifugation (33000g, 10 min, 12
°C). The EtOH was removed from the clear supernatant by rotary
evaporation, and the resulting aqueous solution was lyophilized to
obtain 82.3 g of the raw extract RE, corresponding to a yield of 8.1%
related to the dried plant material.
Fractionation on Sephadex LH-20. RE (32 g) was fractionated
on Sephadex LH-20 (General Electrics, Munich, Germany) using a
column size of 900 × 95 mm i.d. and a step gradient of EtOH (8.8 L),
MeOH (3.5 L), and acetone−H2O (4.0 L, 7:3 v/v), fraction size, 10
mL. Every second fraction was monitored by TLC, and fractions with
comparable composition were combined to afford fractions A−N.
MPLC. Flash chromatography was performed on a Spot Liquid
Chromatography Flash (Armen Industries, Saint-Ave, France) on a
SVP D40 RP18 stationary phase (40−63 μm particle size, 84 g of
stationary phase) (Merck Chimie SAS, Bois, France) at a flow rate of
10 mL/min and a mobile phase gradient elution using H2O and
MeOH. MPLC on MCI gel: stationary phase, MCI CHP-20P, 75−150
μm, 450 Å (Mitsubishi Kasei, Tokyo, Japan); column, 500 × 25 mm
i.d.; mobile phase, binary gradient of H2O and MeOH; flow rate, 6
mL/min.
1
(−3.8), 350 (1.7); H NMR (600 MHz, D2O) δ 8.11 (d, J = 8.3 Hz,
1H, H-6′), 7.86 (s, 1H, H-2′), 7.26 (d, J = 8.6 Hz, 1H, H-5′), 6.62 (s,
1H, H-3), 6.38 (s, 1H, H-6), 5.19 (d, J = 7.8 Hz, 1H, H-1″), 4.55 (t, J
= 8.5 Hz, 1H, H-2″), 3.98 (s, 3H, -OCH3), 3.90 (dd, J = 8.8, 8.2 Hz,
1H, H-3″), 3.82 (dd, J = 2.1, 12.5 Hz, 1H, H-6″a), 3.74 (dd, J = 5.1,
12.9 Hz, 1H, H-6″b), 3.71 (t, J = 9.1 Hz, 1H, H-4″), 3.49 (m, 1H, H-
5″); 13C NMR (150 MHz, D2O) δ 182.55 (C-4), 163.62 (C-2), 156.27
(C-5 and C-7), 154.84 (C-4′), 149.48 (C-9), 139.82 (C-3′), 126.43
(C-6′), 123.84 (C-8), 122.55 (C-1′), 121.07 (C-2′), 113.35 (C-5′),
104.37 (C-10), 103.58 (C-3), 101.9 (C-1″), 99.39 (C-6), 80.27 (C-
2″), 76.08 (C-5″), 70.97 (C-3″), 69.15 (C-4″), 60.41 (C-6″), 51.26
(−OCH3); ESITOFMS m/z 559.0765 (calcd for C22H23O15S,
559.0768).
4′-O-Methylhypolaetin-8-O-β-D-(3″-O-sulfo)glucurono-
pyranoside (9): yellow, amorphous solid; UV (MeCN, H2O) λmax 253,
271, 347; ECD (H2O, λ [nm] (Δϵ), c 0.036) 212 (25.3), 240 (23.7),
Preparative HPLC. Two Waters 515 HPLC pumps, pump control
module II (Waters, Milford, USA), Degasys DG-2410 (Optilab,
Munich, Germany); stationary phase, Eurospher 100-C8 5 μm, 250 ×
8.00 mm i.d. (Knauer, Berlin, Germany); mobile phase, binary gradient
of H2O and MeOH; injection volume, 1 mL; flow rate, 5.5 mL/min;
detection, UV 280 nm.
1
261 (−45.3), 284 (−6.8), 330 (14.9); H NMR (600 MHz, D2O) δ
7.38 (d, J = 8.7 Hz, 1H, H-6′), 7.28 (s, 1H, H-2′), 6.93 (d, J = 8.7 Hz,
1H, H-5′), 6.39 (s, 1H, H-3), 6.24 (s, 1H, H-6), 5.02 (d, J = 8.0 Hz,
1H, H-1″), 4.52 (t, J = 9.1 Hz, 1H, H-3″), 4.10 (d, J = 9.7 Hz, 1H, H-
5″), 3.94 (t, J = 9.0 Hz, 2H, H-2″ and H-4″), 3.91 (s, 3H, −OCH3);
F
DOI: 10.1021/acs.jnatprod.6b00670
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
13C NMR (150 MHz, D2O) δ 181.6 (C-4), 164.26 (C-2), 156.2 (C-5
Autodisplay of HYAL-1 and Inhibitor Testing. This was
performed as described by Orlando et al.18
and C-7), 150.59 (C-4′), 145.01 (C-3′), 122.61 (C-1′), 125.9 (C-8),
120.06 (C-6′), 112.16 (C-2′), 111.58 (C-5′), 106.07 (C-1″), 102.81
(C-3 and C-10), 100.3 (C-6), 83.36 (C-3″), 77.49 (C-5″), 72.30 (C-
2″), 70.57 (C-4″), 55.46 (−OCH3), not detected (C-9 and C-6″);
ESITOFMS m/z 573.0573 (calcd for C22H21O16S, 573.0545).
4′-O-Methylisoscutellarein-8-O-β-D -(3″-O-sulfo)-
glucuronopyranoside (10). yellow, amorphous solid; UV (MeCN,
H2O) λmax 271, 323; ECD (H2O, λ [nm] (Δϵ), c 0.025) 241 (4.9), 268
(−14.5), 304 (7.3); 1H NMR (600 MHz, D2O) δ 7.93 (d, J = 7.1 Hz,
2H, H-2′ and H-6′), 7.00 (d, J = 8.4 Hz, 2H, H-3′ and H-5′), 6.55 (s,
1H, H-3), 6.29 (s, 1H, H-6), 4.99 (d, J = 7.8 Hz, 1H, H-1″), 4.46 (t, J
= 8.5 Hz, 1H, H-3″), 3.96 (t, J = 9.0 Hz, 1H, H-2″), 3.94 (m, 2H, H-4″
and H-5″), 3.90 (s, 3H, -OCH3); 13C NMR (150 MHz, D2O) δ 164.35
(C-2), 162.01 (C-4′), 156.5 (C-5 and C-7), 128.40 (C-2′ and C-6′),
125.5 (C-8), 122.52 (C-1′), 114.31 (C-3′ and C-5′), 105.1 (C-1″),
104.0 (C-10), 102.63 (C-3), 99.8 (C-6), 83.31 (C-3″), 77.4 (C-5″),
72.06 (C-2″), 70.59 (C-4″), 55.43 (−OCH3), not detected (C-4, C-6″,
and C-9); ESITOFMS m/z 557.0575 (calcd for C22H21O15S,
557.0596).
Isolation, Quantitation, and Reverse Transcription of Total
RNA. HaCaT cells were seeded in six-well plates at a density of 7.5 ×
105 cells per well. After incubation with test solutions for specific time
intervals, total cellular RNA was isolated using the RNeasy Mini Kit
(Qiagen) according to the manufacturer’s instructions. RNA quality
and concentration were determined using the μCuvette G1.0 and the
BioPhotometer Plus (both Eppendorf). Transcription into cDNA was
achieved by the use of the QuantiTect reverse transcription kit
(Qiagen) according to the manufacturer’s instructions.
Quantitative Real-Time PCR. Quantitative real-time PCR
(qPCR) was performed using TaqMan gene expression assays
(Applied Biosystems) containing gene-specific primers and probe
(PPIA assay ID Hs04194521_s1; 18S assay ID Hs99999901_s1; hyal-
1 assay ID Hs04185319_m1), the SensiMix II Probe Lo-ROX kit
(Bioline), and the CFX96 Touch Real-Time PCR detection system
(Bio-Rad) according to the manufacturer’s instructions. PPIA and 18S
served as control genes. Data were evaluated by means of the CFX
Manager 3.0 software from Bio-Rad.
Hypolaetin-8-O-β-D-(3″-O-sulfo)glucuronopyranoside (theogran-
din II) (11): yellow, amorphous solid; UV (MeCN, H2O) λmax 256,
270, 352; ECD (H2O, λ [nm] (Δϵ), c 0.063) 200 (−46.7), 228 (28.3),
259 (−45.8), 279 (−2.8), 323 (9.7), 377 (10.5); 1H NMR (400 MHz,
D2O) δ 7.28 (d, J = 8.0 Hz, 1H, H-6′), 7.21 (d, J = 1.1 Hz, 1H, H-2′),
6.74 (d, J = 8.3 Hz, 1H, H-5′), 6.29 (s, 1H, H-3), 6.23 (s, 1H, H-6),
4.99 (d, J = 7.8 Hz, 1H, H-1″), 4.49 (t, J = 9.2 Hz, 1H, H-3″), 4.07 (d,
J = 9.7 Hz, 1H, H-5″), 3.92 (dd, J = 8.1, 9.3 Hz, 1H, H-2″), 3.90 (dd, J
= 8.1, 9.3 Hz, 1H, H-4″); 13C NMR (100 MHz, D2O) δ 181.94 (C-4),
175.26 (C-6″), 164.95 (C-2), 156.53 (C-5), 156.40 (C-7), 149.26 (C-
9), 148.31 (C-4′), 144.35 (C-3′), 125.32 (C-8), 121.98 (C-1′), 120.38
(C-6′), 115.84 (C-5′), 113.07 (C-2′), 105.53 (C-1″), 103.86 (C-10),
102.72 (C-3), 99.53 (C-6), 83.30 (C-3″), 77.68 (C-5″), 72.53 (C-2″),
70.67 (C-4″); ESITOFMS m/z 559.0412 (calcd for C21H19O16S,
559.0388).
Hypolaetin-8-O-β-D-glucopyranosyl-(1‴→4″)-β-D-glucuronopyr-
anoside (12): yellow, amorphous solid; UV (MeCN, H2O) λmax 256,
270, 350; ECD (H2O, λ [nm] (Δϵ), c 0.044) 214 (30.5), 226 (8.7),
240 (27.5), 266 (−48.5), 286 (−2.4), 335 (14.6); 1H NMR (600
MHz, D2O) δ 7.24 (s, 1H, H-2′), 7.19 (d, J = 7.6 Hz, 1H, H-6′), 6.77
(d, J = 8.5 Hz, 1H, H-5′), 6.25 (s, 1H, H-3), 6.09 (s, 1H, H-6), 4.85 (d,
J = 7.9 Hz, 1H, H-1″), 4.60 (d, J = 7.9 Hz, 1H, H-1‴), 4.16 (d, J = 9.9
Hz, 1H, H-5″), 3.98 (dd, J = 2.2, 12.5 Hz, 1H, H-6‴a), 3.95 (t, J = 9.1
Hz, 1H, H-4″), 3.80 (dd, J = 5.6, 12.5 Hz, 1H, H-6‴b), 3.78 (m, 2H,
H-2″ and H-3″), 3.58 (t, J = 9.2 Hz, 1H, H-3‴), 3.55 (m, 1H, H-5‴),
3.48 (t, J = 9.2 Hz, 1H, H-4‴), 3.40 (t, J = 8.7 Hz, 1H, H-2‴); 13C
NMR (150 MHz, D2O) δ 181.35 (C-4), 175.21 (C-6″), 163.92 (C-2),
156.25 (C-5 and C-7), 149.33 (C-9), 147.92 (C-4′), 144.17 (C-3′),
126.92 (C-8), 122.28 (C-1′), 119.9 (C-6′), 115.73 (C-5′), 112.81 (C-
2′), 106.73 (C-1″), 102.22 (C-6, C-10, and C-1‴), 102.11 (C-3), 79.97
(C-4″), 76.38 (C-5″), 75.95 (C-5‴), 75.39 (C-3‴), 74.07 (C-3″),
73.32 (C-2″), 73.14 (C-2‴), 69.37 (C-4‴), 60.53 (C-6‴); ESITOFMS
m/z 641.1393 (calcd for C27H29O18, 641.1348).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00670.
■
S
1H NMR spectra, 13C NMR spectra, and where necessary
the spectra of further NMR experiments for compounds
7, 8, 9, 10, and 12 (PDF)
AUTHOR INFORMATION
Corresponding Author
*Tel: +49-251-83-33379. Fax: +49-251-83-38341. E-mail:
sendkej@uni-muenster.de.
■
ORCID
Jandirk Sendker: 0000-0003-4798-699X
Thomas Hofmann: 0000-0003-4057-7165
Notes
The authors declare the following competing financial
interest(s): CF & HAA are employed by Steigerwald
Arzneimittelwerk GmbH. All other authors have declared no
competing conflicts of interest.
ACKNOWLEDGMENTS
■
The authors are grateful to Dr. J. Koehler and Dr. K. Bergander
for the measurement of NMR spectra and appreciate the help
of Mrs. B. Quandt for the CZE determination of carbohydrates.
REFERENCES
Scopolin-6′-O-α-L-rhamnopyranoside (haploperoside A) (13):
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■
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(−0.8); H NMR (600 MHz, D2O) δ 7.98 (d, J = 9.6 Hz, 1H, H-4),
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4″ and C-5″), 66.09 (C-6′), 56.45 (−OCH3), 16.56 (C-6″);
ESITOFMS m/z 501.1550 (calcd for C22H29O13, 501.1602).
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DOI: 10.1021/acs.jnatprod.6b00670
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