a-Glucosidase inhibitors extracted from the roots of Polygonum multiflorum Thunb

Article abstract of DOI:10.1016/j.fitote.2016.11.009

A novel stilbene glucoside, polygonumnolide D (1), and a novel dianthrone glycoside, polygonumnolide E (2), were isolated from a 70% EtOH extract of the dried roots of Polygonum multiflorum Thunb., together with six known compounds (3–8). Their structures were elucidated by 1D and 2D NMR as well as mass spectroscopy data. The isolated compounds were evaluated for their a-glucosidase inhibitory activities in vitro. Compounds 1, 2 and 5 showed the inhibitory activity against a-glucosidase with the IC₅₀ values of 2.4, 2.7 and 0.3?μM, respectively.

Full text of DOI:10.1016/j.fitote.2016.11.009

a-Glucosidase inhibitors extracted from the roots of Polygonum multiflorum
Thunb
Jian-Bo Yang, Jin-Ying Tian, Zhong Dai, Fei Ye, Shuang-Cheng Ma,
Ai-Guo Wang
PII:
DOI:
Reference:
S0367-326X(16)30584-6
doi:10.1016/j.fitote.2016.11.009
FITOTE 3528
To appear in:
Fitoterapia
Received date:
Revised date:
Accepted date:
17 October 2016
14 November 2016
15 November 2016
Please cite this article as: Jian-Bo Yang, Jin-Ying Tian, Zhong Dai, Fei Ye, Shuang-
Cheng Ma, Ai-Guo Wang, a-Glucosidase inhibitors extracted from the roots of Polygonum
multiflorum Thunb, Fitoterapia (2016), doi:10.1016/j.fitote.2016.11.009
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ACCEPTED MANUSCRIPT
a-Glucosidase inhibitors extracted from the roots of Polygonum
multiflorum Thunb
a
a
b
a
b
Jian-Bo Yang , Jin-Ying Tian , Zhong Dai , Fei Ye , Shuang-Cheng Ma *, Ai-Guo
a
Wang *.
a State Key Laboratory of Bioactive Substance and Function of Natural Medicines,
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking
Union Medical College, Beijing 100050, People′s Republic of China.
b Research and Inspection Center of Traditional Chinese Medicine and
Ethnomedicine, National Institutes for Food and Drug Control, State Food and
Drug Administration, Beijing 100050, People′s Republic of China.
Corresponding Author
*
Corresponding author. Tel: 0086-10-67095272. Fax: 0086-10-67095887.
Email: masc@nifdc.org.cn and wangaiguo@imm.ac.cn

ACCEPTED MANUSCRIPT
Abstract A novel stilbene glucoside, polygonumnolide D (1), and a novel dianthrone
glycoside, polygonumnolide E (2), were isolated from a 70% EtOH extract of the
dried roots of Polygonum multiflorum Thunb., together with six known compounds
(3-8). Their structures were elucidated by 1D and 2D NMR as well as mass
spectroscopy data. The isolated compounds were evaluated for their a-glucosidase
inhibitory activities in vitro. Compounds 1, 2 and 5 showed the inhibitory activity
against a-glucosidase with the IC values of 2.4, 2.7 and 0.3 μM, respectively.
5
0
Keywords: Polygonum multiflorum Thunb.; stilbene glucoside; dianthrone glycoside;
polygonumnolide D; polygonumnolide E; a-glucosidase inhibition

ACCEPTED MANUSCRIPT
1
. Introduction
The incidence of diabetes mellitus (DM), a complex chronic metabolic disorder, is
increasing every year. Diabetes mellitus is associated with age and is characterized by
hyperglycemia. a-Glucosidase is an enzyme that can reduce the absorption of
carbohydrates from the digestive tract and decrease after-meal glucose levels [1, 2].
However, some a-glucosidase inhibitors on the market, such as voglibose and
acarbose, often cause severe gastrointestinal side effects, such as flatulence and
diarrhea [3]. Therefore, there is a great need for natural a-glucosidase inhibitors
derived from Traditional Chinese Medicine (TMC).
Our preliminary screening study revealed that the 70% EtOH extraction of the roots
of Polygonum multiflorum Thunb, exhibited significant a-glucosidase inhibitory
activity with IC value of 3.1 μg/ml. P. multiflorum is among the most important and
5
0
widely used TCM herbs, and has a long history as a traditional folk medicine for
treating coronary heart disease, hyperlipidemia, neurosis, hair loss prevention and
premature graying [4]. Phytochemical studies on P. multiflorum have led to the
isolation of anthraquinones, stilbenes, phenolic acid, phospholipids, flavones [5] and
dianthrones [6]. In this research, bioactivity-guided fractionation of the 70% EtOH
extraction of P. multiflorum roots was carried out. A new stilbene glucoside (1) and a
new dianthrone glycoside (2) were isolated together with six known compounds. We
then determined the a-glucosidase inhibitory activity of the compounds.

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2
. Experimental
2
.1. Plant material
The dried roots of P. multiflorum Thunb were collected at Deqing, in Guangdong
province, China, in October 2012, and were identified by associate Professor Ji Zhang
Research and Inspection Center of Traditional Chinese Medicine and Ethnomedicine,
(
National Institutes for Food and Drug Control, State Food and Drug Administration).
A voucher sample of the roots (No. 060104) has been deposited at the Research and
Inspection Center of Traditional Chinese Medicine and Ethnomedicine, National
Institutes for Food and Drug Control, State Food and Drug Administration, Beijing,
China.
2
.2. Chemicals and instrumentation
a-Glucosidase (Sigma, St. Louis, MO, USA) and PNPG (p-nitrophenyl-alpha-D-glu
copyranoside) (Sigma). Acarbose was purchased from the Research and Inspection
Center of Traditional Chinese Medicine and Ethnomedicine, National Institutes for
Food and Drug Control. Optical rotations were acquired on a Jasco P-2000
polarimeter (Jasco Inc., Tokyo, Japan). UV data were recorded using a Jasco V-650
spectrophotometer (Jasco Inc.). IR spectra were measured on a Nicolet iN 10 Micro
FTIR spectrophotometer (Thermo Nicolet Inc., Waltham, MA, USA). NMR spectra
were recorded on Varian Inova-300, 500 and 600 spectrophotometers (Varian Inc.,
Palo Alto, CA, USA). HR-ESI-MS were obtained using an Agilent 1100
UPLC-Q-TOF mass spectrometer (Agilent Technologies Ltd., Santa Clara, CA, USA).
Column chromatography was performed with silica gel (200–300 mesh; Qingdao
Marine Chemistry Company, Qingdao, China), Sephadex LH-20 (GE Healthcare
Bio-Sciences AB, Uppsala, Sweden) and reversed-phase C silica gel (40–60 µm,
1
8
Alltech, Deerfield, IL, USA). Preparative high-performance liquid chromatography
HPLC) separations were carried out using a Shimadzu LC-10 A system equipped
(
with a YMC-Pack ODS-A column (250 × 20 mm, 5 µm; Kyoto, Japan) and a
Shimadzu SPD-20 A detector (Shimadzu).
2
.3. Extraction and isolation
The dried roots of P. multiflorum (28.0 kg) were refluxed three times with 70%

ACCEPTED MANUSCRIPT
EtOH. The obtained EtOH extracts were concentrated under reduced pressure below
5
0 °C and gave a residue (4.0 kg), which was suspended in 1.5 L of H O and
2
successively partitioned with CH Cl . The H O fraction (3.5 kg) was subjected to
2
2
2
macroporous resin (DM-8) eluting with a gradient of water and EtOH (H O, 25%
2
EtOH, 40% EtOH, 55% EtOH and 95% EtOH) to give five fractions [A–E; fraction A
(
2.0 kg, IC >50 μg/ml); fraction B (62.0 g, IC : 10.9 μg/ml); fraction C (200.0 g,
50 50
IC : 10.7 μg/ml); fraction D (38.0 g, IC : 2.1 μg/ml); fraction E (55.0 g, IC : 6.3
5
0
50
50
μg/ml)]. Fraction D was separated on a RP-18 silica gel column using a step gradient
elution of methanol–water (from 10% to 100% v/v) to give six fractions (A , 0.5 g; A ,
1
2
2
.8 g; A , 15.8 g; A , 4.5 g; A , 7.7 g; A , 3.2 g). Fraction A was purified on a
3 4 5 6 3
Sephadex LH-20 column (100% MeOH) to give three fractions, B –B .
1
3
Fraction B was recrystallized by methanol–water (20:80) and compound 3 (12.5 g)
1
was obtained. Fraction B (96 mg) was further separated and purified by preparative
3
HPLC (MeOH/H2O, 30:70; YMC column, 250 × 20 nm, particle size S-5 µm; 220
nm; flow rate 6.0 mL/min) to yield 4 (8.0 mg, 36.2 min) and 5 (20.0 mg, 43.5 min).
Fraction A was purified on a Sephadex LH-20 column (100% MeOH) to give five
4
fractions, C –C . Fraction C (560 mg) was further separated and purified by
1
5
3
preparative HPLC (MeOH/H2O, 80:20; YMC, 250 × 20 nm, S-5 µm; 220 nm; 6.0
mL/min) to yield 6 (50.0 mg, 49.0 min) and 1 (230.0 mg, 68.0 min). Fraction C (860
4
mg) was further separated and purified by preparative HPLC (CH CN/H O, 40:60;
3
2
YMC, 250 × 20 nm, S-5 µm; 220 nm; 6.0 mL/min) to yield 7 (125.0 mg, 45.0 min)
and 8 (215.0 mg, 57.0 min). Fraction C (380 mg) was further separated and purified
5
by preparative HPLC (CH CN/H O, 50:50; YMC, 250 × 20 nm, S-5 µm; 220 nm; 6.0
3
2
mL/min) to yield 2 (8.0 mg, 35.0 min).
.3.1 Polygonumnolide D (1)
2
2
5
Yellow powder; [α]D + 24.28° (c = 0.6, MeOH); UV (MeOH) λmax (logε): 219
(3.50), 332 (5.66) nm; IR (KBr) νmax: 3374, 2974, 2931, 1606, 1514, 1443, 1343,
−
1
1
13
1
1
8
246, 1173, 1070, 838 cm ; H NMR (CD OD, 600 MHz) and C NMR (CD OD,
3 3
+
+
50 MHz) data, see Table 1; HRESI-MS: m/z 839.2734 [M+H] (calcd for C H O ,
42
47 18
39.2757, error of −2.3 ppm)

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2
.3.2 Polygonumnolide E (2)
25
Yellow powder; [α]D −102° (c = 0.1, MeOH); UV (MeOH) λmax (logε): 209 (4.88),
2
78 (4.27), 350 (4.31) nm; IR (KBr) νmax: 3373, 2975, 1620, 1603, 1566, 1490, 1462,
−
1
1
1
358, 1336, 1294, 1259, 1219, 1176, 1075, 1038, 976, 898, 862 cm ; H NMR
1
3
(
CD CO CD , 600 MHz) and C NMR (CD CO CD , 150 MHz) data, see Table 2;
3 3 3 3
−
-
HRESI-MS: m/z 685.1921 [M−H] (calcd for C H O , 685.1927, error of −0.6
37
33 13
ppm).
.4. Enzyme hydrolysis of compounds 1 and 2
According to the methods shown in previous reports [6, 7], compound 1 (3.0 mg
2
each) was enzymatically hydrolyzed by β-glucosidase (8.0 mg) from almonds (Sigma
Aldrich) at 30 °C for 12 h. The reaction mixtures were extracted with EtOAC three
times (3×10 mL). The aqueous layer was frozen for 12 h and a neutral residue was
successfully obtained. The residue was dissolved in anhydrous pyridine (3 ml) and
L-cysteine methyl ester hydrochloride (1.5 mg) was added. The mixtures were kept at
6
0 °C for 2 h, and were frozen to dryness. Trimethylsilylimidazole (1.0 mL) was then
added to the reaction mixture and kept at 60 °C for another 2 h, and separated by H20
2 mL) and n-hexane (3 × 10 mL). The n-hexane extract was subjected to GC analysis,
(
run on a Agilent 7890A gas chromatograph equipped with an Agilent 19091J-216
HP-5 capillary column (60 m × 0.32 mm × 1.0 μm) and an H flame ionization
2
detector with the following conditions: column temperature, 160–280 °C, increasing,
5
°C/min and maintained at 280 °C for about 20 min; carrier gas, N (1 mL/min);
2
injector and detector temperature, 300 °C; injection volume, 5 µL; and a split ratio of
/30. From the enzymatic hydrolysate of 1, D-glucose was confirmed by comparison
1
of the retention time of the derivative with that of authentic sugar derivatized in a
similar way. Both showed a retention time of 40.5 min. The constituent sugar of
compound 2 was identified by the same method as with 1.
2
.5 a-Glucosidase inhibition assay
According to the modified methods of previous reports [8, 9], the inhibitory activity
of a-glucosidase was determined. A total of 3 mM PNPG (p-Nitrophenyl-alpha-
D-glucopyranoside) (20 μL) and a-glucosidase (20 μL, 1 U/mL in PBS, PH 6.8), PBS

ACCEPTED MANUSCRIPT
(50 μL) and the sample solution (10 μL) were placed in a 96-well microplate and
precultured for 15 min at 37 °C. 0.4 M Na CO (50 μL) was added and the mixture
2
3
was incubated additionally for 35 min. The absorbance of each well was measured at
00 nm with a microplate spectrophotometer. Acarbose was used as the positive
control (see Table 3).
4
3
. Results and discussion
In total, eight compounds (1-8), including two novel compounds (1 and 2), were
isolated and identified based on NMR and mass spectra. By comparison of the
spectral data with reported references, the structures of the known compounds were
confirmed as (E)-2,3,5,4'-tetrahydroxylstilbene-2-β-D-glucopyranoside (3) [10], (Z)-2,
3
2
,5,4'-tetrahydroxylstilbene-2-β-D-glucopyranoside (4) [11], rhaponticin (5) [12], (E)-
,3,5,4'-tetrahydroxylstilbene-2-β-D-(3′′-O- monogalloylesters)-glucoside (6) [13], N-
trans-feruloyl tyramine (7) [14], N-trans-feruloyl-3-methyldopamine (8) [14] based
on the spectroscopic analysis and comparison with literature data (Fig. 1).
Compound 1 was obtained as a yellow amorphous powder. Its HR-ESI-MS
+
exhibited a molecular ion peak at m/z 839.2734 [M+H] , corresponding to the
−1
molecular formula C H O . Its IR spectrum showed absorption bands at 3374 cm
4
2
46 18
−
1
1
(
OH), and 1606, 1514 and 1443 cm (phenyl). The H NMR spectrum (CD OD,
3
1
1
Table 1) and H- H COSY spectra (Fig. 2) of 1 exhibited signals for two sets of
AA′BB′ systems [δ 7.41 (4H, d, J = 8.4 Hz, H-2a/2b), 6.72 (4H, d, J = 8.4 Hz,
H
H-3a/3b)], two sets of uncoupled aromatic protons [δ 6.69/6.67 (2H, s, H-14a/14b)],
H
two trans-coupled vinylic protons [δ 7.64 (2H, d, J = 16.2 Hz, H-8a/8b), 6.89 (2H, d,
H
J = 16.2 Hz, H-7a/7b)], one set of an AX system [δ 5.13 (1H, q, J = 7.8 Hz, H-15a),
3
H
1
.70 (3H, d, J = 7.8 Hz, H-16a)] and two β-glucopyranosyl anomeric protons [δ 4.51
H
(1H, d, J = 8.4 Hz, H-1a′ or 1b′) and 4.49 (1H, d, J = 7.8 Hz, H-1a′ or 1b′)].
1
3
The C NMR (CD OD, Table 1) and HSQC spectra of 1 revealed signals for 42
3
carbons. Comparison of the 1D NMR spectrum of 1 with those of
E)-2,3,5,4'-tetrahydroxystilbene-2-β-D-glucopyranoside [10] suggested 1 could be a
(
symmetrical stilbene glucoside dimer linked via a CH(CH ) bridge. Interpretation of
3
HMBC data (Fig. 2) then led to the proposed structure of 1. In the HMBC spectrum,
long-range correlations were observed between H-7a/7b (δ 6.89) and C-9a/9b (δC
H
1
29.9), C-2a/2b (δ 127.7), C-6a/6b (δ 127.7), confirming that C-7a/7b were linked
C C
to C-1a/1b of rings A/A′. The observed HMBC correlations of H-8a/8b (δ 7.64) with
H

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C-1a/1b (δ 129.4), C-10a/10b (δ 137.0/136.9) and C-14a/14b (δ 102.5) indicated
C
C
C
C-8a/8b were linked to C-9a/9b of rings B/B′. The observed HMBC correlations of
H-15a (δ 5.13) with C-16a (δ 16.8), C-11a/11b (δ 152.3/152.2), C-12a/12b (δ
C
H
C
C
1
17.7/117.3) and C-13a/13b (δ 148.1/147.8) indicated that C-15a was linked to
C
C-13a/13b of rings B/B′, which was further confirmed by the observed HMBC
correlations of H-16a (δ 1.70) with C-15a (δ 26.7) and C-12a/12b (δ 117.7/117.3).
H
C
C
The HMBC correlations (Fig. 2) between H-1a′/1b′ (δ 4.51/4.49) and C-10a/10b (δC
H
1
36.9/137.0) suggested that the sugar moieties in 1 were attached at C-10a/10b.
The β-glucopyranosyl configurations were inferred from the coupling constant
JH-1′′, H-2′′ > 7.5 Hz) [7], while the D-glucosyl stereochemistry was determined on the
3
(
basis of the enzymatic hydrolysis with β-glycoside hydrolase, followed by GC
analysis of its corresponding trimethylsilylated L-cysteine adduct. Accordingly, the
structure of 1 was clarified as shown (Fig. 1) and given the trivial name
polygonumnolide D.
Compound 2 was obtained as a yellow powder. Its molecular formula, C H O ,
37
34 13
−
was deduced from HR-ESI-MS from the peak at m/z 685.1921 [M−H] , which
corresponds to 21 indices of hydrogen deficiency. The IR spectrum showed strong
−
1
absorption bands at 1620 and 1603 cm that were assigned to carbonyl groups, a
−
1
peak from hydroxyl groups at 3373 cm and a peak from aromatic ring
−
1
functionalities at 1490 cm . The UV spectrum showed absorption maxima at 209,
2
78 and 350 nm, which were very close to those of previously reported dianthrone
1
1
1
derivatives [6, 15, 16]. The H NMR spectrum (CD CO CD , Table 2) and H- H
3
3
COSY spectra (Fig. 2) showed the signals of eight meta-coupled aromatic protons [δH
.66 (1H, s, H-2), 6.20 (1H, br s, H-4), 6.60 (1H, br s, H-5), 7.08 (1H, d, J = 2.4 Hz,
H-7), 6.69 (1H, s, H-2′), 6.45 (1H, br s, H-4′), 6.00 (1H, br s, H-5′), and 6.29 (1H, d, J
2.4 Hz, H-7′)], two methyl groups [δ 2.26 (3H, s, Me-3) and 2.31 (3H, s, Me-3′)],
6
=
H
one methoxy group at δ 3.89 (3H, s, OCH -6), two vicinal methine protons [δ 4.67
H
3
H
(1H, d, J = 3.6 Hz, H-10) and 4.59 (1H, d, J = 3.6 Hz, H-10′)], and one
β-glucopyranosyl anomeric proton at δ 4.74 (1H, d, J = 7.8 Hz, H-1′′).
H
Comparison of the 1D NMR spectrum of 2 with those of physcion dianthrones,
emodin dianthrones, and physcion-emodin dianthrones [17-19] suggested 2 could be a
1
3
C-10/C-10′ isomer of a physcion-emodin dianthrone glycoside. The C NMR and
DEPT spectra (Table 2) exhibited 37 carbon signals, including 31 carbon signals of
physcion-emodin dianthrones that were categorized by DEPT and HSQC techniques

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into 18 quaternary carbons, including with two carbonyl groups [δ 191.1 (C-9′) and
C
1
88.3 (C-9)], 10 methine groups [δ 117.3 (C-2), 121.5 (C-4), 110.7 (C-5), 107.1
C
(C-7), 117.7 (C-2′), 122.3 (C-4′), 110.3 (C-5′), 102.9 (C-7′), 57.5 (C-10) and 56.4
(
C-10′)], two methyl groups [δ 21.9 (Me-3) and 22.1 (Me-3′)], one methoxy group δC
C
5
7
6.5 (OMe-6)] and six carbon signals characteristic of glucose [δ 106.5, 75.0, 78.7,
C
1.5, 77.5, 62.9].
The HMBC spectrum (Fig. 2) of 2 showed long-range correlations from H-2 (δH
.66) to C-1 (δ 162.4), C-4 (δ 121.5), and C-1a (δ 117.3), and from Me-3 (δ 2.26)
6
C
C
C
H
to C-2 (δ 117.3), C-3 (δ 146.9), and C-4 (δ 121.5), indicating the presence of an A
C
C
C
ring. The HMBC correlations between H-7 (δ 7.08) and C-5 (δ 110.7), C-6 (δ
H
C
C
1
1
65.0), C-8 (δ 161.8), and C-8a (δ 117.6), and from OMe-6 (δ 3.89) to C-6 (δ
C C H
C
65.0) indicated the presence of a B ring. Meanwhile, the HMBC correlations from
H-2′ (δ 6.68) to C-1′ (δ 162.8), C-4′ (δ 122.3), and C-1a′ (δ 115.4), and from
H
C
C
C
Me-3′ (δ 2.31) to C-2′ (δ 117.7), C-3′ (δ 147.8), and C-4′ (δ 122.3) indicated the
H
C
C
C
presence of an A′ ring. The HMBC correlations from H-7′ (δ 6.30) to C-5′ (δ 110.3),
H
C
C-6′ (δ 165.2), C-8′ (δ 165.6), and C-8a′ (δ 111.1) indicated the presence of an B′
C
C
C
ring. The configuration of the C-10/C-10′ junction of the two anthronyl moieties was
deduced from the HMBC correlations observed between the proton at δ 4.67 (1H, d,
H
J = 3.6 Hz, H-10) and C-1a (δ 117.3), C-4 (δ 121.5), C-5 (δ 110.7), C-8a (δ 117.6),
C
C
C
C
and C-10′ (δ 56.4) and between the proton at δ 4.59 (1H, d, J = 3.6 Hz, H-10′) and
C
H
C-1a′ (δ 115.4), C-4′ (δ 122.3), C-5′ (δ 110.3), C-8a′ (δ 111.1), and C-10 (δ 57.5).
C
C
C
C
C
The HMBC correlations between δ 4.74 (1H, d, J = 7.8 Hz, H-1′′) and C-8 (δ 161.8)
H
C
suggested that the sugar moiety in 2 was attached at C-8. The β-glucopyranosyl
3
configuration was inferred from the coupling constant ( JH-1′′, H-2′′ = 7.8 Hz) [7], while
the D-glucosyl stereochemistry was determined on the basis of the enzymatic
hydrolysis with β-glycoside hydrolase, followed by GC analysis of its corresponding
trimethylsilylated L-cysteine adduct. Thus, the planar structure of 2 was assigned as
physcion-emodin-8-O-β-D-glucopyranoside dianthrone.
The relative structure of dianthrone derivatives was unable to be confirmed by
NOESY experiments and constants between H-10 and H-10′ [20-22]. According to
the reports [6, 23, 24], the H-4 (δ 6.20) and H-5′ (δ 6.00) peaks of 2 are more
H
H
upfield than the H-5 (δ 6.59) and H-4′ (δ 6.45) peaks because of the shielding effect
H
H
from A′/B rings, suggesting the relative structure of 2 was confirmed as a cis H-10/10′
dianthrone. Accordingly, the structure of 2 was clarified as shown (Fig. 1) and given

ACCEPTED MANUSCRIPT
the trivial name polygonumnolide E.
Meanwhile, dianthrone derivatives, such as polygonumnolides C1-C4, emodin
dianthrones [6] and polygonumnolide E (2), were firstly isolated from P. multiflorum
and anthraquinones were widely distributed in Polygonum species. Accordingly,
postulated biogenetic relationship between dianthrones, including some possible new
compounds, and anthraquinones was speculated as Scheme 1.
The isolated compounds were also subjected to an evaluation of their a-glucosidase
inhibitory activities in vitro. Compounds 1-6 showed the inhibitory activity against
a-glucosidase with the IC values of 2.4, 2.7, 14.9, 8.6, 0.3 and 11.1 μM, respectively.
50
Among them, compounds 1 and 3-6 belong to stilbenes. According to the literature
25], stilbenes showed diverse bioactivities, such as antioxidant, anti-inflammatory,
[
anticancer and anti-diabetic activity. Thus, stilbenes and dianthrone derivatives may
be given more attentions in order to obtain a-glucosidase inhibitors from P.
multiflorum.

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Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
The authors are grateful to the members of the analytical group of the State Key
Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of
Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical
College, Beijing, China, for measuring the spectroscopic data. This Project was
financially supported by the National Natural Science Foundation of China (Grant No.
th
8
1173506) and the 12 Five Year National significant new drugs creation feature
subjects-traditional Chinese medicine quality safety evaluation and risk control
technology platform (2014ZX09304307-002).

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Appendix A. Supplementary data
1
13
Spectroscopic data for the new compounds (1 and 2) including H, C and 2D NMR,
IR and ESI-MS/MS data are provided in the supporting information.

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(

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Fig. 1. Structures of compounds 1 and 2 from the roots of Polygonum multiflorum
Thunb.

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1
1
Fig 2. Key H– H COSY and HMBC correlations of compounds 1 and 2.

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Scheme 1. Hypothetical Biogenetic Relationship between dianthrones and
anthraquinones

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Table 1
1
13
H NMR (600 MHz) and C NMR (150 MHz) data for 1 were measured in CD OD
3
Position
1
δH (J in Hz)
δC
1
2
3
4
5
6
7
8
9
1
a/1b
a/2b
a/3b
a/4b
a/5b
a/6b
a/7b
a/8b
a/9b
0a/10b
129.4
7.41, d (8.4)
6.72, d (8.4)
127.7
115.0
156.9/156.8
115.0
127.7
6.89, d (16.2)
7.64, d (16.2)
128.4/128.3
119.9
129.9
137.0/136.9
11a/11b
152.3/152.2
1
1
2a/12b
3a/13b
117.7/117.3
148.1/147.8
1
1
1
1
4a/14b
5a
6.69,s; 6.67,s
102.5
26.7
5.13, q (7.8)
6a
1.70, d (7.8)
16.8
a′/1b′
4.51, d (8.4); 4.49, d (7.8)
106.7
2
3
4
5
6
a′/2b′
a′/3b′
a′/4b′
a′/5b′
a′/6b′
3.53-3.57, m
74.1
3.41, dd (8.4, 9.6)
3.48-3.51, m
76.4
69.4
3.22-3.25, m
76.8/76.7
60.8/60.7
3.81, dd (2.4, 12.0), 3.78, dd (2.4, 12.0),
.74, dd (4.8, 12.0), 3.73, dd (4.2, 12.0)
3

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Table 2
1
13
H NMR (600 MHz) and C NMR (150 MHz) data for 2 were measured in CD COCD
3
3
2
2
Position
Position
δC
δH (J in Hz)
δC
δH (J in Hz)
1
2
3
162.4
117.3
146.9
1′
2′
3′
162.8
117.7
147.8
6.66, s
6.68, s
4
5
121.5
110.7
6.20, br s
6.60, br s
4′
5′
122.3
110.3
6.45, br s
6.00, br s
6
7
8
9
165.0
107.1
161.8
188.3
57.5
6′
165.2
102.9
165.6
191.1
56.4
7.08, d (2.4)
4.67, d (3.6)
7′
6.30, d (2.4)
4.59, d (3.6)
8′
9′
1
1
4
5
8
0
a
a
a
a
10′
1a′
4a′
5a′
8a′
117.3
142.2
145.4
117.6
115.4
140.7
144.4
111.1
3
6
-Me
21.9
56.3
2.26 s
3′-Me
22.1
2.31 s
-0Me
3.89, s
1′′
2′′
106.5
75.0
4.74, d (7.8)
3.46-3.49, m
OH-1
12.15, s
OH-8′
11.94, s
11.79, s
3′′
78.7
3.56-3.59, m
OH-1′
4′′
5′′
71.5
77.5
3.43-3.46, m
3.55-3.57, m
OH-6
3
3
.98, dd (1.2, 12);
.74, dd (6.0,12)
OH-6′
9.75, s
6′′
62.9

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Table 3
a-glucosidase inhibitory activity of the isolated compounds (n=3)
a
Compound
IC50 (μM)
b
Acarbose
50.04
1
2
3
4
5
6
7
8
2.4
2.7
14.9
8.6
0.3
11.1
26.6 % inhibition at 10 μM
16.1 % inhibition at 10 μM
a
IC is the concentration required to produce 50 % inhibition of the enzyme activity.
5
0
b
Acarbose as a positive control

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