Polyphenols from Euphorbia pekinensis Inhibit AGEs Formation in Vitro and Vessel Dilation in Larval Zebrafish in Vivo

Article abstract of DOI:10.1055/s-0043-120447

To identify active compounds in the roots of Euphorbia pekinensis for treatment of diabetic complications, an active column fraction from a 70% EtOH extract of E. pekinensis root was purified by preparative reversed-phase high-performance liquid chromatography, leading to the isolation of a new ellagic acid derivative, 3,3′-di- O -methylellagic acid 4- O -(6- O -galloyl)- β -D-galactopyranoside (1), along with three known compounds, geraniin (2), 3,3′-di- O -methylellagic acid 4- O - β -D-xylopyranoside (3), and ellagic acid 3,3′-dimethyl ether (4). The structure of the new compound was established by extensive spectroscopic studies and chemical evidence. The inhibitory effects of isolated compounds 1 - 4 on advanced glycation end-products (AGEs) formation were examined. All compounds exhibited considerable inhibition of AGEs formation and IC ₅₀ values of 0.41 - 12.33 μM, compared with those of the positive controls aminoguanidine (IC ₅₀ = 1122.34 μM) and quercetin (IC ₅₀ = 27.80 μM). In addition, the effects of 2 and 4 on the dilation of hyaloid-retinal vessels induced by high glucose (HG) in larval zebrafish were investigated; both compounds significantly reduced the HG-induced dilation of hyaloid-retinal vessels relative to the HG-treated control group.

Full text of DOI:10.1055/s-0043-120447

Original Papers
Polyphenols from Euphorbia pekinensis Inhibit AGEs Formation
In Vitro and Vessel Dilation in Larval Zebrafish In Vivo
Authors
1
2
1
Ik-Soo Lee , Seung-Hyun Jung , Jin Sook Kim
Affiliations
ABSTRACT
1
2
KM-Based Herbal Drug Research Group, Herbal Medicine
Research Division, Korea Institute of Oriental Medicine,
Daejeon, Republic of Korea
To identify active compounds in the roots of Euphorbia peki-
nensis for treatment of diabetic complications, an active col-
umn fraction from a 70% EtOH extract of E. pekinensis root
was purified by preparative reversed-phase high-performance
liquid chromatography, leading to the isolation of a new ella-
gic acid derivative, 3,3′-di-O-methylellagic acid 4-O-(6′′-O-
galloyl)-β-D-galactopyranoside (1), along with three known
compounds, geraniin (2), 3,3′-di-O-methylellagic acid 4-O-β-
D-xylopyranoside (3), and ellagic acid 3,3′-dimethyl ether (4).
The structure of the new compound was established by exten-
sive spectroscopic studies and chemical evidence. The inhib-
itory effects of isolated compounds 1–4 on advanced glyca-
tion end-products (AGEs) formation were examined. All com-
pounds exhibited considerable inhibition of AGEs formation
and IC50 values of 0.41–12.33 µM, compared with those of
the positive controls aminoguanidine (IC50 = 1122.34 µM)
and quercetin (IC50 = 27.80 µM). In addition, the effects of 2
and 4 on the dilation of hyaloid-retinal vessels induced by high
glucose (HG) in larval zebrafish were investigated; both com-
pounds significantly reduced the HG-induced dilation of hya-
loid-retinal vessels relative to the HG-treated control group.
Division of Marine-Bio Research, National Marine Biodiver-
sity Institute of Korea, Seocheon-gun, Republic of Korea
Key words
Euphorbia pekinensis, Euphorbiaceae, polyphenols, diabetic
complications, advanced glycation end‑products, vessel dila-
tion
received July 25, 2017
revised
September 11, 2017
accepted September 21, 2017
Bibliography
DOI https://doi.org/10.1055/s-0043-120447
Published online November 17, 2017 | Planta Med 2018; 84:
1
76–181 © Georg Thieme Verlag KG Stuttgart · New York |
ISSN 0032‑0943
Correspondence
Dr. Jin Sook Kim
KM Convergence Research Division, Korea Institute of Oriental
Medicine
Daejeon, 34054, Republic of Korea
Phone: + 82428689465, Fax: + 82428689471
jskim@kiom.re.kr
noids, diterpenes and triterpenes, have been isolated from E. peki-
nensis, and their pharmacological activities have been verified in
vitro [6–13].
Introduction
Euphorbia pekinensis Rupr. (Euphorbiaceae), a species in the genus
Euphorbia, which comprises approximately 2000 species, is a pe-
rennial herb that is distributed widely, particularly in tropical and
subtropical regions of southwestern and eastern America and in
Africa, Asia, and certain parts of Europe. It is considered one of
the 50 most important medicinal herbs used in traditional Chi-
nese medicine [1]. The root of this plant has been used in China
for thousands of years for the treatment of edema, fullness of
the chest, viscous sputum, epilepsy, dyspnea, ascites, and tuber-
cular disease [2] and has been shown to possess various pharma-
cological activities, including those attributable to cytotoxic, anti-
viral, and anti-inflammatory properties [3–5]. Numerous bio-
active substances, such as phenolics, hydrolysable tannins, flavo-
In our ongoing efforts to identify effective, naturally sourced
therapeutic agents for the treatment of diabetic complications,
we found a 70% EtOH extract of E. pekinensis root exhibited con-
siderable inhibition of advanced glycation end-products (AGEs)
formation. This prompted us to investigate active compounds in
the roots of E. pekinensis. This report describes the isolation and
structural elucidation of active compounds (1–4) from the roots
of E. pekinensis, as well as their inhibitory effects on AGEs forma-
tion. We further investigated the effects of two isolates (2 and 4)
on the high glucose (HG)-induced dilation of hyaloid-retinal ves-
sels in larval zebrafish.
Lee IS et al. Polyphenols from Euphorbia… Planta Med 2018; 84: 176–181
1
76

▶
Fig. 1 Gel chromatography of a 70% EtOH extract of E. pekinensis root, performed with a Diaion HP-20 column (A), and AGEs formation inhibitory
activity of column fractions A–D (B).
carbonyl carbons due to two α,β-unsaturated lactones (δC 158.4
and 158.3) and an ester carbonyl group (δC 165.7), 18 aromatic
carbons, two methoxy carbons (δC 61.6 and 61.0), and six car-
bons for a 6-deoxysugar. The six carbon signals at δC 101.2,
76.1, 73.9, 73.3, 69.6, and 63.1 and an anomeric proton signal
at δH 5.28 of 1 were typical of a galactose unit, which was identi-
fied as D-galactose by GC analysis of the acid hydrolysate. More-
over, the large coupling constant (J = 6.8 Hz) of the anomeric pro-
ton indicated that the galactose unit was linked in a β-configura-
tion [16]. The complete assignment of the chemical shifts of 1
and its substitution pattern were made by various 2D NMR tech-
niques, such as H- H COSY, HSQC, HMBC, and NOESY. The loca-
tion of two methoxy groups at C-3 and C-3′ on the ellagic acid
structure was deduced from the HMBC correlations of δH 4.05
with C-3 (δC 141.7) and δH 4.06 with C-3′ (δC 140.2; ▶ Fig. 3).
The HMBC correlation between the anomeric proton at δH 5.28
(H-1′′) and C-4 (δC 151.2) clearly indicated that the galactose unit
was linked to C-4 of the di-O-methylellagic acid structure. This
linkage was further confirmed by the observation of an NOE cor-
relation between the anomeric proton (δH 5.28) and H-5 (δH
7.85; ▶ Fig. 3). A galloyl group was determined to be adjacent to
the 6-position of the galactose moiety by the downfield-shifted H-
6′′ protons (δH 4.45 and 4.27). HMBC correlations from the pro-
ton H-6′′ of the galactose moiety to C-7′′′ (δC 165.7, carbonyl
group) and C-1′′ (δC 119.2) were in accordance with this conclu-
sion. Hence, the structure of 1 was established as 3,3′-di-O-meth-
ylellagic acid 4-O-(6′′-O-galloyl)-β-D-galactopyranoside.
Results and Discussion
The 70% EtOH extract of E. pekinensis root that significantly inhib-
ited AGEs formation [IC50 = 3.00 µg/mL] was subjected to Diaion
HP-20 column chromatography and divided into four fractions
(
A–D) based on TLC results (▶ Fig. 1A). The AGEs formation inhib-
itory activity of these fractions was evaluated; fraction C exhibited
the strongest activity, with an IC50 value of 0.44 µg/mL
(
▶ Fig. 1B). HPLC analysis of this fraction at 254 nm produced a
chromatogram with one small peak (I) and three large peaks (II–
IV; ▶ Fig. 2A). Fraction C was further separated by preparative re-
versed-phase HPLC, leading to the isolation of four compounds
1
1
(
1–4). Based on the comparison of physicochemical and spectral
data on these compounds with those in the literature, three
known compounds were identified: geraniin (2), 3,3′-di-O-meth-
ylellagic acid 4-O-β-D-xylopyranoside (3), and ellagic acid 3,3′-di-
methyl ether (4; ▶ Fig. 2B).
Compound 1 was obtained as an amorphous white powder
with the molecular formula C29H24O17, as established by
HRESIMS, based a molecular ion peak at m/z 667.0902 [M+Na]+.
Characteristic UV absorption maxima (at 250, 280, and 355 nm)
suggested the presence of an ellagic acid moiety [14, 15]. 1H-
and 13C‑NMR data suggested that compound 1 was di-O-methyl-
ellagic acid monoglycoside with a galloyl group (▶ Table 1). The
1H‑NMR spectrum exhibited characteristic peaks for two aromatic
proton singlets [δH 7.85 and 7.54 (each 1H, s)] of an ellagic acid
moiety, a galloyl group [δH 6.86 (2H, s)], two methoxy groups [δH
4
.05 and 4.06 (each 3H, s)], and sugar proton signals. The
To assess the effects of the isolated compounds (1–4) on dia-
betic complications, the inhibitory effects of these compounds on
AGEs formation were examined in vitro using commercially avail-
13C‑NMR data, combined with DEPT (distortionless enhancement
of polarization transfer) data, exhibited 27 carbon signals, three
Lee IS et al. Polyphenols from Euphorbia… Planta Med 2018; 84: 176–181
1
77

Original Papers
▶
Fig. 2 HPLC profile (at 254 nm) of the column fraction C from an extract of E. pekinensis root (A) and chemical structures of isolated compounds
(
1–4) from column fraction C (B).
able aminoguanidine, the first AGEs inhibitor used for the treat-
ment of diabetic nephropathy [17], and quercetin, a natural com-
pound, as positive controls. As shown in ▶ Table 2, all tested com-
pounds exhibited considerable inhibition of AGEs formation, with
IC50 values ranging from 0.41 to 12.33 µM, compared with those
ner compared with the HG-treated control group (▶ Fig. 4). At the
highest concentration used (20 µM), these compounds reduced
the diameters of hyaloid-retinal vessels with HG-induced dilation
by about 82% and 84%, respectively, relative to the HG-treated
control group. Treatment with a vascular endothelial growth fac-
tor receptor (VEGFR) tyrosine kinase inhibitor, VEGFR-2/Flt3/c-Kit
inhibitor, at a concentration of 1 µM, used as a positive control, re-
duced HG-induced dilation of hyaloid-retinal vessels by 77%.
Chronic hyperglycemia plays a crucial role in the initiation of
diabetic complications through various hyperglycemia-induced
metabolic and hemodynamic derangements [19–21]. Of various
metabolic derangements implicated in the pathogenesis of dia-
betic complications, the accumulation of AGEs, heterogeneous
molecules derived from non-enzymatic glycation between amino
acid residues and oxidative derivatives of glucose or pentose, are
among the most important. AGEs induce irreversible structural
and functional changes in proteins, such as collagen, elastin, and
albumin. These changes are believed to contribute, in part, to the
development and progression of diabetic complications [22]. Due
to these potential roles of AGEs in diabetic complications, the de-
velopment and investigation of AGE inhibitors, particularly natural
anti-AGE agents without side effects, may provide insight leading
to the development of a therapeutic approach to delay or prevent
diabetic complications [23]. Several studies have been conducted
to identify medical herbs and dietary plants that inhibit protein
glycation. These plants are rich in flavonoids or polyphenolic sub-
stances, which are reported to reduce AGEs formation [24–26]. In
the current study, which sought to identify effective agents for di-
abetic complications derived from E. pekinensis root, several poly-
of aminoguanidine (IC50 = 1122.34 µM) and quercetin (IC50
=
2
7.80 µM). Of the compounds tested, geraniin (2), a hydrolyzable
tannin, was the strongest inhibitor of AGE formation, with an IC50
value of 0.41 µM. Its activity was 20 times more effective than
that of the other isolates (1, 3, and 4), which contained ellagic ac-
id moieties, suggesting that garaniin contributes to a major por-
tion of the AGEs formation inhibitory effect of the active column
fraction A3 from an extract of E. pekinensis root.
Retinal vessel dilatation is a clinically and pathologically noted
retinal change associated with diabetic retinopathy [18]. Thus,
compounds 2 and 4, which potently inhibited AGEs formation in
vitro, were examined to determine their potential effects on reti-
nopathy in vivo in a diabetic zebrafish model. In this study, the
change in hyaloid-retinal vessel dilation was assessed in transgen-
ic zebrafish embryos expressing enhanced green fluorescent pro-
tein (EGFP) in the vasculature under HG conditions. The toxic ef-
fects on zebrafish were examined initially, and the results indi-
cated that compounds 2 and 4 were not toxic at concentrations
up to 20 µM (data not shown). Thus, to evaluate the effects of 2
and 4 on the HG-induced dilation of hyaloid-retinal vessels,
flk:EGFP transgenic zebrafish embryos were treated with these
compounds at concentrations of 5, 10, and 20 µM. Both com-
pounds significantly reduced the diameters of hyaloid-retinal ves-
sels with HG-induced dilation in a concentration-dependent man-
Lee IS et al. Polyphenols from Euphorbia… Planta Med 2018; 84: 176–181
1
78

▶
Table 1 1H- (400 MHz) and 13C‑NMR (100 MHz) and HMBC data
for 1 (in DMSO-d6).
▶ Table 2 Inhibitory effects of compounds 1−4 against AGEs for-
mation.
C
1
2
3
4
5
δc
δH (J in Hz)
HMBC (H → C)
Compounds
IC50 (95% CI) µMa
114.4
141.0
141.7
151.2
112.1
1
10.23 (9.64–10.82)
0.41 (0.40–0.42)
2
3
12.33 (12.06–12.60)
8.94 (7.98–9.90)
4
7.85 s
112.8, 114.4, 141.0,
Aminoguanidineb
1122.34 (1112.45–1132.21)
27.80 (26.52–29.08)
1
41.7, 151.2, 158.3
Quercetinb
6
7
112.8
158.3
111.1
141.9
140.2
152.9
111.6
a
Results are expressed as IC50 values and 95% confidence intervals
95% CI) and IC50 indicates the concentration (µM) at which the inhibi-
(
1
2
3
4
5
′
tion percentage of the AGEs formation was 50%. The values were de-
termined by regression analysis. b Positive controls.
′
′
′
′
phenols were isolated from an active column fraction of an extract
of this root. Although E. pekinensis is classified as a toxic herb in
Chinese medicine, reports suggest that this toxicity is associated
with the presence of many diterpenoids in this plant [10–13].
Thus, our results suggest that active polyphenols or a polyphe-
nol-rich column fraction of an extract of E. pekinensis root would
have beneficial uses in the development of therapeutic and/or
preventive agents for diabetic vascular complications and other
related diseases.
7.54 s
111.1, 112.1, 140.2,
41.9, 152.9, 158.4
1
6
7
1
2
3
4
5
6
′
112.1
158.4
101.2
73.3
′
′′
′′
′′
′′
′′
′′
5.28 d (6.8)
73.3, 151.2
73.9, 101.2
69.6, 73.3
3.87
73.9
3.79
69.6
3.41
73.9, 76.1
76.1
3.40
63.1, 69.6
63.1
4.27 dd (5.2, 12.0),
76.1, 119.2, 165.7
Materials and Methods
4
.45 dd (2.1, 12.0)
1
2
′′′
′′′
119.2
108.6
General experimental procedures
6.86 s
119.2, 138.4, 145.4,
Optical rotations were measured using the JASCO P-2000 digital
polarimeter. UV spectrum was recorded using the JASCO V-550
UV/VIS spectrometer. 1H‑NMR (400 MHz) and 13C‑NMR
1
65.7
3
4
5
6
′′′
′′′
′′′
′′′
145.4
138.4
145.4
108.6
(
100 MHz) spectra were obtained using the Bruker DRX-400 spec-
trometer with tetramethylsilane as an internal standard. 2D NMR
experiments (COSY, HSQC, HMBC, and NOESY) were performed
on the Bruker Avance 500 NMR spectrometer. HRESIMS was per-
formed using the Shimadzu LCMS‑IT‑TOF spectrometer. Column
chromatography was performed using Diaion HP-20 (Supelco).
TLC was performed on pre-coated silica gel 60 F254 (0.25 mm,
Merck) and RP-18 F254s plates (0.25 mm, Merck). Spots were de-
tected by UV light (254 nm) and spraying of 10% H SO followed
6.86 s
119.2, 138.4, 145.4,
65.7
1
7
3
3
′′′
165.7
61.6
61.0
-OMe
′-OMe
4.06 s
4.05 s
61.6
61.0
2
4
by heating. The HPLC analysis was performed on an Agilent 1200
HPLC system with a binary pump (G1312A), a vacuum degasser
(
G1322A), a thermostatted column compartment (G1316A), a
multiple wavelength detector (G1365B, MWD), and an auto-
sampler (G1329A), using a Luna C18 (250 × 10 mm, 5.0 µm,
Phenomenex) column.
Plant material
The roots of E. pekinensis were purchased from Namseongdang
herbal medicine store (Daegu, Republic of Korea). The origin of
the herbal medicine was identified by Prof. J. H. Kim, Gachon Uni-
versity, Republic of Korea, and a voucher specimen (KIOM-EUPE)
has been deposited in the Herbarium of Korea Institute of Oriental
Medicine, Republic of Korea.
1
1
▶
Fig. 3 Key H- H COSY, HMBC, and NOE correlations of com-
pound 1.
Lee IS et al. Polyphenols from Euphorbia… Planta Med 2018; 84: 176–181
1
79

Original Papers
Extraction, fractionation, and isolation
The air-dried roots of E. pekinensis (1 kg) were extracted using 70%
aqueous EtOH (three times, with 10 L each time) at room temper-
ature for 7 d, filtered, and concentrated to yield a 70% EtOH ex-
tract (216 g, 21.6% yield). The extract (20 g) was subjected to
Diaion HP-20 column chromatography (60 × 10 cm) and eluted
with a gradient solvent system consisting of (A) MeOH and (B)
water: 100% B (2 L), 80% B (2 L), 60% B (2 L), 40% B (2 L), 20% B
(
2 L), and 100% A (2 L). The resulting portions from column chro-
matographic separation were combined into four fractions (A,
.2 g; B, 3.3 g; C, 6.1 g; D, 1.8 g) based on TLC results. In this bio-
8
assay-guided study, the most active column fraction A3 was fur-
ther purified by extensive preparative reversed-phase HPLC
[
5
Agilent 1200 HPLC system; Luna C18 column (250 × 10 mm,
.0 µm), Phenomenex; gradient from 20–60% MeOH in water
over 50 min; UV detection, 254 nm; flow rate, 2.5 mL/min] to ob-
tain compounds 1 (tR: 29.78 min, 10 mg), 2 (tR: 31.42 min,
1
4
9
80 mg), 3 (tR: 36.36 min, 105 mg), and 4 (tR: 40.94 min,
2 mg). The purity of the isolated compounds ranged from
8.0% to 99.5%, as determined by analytical HPLC [Agilent 1200
HPLC system, Luna C18 column (250 × 4.6 mm, 5.0 µm), Pheno-
menex; 40% acetonitrile in water; UV detection, 254 nm; flow
rate, 1.0 mL/min].
3
,3′-Di-O-methylellagic acid 4-O-(6′′-O-galloyl)-β-D-galacto-
2
5
pyranoside (1): white powder; [α]
D
+ 26.0 (c 0.1, MeOH); UV
1
(
MeOH) λmax 250, 280, and 355 nm; H- and 13C‑NMR, see ▶ Ta-
ble 1; HRESIMS m/z 667.0902 [M+Na] (calcd for C29H24O17Na+,
+
6
67.0906).
Acid hydrolysis of compound 1
▶
Fig. 4 Effects of compounds 2 and 4 on dilation of hyaloid-retinal
vessels in a HG-induced diabetic retinopathy model. Hyaloid-retinal
vessels of flk:EGFP transgenic zebrafish from the following groups:
A untreated normal (NOR); B HG-treated control (HG); C HG-
treated specimens receiving 1 µM VEGFR-2/Flt3/c-Kit inhibitor
(VEGFR in.); HG-treated specimens receiving (D) 5 µM, (E) 10 µM,
and (F) 20 µM compound 2; and HG-treated specimens receiving
Compound 1 (2 mg) in 10% HCl/dioxane (1:1, 1 mL) was heated
separately at 80°C for 3 h in a water bath. The mixture was neu-
tralized with Ag CO , filtered, and then extracted with ethyl ace-
2
3
tate (20 mL). The aqueous layer was evaporated, and the residue
was treated with L-cysteine methyl ester hydrochloride (2 mg) in
pyridine (0.5 mL) at 60°C for 1 h. After the reaction was complete,
the solution was treated with acetic anhydride (3 mL) at 60°C for
(
G) 5 µM, (H) 10 µM, and (I) 20 µM compound 4. J Data are dis-
played as mean artificial units (AU) for vessel diameters. The diam-
eters of hyaloid-retinal vessels were measured at locations proximal
to the optic disc (red circle). Scale bar = 50 µm. The hyaloid vessel
diameter of each lens was measured three times and the experi-
ment was performed in triplicate. ### p < 0.001 vs. NOR, ** p <
1
h. Authentic sample was prepared using the same procedure.
The acetate derivatives were then subjected to GC analysis using
a GC-2010 device (Shimadzu) with a flame ionization detector
and TC-1 capillary column (0.25 mm × 30 m; GL Sciences, Inc.) us-
ing the following parameters: column temperature, 230°C; pro-
0
.01 vs. HG, *** p < 0.001 vs. HG.
grammed increase, 38°C/min; carrier gas, N (1 mL/min); and in-
2
jection and detector temperature, 270°C. The sugar derivative
thus obtained showed a retention time of 17.30 min, identical to
that of authentic D-galactose.
HT; excitation wavelength, 350 nm, emission wavelength,
450 nm). The AGEs assay was performed in triplicate. The IC50
was estimated from the least-squares regression line of the loga-
rithmic concentration plotted against remaining activity using the
GraphPad 5.0 Prism software.
Inhibition of AGEs formation assay
In accordance with a well-established method, a reaction mixture
of BSA (10 mg/mL, 700 µL; Sigma) in 50 mM phosphate buffer
Measurement of vessel dilation in larval zebrafish
(
pH 7.4) with 0.02% sodium azide (Sigma) was added to 0.2 M
All experimental protocols for animal care and use were approved
by the local ethics board (South Korean Institute of Oriental Med-
icine Animal Care and Use Committee) Committee (Approval date
and number; April 20, 2015 and 15–020, respectively), and ani-
mal husbandry and procedures were performed in accordance
with institutional guidelines. Adult zebrafish were maintained
under standard conditions at 28.5°C under a 14-h light/10-h dark
fructose and glucose (100 µL). In screw-cap tubes (1.5 mL), the re-
action mixture was then mixed with 200 µL of serially diluted com-
pounds or aminoguanidine (99% purity; Sigma) or quercetin
(
99.5% Purity; Sigma). After incubating at 37°C for 7 d, the fluo-
rescent reaction products (200 µL) were transferred to 96-well
plates and assayed on a spectrofluorometric detector (Synergy
Lee IS et al. Polyphenols from Euphorbia… Planta Med 2018; 84: 176–181
1
80

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day-old flk:EGFP embryos were placed in a 24-well plate (five em-
bryos per well) and maintained in 2 mL egg water containing
[
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Hyaloid vessel diameter was used to calculate the percentage in-
hibition of sample in HG-treated embryos according to the follow-
ing formula:
[
[
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where C = hyaloid vessel diameter (AU) of the control embryos;
HG = hyaloid vessel diameter (AU) of the HG-treated embryos;
and T = hyaloid vessel diameter (AU) of the HG-treated embryos
treated with sample. The hyaloid vessel diameter of each lens
was measured three times and the experiment was performed in
triplicate. Statistical significance was assessed using one-way anal-
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