Ellagitannins and oligomeric proanthocyanidins of three polygonaceous plants

Article abstract of DOI:10.3390/molecules26020337

The aim of this study was to characterize hydrolyzable tannins in Polygonaceous plants, as only a few plants have previously been reported to contain ellagitannins. From Persicaria chinensis, a new hydrolyzable tannin called persicarianin was isolated and characterized to be 3-O-galloyl-4,6-(S)-dehydrohexahydroxydiphenoyl-D-glucose. Interestingly, acid hydrolysis of this compound afforded ellagic acid, despite the absence of a hexahydroxydiphenoyl group. From the rhizome of Polygonum runcinatum var. sinense, a large amount of granatin A, along with minor ellagitannins, helioscpoinin A, davicratinic acids B and C, and a new ellagitannin called polygonanin A, were isolated. Based on 2D nuclear magnetic resonance (NMR) spectroscopic examination, the structure of polygonanin A was determined to be 1,6-(S)-hexahydroxydiphenoyl-2,4-hydroxychebuloyl-β-D-glucopyranose. These are the second and third hydrolyzable tannins isolated from Polygonaceous plants. In addition, oligomeric proanthocyanidins of Persicaria capitatum and P. chinensis were characterized by thiol degradation. These results suggested that some Polygonaceous plants are the source of hydrolyzable tannins not only proanthocyanidins.

Full text of DOI:10.3390/molecules26020337

molecules
Article
Ellagitannins and Oligomeric Proanthocyanidins of Three
Polygonaceous Plants
Yun-Qiu Li 1,2, Masako Kitaoka 3, Juri Takayoshi 3, Ya-Feng Wang 4, Yosuke Matsuo 1 , Yoshinori Saito 1,
Yong-Lin Huang 4, Dian-Peng Li 4, Gen-ichiro Nonaka 5, Zhi-Hong Jiang 6 and Takashi Tanaka 1,
*
1
Department of Natural Product Chemistry, Graduate School of Biomedical Sciences, Nagasaki University,
1-14 Bunkyo-machi, Nagasaki 852-8521, Japan; leeyq88@126.com (Y.-Q.L.); y-matsuo@nagasaki-u.ac.jp (Y.M.);
saiyoshi@nagasaki-u.ac.jp (Y.S.)
College of Medical Laboratory Science, Guilin Medical University, 109, Huancheng North 2 Road,
Guilin 541004, China
Department of Natural Product Chemistry, School of Pharmaceutical Sciences, Nagasaki University,
1-14 Bunkyo-machi, Nagasaki 852-8521, Japan; bb00509015@cc.nagasaki-u.ac.jp (M.K.);
bb30217021@ms.nagasaki-u.ac.jp (J.T.)
Guangxi Key Laboratory of Functional Phytochemicals Research and Utilization, Guangxi Institute of Botany,
Guilin 541006, China; wyf@gxib.cn (Y.-F.W.); hyl@gxib.cn (Y.-L.H.); ldp@gxib.cn (D.-P.L.)
Usaien Pharmaceutical Company, Ltd., 1-4-6 Zaimoku, Saga 840-0055, Japan; usaien@lily.ocn.ne.jp
Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa,
Macau 999078, China; zhjiang@must.edu.mo
2
3
4
5
6
*
Correspondence: t-tanaka@nagasaki-u.ac.jp; Tel.: +81-95-819-2432
Abstract: The aim of this study was to characterize hydrolyzable tannins in Polygonaceous plants, as
only a few plants have previously been reported to contain ellagitannins. From Persicaria chinensis, a
new hydrolyzable tannin called persicarianin was isolated and characterized to be 3-O-galloyl-4,6-(S)-
dehydrohexahydroxydiphenoyl-D-glucose. Interestingly, acid hydrolysis of this compound afforded
ellagic acid, despite the absence of a hexahydroxydiphenoyl group. From the rhizome of Polygonum
runcinatum var. sinense, a large amount of granatin A, along with minor ellagitannins, helioscpoinin
A, davicratinic acids B and C, and a new ellagitannin called polygonanin A, were isolated. Based
on 2D nuclear magnetic resonance (NMR) spectroscopic examination, the structure of polygonanin
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Li, Y.-Q.; Kitaoka, M.;
Takayoshi, J.; Wang, Y.-F.; Matsuo, Y.;
Saito, Y.; Huang, Y.-L.; Li, D.-P.;
Nonaka, G.-i.; Jiang, Z.-H.; et al.
Ellagitannins and Oligomeric
Proanthocyanidins of Three
A was determined to be 1,6-(S)-hexahydroxydiphenoyl-2,4-hydroxychebuloyl-β-D-glucopyranose.
Polygonaceous Plants. Molecules 2021,
26, 337. https://doi.org/10.3390/
molecules26020337
These are the second and third hydrolyzable tannins isolated from Polygonaceous plants. In addition,
oligomeric proanthocyanidins of Persicaria capitatum and P. chinensis were characterized by thiol
degradation. These results suggested that some Polygonaceous plants are the source of hydrolyzable
tannins not only proanthocyanidins.
Academic Editor: Luca Rolle
Received: 10 December 2020
Accepted: 7 January 2021
Published: 11 January 2021
Keywords: ellagitannin; hydrolyzable tannin; proanthocyanidin; Persicaria; Polygonum; Polygo-
naceae
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional clai-
ms in published maps and institutio-
nal affiliations.
1. Introduction
Ellagitannins are hydrolyzable tannins containing hexahydroxydiphenoyl (HHDP)
groups and related acyl groups. Recently, their high structural diversity and biological
activities have attracted intense interest amongst natural product chemists [1–5]. Compared
to proanthocyanidins (synonym (syn.) condensed tannins), distribution of ellagitannins
in the plant kingdom is relatively limited. Among Polygonaceous plants, only 2,3,4-tri-O-
Copyright: © 2021 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
galloyl-1,6-(S)-HHDP-
(syn. Polygonum capitatum) [
reported [ ]. More recently, the presence of geraniin and chebulagic acid in the aerial parts
β-D-glucose (davidiin,
1) has been isolated from Persicaria capitatum
6]; its biological activity and metabolism have also been
7–9
of Persicaria chinense var. hispidum was suggested by the use of high-performance liquid
chromatography (HPLC)-mass spectroscopy (MS) [10]. In the course of chemical studies
on ellagitannins, we analyzed aqueous CH3CN extracts of eight Polygonaceous plants,
Molecules 2021, 26, 337. https://doi.org/10.3390/molecules26020337
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 337
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Reynoutria japonica, Persicaria perfoliata, P. longiseta, P. lapathifolia, P. capitata, P. chinensis,
P. filiformis, and P. thunbergii, by HPLC-diode array detector (DAD) analysis (Figure S1,
Supplementary Materials). In the study, only the extracts of P. capitata and P. chinensis
showed peaks with UV absorption characteristic to ellagitannins (Figure 1). The results
were consistent with the above-mentioned studies; the major ellagitannin of P. capitata
was davidiin (1), and P. chinensis contains geraniin (2) (Figure 2). In addition, we found
that the rhizome of Polygonum runcinatum var. sinense, a Chinese perennial plant used
as a folk medicine for detoxification and hemostasis, contains ellagitannins as the major
constituents. This paper describes the isolation of phenolic substances from these three
plants and structural determination of the two new ellagitannins obtained from Persicaria
chinensis and Polygonum runcinatum var. sinense. In addition, proanthocyanidin oligomers
from Persicaria capitata and Persicaria chinensis were chemically characterized.
(a)
1
(b)
(c)
7
CH-09 [MaxABS (200.0--550.0 nm)]
tsurusoba - CH5
Euphorbia - CH5
1800000
1600000
1400000
1200000
1000000
800000
600000
400000
200000
600000
Fl
2
Fl
400000
200000
0
1000000
Fl
3
Fl
-0
0
0.0
0
10.0
10
20.0
20
30.0
Retention Time [min]
40.0
40
50.0
50
60.0
min
0.0
10.0
20.0
40.0
50.0
60.0
330.00
40
50
min
0.0
10.0
20.0
30.0
30
40.0
60.0
0
10
20
40
550.00
min
0
10
20
30
Retention Time [min]
Figure 1. HPLC profiles of 60% CH3CN extracts of (a) Persicaria capitata, (
b
) Persicaria chinensis and (
c) Polygonum runcinatum
var. sinense. 1: davidiin, 2: geraniin, 3: persicarianin, 7: granatin A and Fl: flavonoid glycosides.
HO HO OH OH
HO HO OH OH
R
HO
OH
HO
(S)-HHDP
OH
S
OH
OH
O
O
(R)-HHDP
O
O
O
O
O
OH
O
O
HO
O
O
OH
OH
O
O
HO
O
O
OH
O
O
O
O
O
O
O
H
O
galloyl
O
HO
H
HO
O
HO
HO
OH
OH
HO
HO
OH
O
O
HO
OH
O
HO
OH
(R)-DHHDP
OH
1
2
Figure 2. Structures of davidiin (1) from Persicaria capitata and geraniin (2) from Persicaria chinensis.
2. Results and Discussion
2.1. Polyphenols of Persicaria capitata
The EtOH extract of Persicaria capitata whole plant, collected in Shui Cheng, Guizhou,
China, was fractionated by Diaion HP20SS and further separated using Sephadex LH-
20 and Chromatorex ODS to give davidiin (1) as the major constituent (7% from the
extract), along with minor compounds, which were identified as 2,3-di- [11], 2,4-di- [12],
2,3,4-tri- [13], and 2,3,6-tri-O-galloyl glucoses [14], 6’-O-galloyl arbutin [15], 4-hydroxy-3-
methoxyphenol 1-O-
β
-D-(6’-O-galloyl)-glucopyranoside [16], 4-hydroxy-2-methoxyphenol
-D-(6’-O-galloyl)-glucopyranoside [16], quercetin 3-O- -D-glucoside [17], quercetin
-L-rhamnopyranoside [17], quercetin 3-O- -L-(3”-O-galloyl)-rhamnopyranoside [18],
1-O-β
β
3-O-
α
α
ellagic acid, and quercetin.

Molecules 2021, 26, 337
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2.2. Polyphenols from Persicaria Chinensis
Similar separation of an aqueous acetone extract of the fresh aerial part of Persi-
caria chinensis collected in Nagasaki, Japan, afforded 1-O- [19] and 1,2,6-tri-O-galloyl-
D-glucoses [20], geraniin ( ) [21], quercetin 3-O-(2”- -rhamnopyranosyl)- -glucuronop
yranoside [22], and a new ellagitannin called persicarianin. Persicarianin ( ) was obtained
β-
2
α
β
3
as a brown amorphous powder, and high-resolution electrospray ionization time of flight
(HRESITOF) MS indicated a molecular formula of C27H22O19 (m/z: 673.0689, calculated
for C27H22O19Na: 673.0653). The 1H NMR spectrum showed signals arising from
β
α
- and
-hexopyranoses, and it is apparent that the sugar is a 4C1-glucopyranose based on the
large coupling constants of the pyranose ring proton signals (J2,3, J3,4, J4,5 = 8–10 Hz). The
low field shifts of H-3 [ 5.63 ( -H-3), 5.40 ( -H-3)], H-4 [5.40 ( -H-4), 3.94 ( -H-4)], and
H-6 [4.94, 3.95 ( -H-6), 5.40, 3.92 ( -H-6)] indicates acylation of these positions. The acyl
δ
α
β
α
β
α
β
groups were shown to be a galloyl and a dehydrohexahydroxydiphenoyl (DHHDP) group
by 13C NMR spectroscopy, which exhibited signals attributable to an aliphatic methine
(δ 43.0), a double bond (δ 151.7 and 130.8), a ketone (δ 192.0), and two acetal carbons (δ
91.5 and 96.5), constructing a hydrated cyclohexenetrione ring of the DHHDP group. The
large chemical shift differences of the glucose H-6 methylene proton signals in the 1H NMR
spectrum suggested that the DHHDP esters bridges between the C-4 and C-6 hydroxy
groups [23]. This was confirmed by heteronuclear multiple bond coherence (HMBC) cor-
relations of the glucose H-6 and H-4 to the DHHDP C-7’ and C-7 ester carbonyl carbons,
respectively (Figure 3). The configuration of the DHHDP methine carbon (C-1’) was con-
cluded to be S, based on the appearance of negative and positive Cotton effects at 213 nm
and 234 nm, respectively [24]. The location of the galloyl group was determined to be at
the glucose C-3 hydroxy group by observation of HMBC correlations between the glucose
H-3 and galloyl C-7. The D configuration of the glucosyl moiety was determined via acid
hydrolysis followed by HPLC analysis of the thiazolidine derivatives prepared by reaction
with L-cysteine and o-tolylisothiocyanate [25
characterized to be 3-O-galloyl-4,6-(S)-DHHDP-D-glucose (
mentioned hydrolysis products, before condensation with cysteine, revealed production of
gallic acid ( ), ellagic acid ( ), and brevifolin carboxylic acid ( ) (Figure S2, Supplementary
Materials). Ellagic acid is a bislactone form of the HHDP group, and therefore, a reduction
product of the DHHDP ester of . Similar production of from DHHDP groups on acid
hydrolysis have been also observed for and related ellagitannins, and the reaction was
deduced to be a redox disproportionation [27 28]. Ellagitannins were originally defined as
hydrolyzable tannins which afford upon hydrolysis, and is usually considered to be
,
26]. Based on this evidence, persicarianin was
3
). HPLC analysis of the afore-
4
5
6
3
5
2
,
5
5
originated from HHDP groups. In this context, despite the absence of a HHDP group,
also an ellagitannin.
3 is
O
3'
O
HO
7"
OH
OH
O
6'
HO
OH
O
4
O
6
H1'
HO
6
1
CO2H
OH
O
HO
OH
+
O
O
5% H2SO4
105 °C, 5 h
7
O
O
HO
O
7
+
+
D-glucose
3
2
OH
OH
OH
O
O
3
O
O
HO
(S)-DHHDP
O
CO2H
OH
OH
OH
OH
OH
OH
3
4
5
6
Figure 3. Selected heteronuclear multiple bond coherence (HMBC) correlations and acid hydrolysis of 3-O-galloyl-4,6-(S)-
DHHDP-D-glucose (3).

Molecules 2021, 26, 337
4 of 11
2.3. Hydrolyzable Tannin from Polygonum Runcinatum var. Sinense
Reverse-phase HPLC analysis of the rhizome of Polygonum runcinatum var. sinense
showed a prominent peak arising from a principal phenolic component (Figure 1c), which
was isolated by Diaion HP20SS column chromatography and identified as granatin A (
1D and 2D NMR spectroscopic analysis [29]. Furthermore, the acetonyl derivative 7a was
prepared by treatment of with aqueous acetone containing HCO2NH4, and spectroscopic
comparison of 7a with those of an authentic sample provided further evidence for the
structure of 30]. In the purification process of (Figure 4), four minor ellagitannins
were isolated by a combination of column chromatography using Chromatorex ODS and
Toyopearl Butyl-650M to give helioscopinin A ( ) [31], davicratinic acids B ( ) and C
(10) [32], and a new ellagitannin called polygonanin A.
7) by
7
7
[
7
8
9
HO HO OH OH
HO OH
HO HO OH OH
HO HO OH
OH
OH
HO
OH
HO
OH
OH
O
O
O
galloyl =
OH
O
O
O
O
O
O
O
O
O
O
O-R
O
OH
O
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
H
H
O
5'
H
O
H
O
OH
3'
2'
HO
HO
HO
OH
O
HO
HO
O
OH
OH
HO
HO
O
HO
O
H
OH
O
1'
OH
HO
O
OH
CO2H
OH
O
9 : 2' -H
10: 2' -H
7 : R = H
8 : R = galloyl
7a
Figure 4. Structures of 7–10 and acetonyl derivative 7a.
The 1H and 13C NMR spectra of polygonanin A (11) was related to those of 9 and 10,
indicating 1,2,4,6-acylated glucopyranose with a HHDP group. The location of the HHDP
group at the 1,6-positions of glucose was confirmed by HMBC correlations of glucose H-1
(δH 5.95, s) and H-6 [δH 4.99 (t, J = 11.1 Hz), 4.08 (dd, J = 5.3, 11.1 Hz)] with the HHDP ester
carbonyl carbons (δC 166.4, 168.2). The atropisomerism of the HHDP was shown to be an S
configuration by appearance of positive and negative Cotton effects at 242 nm and 262 nm,
respectively, in the electronic circular dichroism (ECD) spectrum. The configuration is
the same as that of
2,3, J3,4, and J4,5) were <2 Hz. This was similar to those of 7, 9, and 10, but different to
those of 1 (J1,2 = 3.2 Hz, J2,3 = 8.5 Hz, J3,4 = 7.7 Hz, and J4,5 = 2.9 Hz), suggesting that the
glucopyranoses of
10, and 11 adopt a 1C4 conformation, whereas the glucopyranose
core of with a 2,3,4-trigalloyl structure adopts a boat conformation (Figure 1). The NMR
1, 7, 9, and 10. Coupling constants of the pyranose ring protons (J1,2,
J
7, 9,
1
signals indicated that the 2,4-acyl group of 11 was composed of 3 carboxyl carbons (δC
172.3 (C-1’), 172.0 (C-6’), 166.1 (C-7’)), an oxygenated tertiary carbon (δC 76.3 (C-4’)), an
oxygenated methine (δC 66.3 (C-2’)), a methylene (δC 41.6 (C-5’)), and a benzylic methine
(
δC 49.2 (C-3’)), along with a trihydroxy benzoyl moiety. Taking the molecular formula
C34H26O24 indicated by HR-fast atom bombardment (FAB) MS into account, these building
blocks of the 2,4-acyl group suggested that is generated by addition of H2O to the
double bond of or 10 and rearrangements of lactone formation. This was supported by
8
9
HMBC correlations, as illustrated in Figure 5. The planar structure of this acyl group is the
same as the 4’-hydroxychebuloyl group of an ellagitannin isolated from a Euphorbiaceous
plant [33], and the chemical shifts of the aliphatic proton and carbon signals in the literature
(
δH 4.84 (d, J = 5 Hz, H-2’), 4.90 (d, J = 5 Hz, H-3’), 3.28 and 3.43 (each d, J = 18 Hz, H-5’);
δC 68.5 (C-2’), 47.3 (C-3’), 77.6 (C-4’), 42.5 (C-5’)) are similar to those of 11 δH 4.83 (br
s, HChe H-2’) and 4.88 (d, J = 2.8 Hz, HChe H-3’), 3.35 and 2.78 (each d, J = 15.9 Hz,
(

Molecules 2021, 26, 337
5 of 11
HChe H-5’); δC 66.3 (C-2’), 49.2 (C-3’), 76.3 (C-4’), 41.6 (C-5’)). The configuration of C-2’,
C-3’, and C-4’ was concluded to be S*, R*, and S* based on the nuclear Overhauser effect
spectroscopy (NOESY) correlations between the H-2’, H-3’ and H-5’ of 11. The most stable
conformation of 11 obtained by computational calculation along with NOESY correlations
is shown in Figure 5b. The NOESY correlation between H-2’ and one of the H-5’ methylene
protons confirmed the relative configurations of C-2’ – C-5’. From the biogenesis illustrated
Scheme 1, the configuration of the benzylic methines of the acyl groups of 11 was deduced
to be the same as that of the DHHDP group of
5.14) resonated at a much lower field compared to those of 7a
δH 4.44). This could be due to the deshielding effect of the ester carbonyl group attached
7
. Interestingly, the glucose H-3 of 11
(δH
(
δH 4.58), δH 4.40) and 10
9 (
(
to the glucose C-2 hydroxy group (Figure 5b), suggesting that hydroxylation at C-4’ of the
2,4-acyl group affects the conformation of the macrocyclic ester structure.
OH
HO HO
HO
OH
OH
O
O
O
O
OH
O
O
O
O
O
CO2H
H
OH
H
OH
HO
HO
HMBC (H to C)
O
O
(a)
(b)
Figure 5.
(a) Structure and selected HMBC correlations for 11. (b) A partial structure of the most
stable conformer of 11 and nuclear Overhauser effect spectroscopy (NOESY) correlations of the
glucose and 2,4-acyl group.
2.4. Proanthocyanidins of Persicaria Capitata and Persicaria Chinensis
In addition to hydrolyzable tannins, oligomeric proanthocyanidins are also important
constituents of Persicaria capitata and Persicaria chinensis. The oligomers were detected as
broad humps on the HPLC baseline; thiol degradation using 2-mercaproethanol was used
to characterize the structural components [34] (Figure S3, Supplementary Materials). HPLC
analysis of the degradation products obtained from proanthocyanidins of Persicaria capitata
exhibited peaks attributable to 4β-(2-hydroxyethylsulfanyl) derivatives of epicatechin and
epicatechin-3-O-gallate originating from extension units, accompanied by small peaks
of catechin, epicatechin, and epicatechin gallate arising from terminal units. Oligomeric
proanthocyanidins of P. chinensis yielded 2-hydroxyethylsulfanyl derivatives of epicatechin,
epigallocatechin, epicatechin-3-O-gallate and epigallocatechin-3-O-gallate originating from
extension units, along with the free form of epicatechin-3-O-gallate originating from the
terminal unit. The results indicates that proanthocyanidins in these two plants belonging
the same genus Persicaria are composed of different flavan-3-ol units (Figure 6). In contrast,
high-molecular weight polyphenols obtained from the rhizome of Polygonum runcinatum
var. sinense did not yield 2-hydroxyethylsulfanyl derivatives on thiol degradation, indicat-
ing that the polyphenols are not proanthocyanidins. The 13C NMR spectrum suggested
that the polyphenols are oligomeric hydrolyzable tannins. Further investigations are now
underway.

Molecules 2021, 26, 337
6 of 11
O
O
H
O
HO
O
OH
HO
O
H
OH
H+
4
O
O
O
O
O
O
H
H
H
H2O
+ H2O
HO
HO
CO2H
HO
O
HO
HO
O
H
HO
OH
H
OH
O
HO
HO HO
OH
CO2H
H
CO2H
CO2H
9, 10
+ H2O
O
O
H
CO2H
O
O
H
CO2H
H2O
OH
H
HO
HO
OH
H
HO
HO
HO
OH
HO
O
CO2H
O
11
Scheme 1. Plausible production mechanism of 9–11 from 4.
R1
OH
HO
O
OH
R1
O-R2
O
OH
HO
OH
OH
extension units
n
OH
OH
O-R2
Persicaria capitata
R1 = H
R2 = H or galloyl
OH
HO
O
Persicaria chinensis
R1 = H or OH
terminal units
O-R2
R2 = H or galloyl
OH
Figure 6. Structures of oligomeric proanthocyanidins of Persicaria capitata and P. chinensis suggested
by thiol degradation.
3. Materials and Methods
3.1. General Information
Optical rotations were measured on a JASCO P-1020 digital polarimeter (JASCO,
Tokyo, Japan). IR spectra were measured on a JASCO FT/IR 410 spectrophotometer.
Ultraviolet (UV) spectra were obtained on a JASCO V-560 UV/VIS spectrophotometer.
1
ECD spectra were measured with a JASCO J-725N spectrophotometer. H- and 13C-NMR
spectra were recorded on a Varian Unity plus 500 spectrometer (Agilent Technologies, Santa
Clara, CA, USA) operating at 500 MHz and 126 MHz for the 1H and 13C nuclei, respectively.
1H- and 13C-NMR spectra were also recorded on a JEOL JNM-AL400 spectrometer (JEOL

Molecules 2021, 26, 337
7 of 11
Ltd., Tokyo, Japan) operating at 400 and 100 MHz for the 1H and 13C nuclei, respectively.
Coupling constants (J) were expressed in hertz and chemical shifts (δ) are reported in ppm
with the solvent signal used as a standard (pyridine-d5: δH 7.19, δC 123.5 and methanol-d4:
δH 3.31, δC 49.0). FAB-MS data were recorded on a JMS700N spectrometer (JEOL Ltd.,
Tokyo, Japan) using m-nitrobenzyl alcohol or glycerol as the matrix. ESI-TOF-MS data
were recorded on a quadrupole (Q)-TOF LC/MS (Agilent 6550, Agilent Technologies,
Santa Clara, CA, USA). Column chromatography was performed using Sephadex LH-20
(25–100 µm, GE Healthcare UK Ltd., Little Chalfont, UK), Diaion HP20PSS (Mitsubishi
Chemical Co., Tokyo, Japan), Chromatorex ODS (Fuji Silysia Chemical Ltd., Kasugai,
Japan), and Toyopearl butyl-650M (Tosoh Corporation, Tokyo, Japan) columns. TLC
was performed on precoated Kieselgel 60 F254 plates (0.2 mm thick, Merck, Darmstadt,
Germany) with CHCl3-MeOH-H2O (10:3:0.5 or 7:3:0.5, v/v) and toluene-ethyl formate-
formic acid (1:7:1, v/v) mixtures being used as the eluents. The spots were detected using
UV illumination and by spraying with a 5% H2SO4 solution followed by heating. Analytical
HPLC was performed on a Cosmosil 5C18-ARII (Nacalai Tesque, Kyoto, Japan) column
(250 mm
×
4.6 mm, i.d.) with a gradient elution of 4–30% (39 min) and 30–75% (15 min)
CH3CN in 50 mM H3PO4 at 35 ◦C (flow rate, 0.8 mL/min; detection, JASCO photodiode
array detector MD-2010).
3.2. Plant Material
The whole plant of Persicaria capitata was collected in Shui Cheng, China, in 2014, and
in Nagasaki, Japan, in 2017. The aerial part of Persicaria chinensis were collected in Nagasaki,
Japan, in 2014. Voucher specimens were deposited at the Nagasaki University Graduate
School of Biomedical Sciences. Fresh rhizome of Polygonum runcinatum var. sinense were
purchased in a medicinal plant market in Guangxi, China. A voucher specimen was
deposited at the Guangxi Institute of Botany. Aerial parts of Reynoutria japonica, Persicaria
perfoliata, P. longiseta, P. lapathifolia, P. filiformis, and P. thunbergii were collected in Nagasaki,
Japan.
3.3. HPLC Analysis
Fresh aerial parts (1.0 g) of Reynoutria japonica, Persicaria perfoliata, P. longiseta, P.
lapathifolia, P. capitata (syn. Polygonum capitatum), P. chinensis (syn. Polygonum chinense),
P. filiformis, and P. thunbergii were extracted with 60% CH3CN (20 mL) and analyzed by
HPLC (Figure S1, Supplementary Materials).
3.4. Extraction and Separation
3.4.1. Persicaria Capitata
The EtOH extract (200 g) of P. capitata was suspended in 50% MeOH and insoluble
material was removed by filtration. The soluble part (132 g) was fractionated by Diaion
HP20SS column chromatography (8 cm i.d.
×
35 cm) with 0–100% MeOH (10% stepwise
gradient elution, each 500 mL) to give 8 fractions (fr.): fr. 1 (84.5 g), fr. 2 (2.2 g), fr. 3
(3.9 g), fr. 4 (9.5 g), fr. 1–5 (14 g), fr. 1–6 (4.3 g), fr. 1–7 (6.9 g), and fr. 1–8 (2.1 g). Fr. 3 was
subjected to a combination of column chromatography using Sephadex LH-20 (0–100%
MeOH), Avicel cellulose (2% AcOH), and Diaion HP20SS (0–50% MeOH) to give 2,3-di-O-
galloyl-D-glucose (719 mg) and 2,4-di-O-galloyl-D-glucose (101 mg). Similar separation
of fr. 4 by chromatography using Sephadex LH-20 and Chromatorex ODS (0–50% MeOH,
5% stepwise gradient) to yield 2,3,4-tri-O-galloyl-D-glucose (2.26 g), 6-O-galloyl arbutin
(79 mg), 4-hydroxy-3-methoxyphenol 1-O-
4-hydroxy-2-methoxyphenol 1-O-
-D-(60-O-galloyl)-glucopyranoside (46 mg). Fr. 6 was
purified by precipitation from water to give davidiin ( ) (13 g). Fr. 7 was subjected to
a column chromatography on Sephadex LH-20 (60–100% MeOH), Diaion HP20SS, and
Chromatorex ODS to give quercetin 3-O- -rhamnoside (2.5 g), quercetin (719 mg), quercetin
3-O- -D-glucopyranoside (182 mg), quercetin 3-O- -L-(3”-O-galloyl)-rhamnoside (6.7 mg).
The insoluble part of the extract (68 g) was washed with CHCl3–MeOH (1:1, v/v) to
β
-D-(60-O-galloyl)-glucopyranoside (37 mg),
β
1
α
β
α

Molecules 2021, 26, 337
8 of 11
remove non-polar substances and a part (5 g) of the residue (31.5 g) was subjected to
size-exclusion column chromatography using a Sephadex LH-20 column (4 cm i.d.
×
45 cm) with 7 M urea:acetone (2:3, v/v, containing conc. HCl at 5 mL/L) to give fractions
containing oligomeric polyphenols and compounds with low-molecular weight [35]. The
fractions were separated by Diaion HP20SS column chromatography to give oligomeric
proanthocyanidins (707 mg) and ellagic acid (54 mg).
3.4.2. Persicaria Chinensis
The fresh aerial part of P. chinensis (1.18 kg) was extracted with 60% aqueous acetone
(3 L) three times. The extract was concentrated and the resulting insoluble precipitates
were removed by filtration. The aqueous filtrate was applied to a column of Diaion HP20SS
(7 cm i.d.
give 11 fractions. Crystallization of fr. 3 from H2O yielded 1-O-galloyl-
(301 mg). Separation of fr. 7 (4.61 g) by Sephadex LH-20 (0–100% MeOH) gave polymeric
proanthocyanidins (1.37 g), (1.37 g), and 1,2,6-tri-O-galloyl- -D-glucopyranose (76 mg). Fr.
8 (11.2 g) was subjected to Sephadex LH-20 (0–100% MeOH and then MeOH–H2O–acetone,
×
50 cm) with 0–100% MeOH (10% stepwise gradient elution, each 500 mL) to
β-D-glucopyranose
3
β
8:1:1 and 3:1:1) to give quercetin 3-O-(2”-
(481 mg) and 2 (553 mg).
α-rhamnopyranosyl)-β-glucuronopyranoside
3.4.3. Polygonum Runcinatum var. Sinense
The dried root of Polygonum runcinatum var. sinense (805 g) was extracted with 60%
acetone (4 L) twice and then MeOH. The extracts were combined and concentrated, and
the aqueous solution was applied to a Diaion HP20SS column (8 cm i.d.
0–100% MeOH (10% stepwise gradient elution, each 500 mL) to give 13 fractions. HPLC
analysis showed that fr. 4–10 (total 159 g) contained as the major component. Fr. 3
(29.7 g) was separated by Sephadex LH-20 column chromatography (6 cm i.d.
with 0–100% MeOH to give (6.68 g), 10 (1.44 g), and a subfraction containing
×
35 cm) with
7
×
31 cm)
and 11
7
9
.
Further chromatography of the subfraction using Chromatorex ODS (0–50% MeOH) and
Toyopearl butyl-650M (0–50% MeOH) to yield (490 mg) and 11 (19 mg). Fr. 11 (40.2 g)
9
was separated by Sephadex LH-20 (60–100% MeOH), Sephadex LH-20 [7 M urea:acetone
(2:3, v/v, containing conc. HCl at 5 mL/L)], and Diaion HP20SS column chromatography to
afford
8 (990 mg) and a mixture of oligomeric polyphenols detected at origin on TLC plate.
3.5. Spectroscopic Data
3.5.1. Persicarianin (3)
Brown amorphous powder, [
1614, 1533, 1450, UV λmax (MeOH) nm (log ε): 271 (4.06), 218 (4.59), HRESIMS m/z: 673.0690
(Calculated for C27H22O19Na: 673.0653), ECD (MeOH) ∆ε (nm):
α
]
+43.8 (c 0.1, MeOH), IR
ν
cm−1: 3412, 1728, 1708,
25.16 (213), +6.68 (234),
max
D
−
1
−
5.47 (283), +2.36 (374). H-NMR (acetone-d6, 500 MHz) δH: 7.19 (s,
-galloyl-2,6), 6.76 (s,
α
-galloyl-2,6), 7.18 (s,
-DHHDP-3), 6.37 (s, -DHHDP-3), 5.63
-glc H-3), 5.40 (t, J = 9.4, -glc H-4), 5.32
-glc H-6), 4.94 (dd, J = 4.8, 10.7 Hz, -glc
-DHHDP-1’), 4.63 (s, -DHHDP-1’), 4.25 (m,
-glc H-6), 3.89 (dd, J = 9.8, 3.4 Hz,
-glc H-2), 13C NMR (acetone-d6, 125 MHz) δC: 192.0 (
-DHHDP-7), 166.9 (galloyl-7), 164.6 ( -DHHDP-7’), 151.7 (
-DHHDP-4), 145.9 (galloyl-3,5), 142.3 ( -DHHDP-6), 138.8 (galloyl-4), 135.4
-DHHDP-3’), 124.1 ( -DHHDP-2), 121.7 (galloyl-1), 112.7
-DHHDP-1), 110.1 (galloyl-2,6), 107.7 ( -DHHDP-3), 107.6 ( -DHHDP-3), 97.8 ( -glc
-DHHDP-5’), 93.6 ( -glc C-1), 91.5 ( -DHHDP-6’), 75.6 ( -glc C-3), 74.1
-glc C-4), 74.0 ( -glc C-4), 73.7 ( -glc C-2), 73.3 ( -glc C-3), 71.2 ( -glc C-2), 68.3 ( -glc
C-5), 66.9 (α-glc C-6), 66.4 (β-glc C-6), 64.4 (α-glc C-5), 43.0 (α,β-DHHDP-1’).
β
α
,
β
-DHHDP-6”), 6.38 (s,
α
β
(dd, J = 9.8, 9.5 Hz,
α
-glc H-3), 5.42 (t, J = 9.4 Hz,
β
α,β
(d, J = 3.4 Hz,
H-6), 4.88 (d, J = 7.8 Hz,
-glc H-5), 3.95 (m, -glc H-5,
(dd, J = 9.4, 7.8 Hz,
α
-glc H-1), 5.01 (dd, J = 4.8, 10.7 Hz,
-glc H-1), 4.65 (s,
-glc H-6,
α
α
β
α
β
α
α
α
β
α
-glc H-2), 3.66
β
α,β-DHHDP-4’),
α,β-DHHDP-2’),
168.1 (
146.0 (
α
,
β
α
,β
α
,
β
α,β
α,β
(
α
,β-DHHDP-5), 130.8 (
α
,
β
(
α
,β
β
α
β
H-1), 96.5 (
α
,β
α
α
,
β
β
(α
β
β
α
α
β

Molecules 2021, 26, 337
9 of 11
3.5.2. Polygonanin A (11)
Tan amorphous powder, [
α
]
−
51.3◦ (c 0.1, MeOH), FABMS m/z: 841 (M + Na)+, 819
D
(M + H)+, HRFABMS m/z: 819.0891 (M + H)+ (Calcd for C34H27O24: 819.0887), IR max cm-1
ν
(neat): 3412, 1719, 1616, 1445, 1313, 1223, 1190. UV
λ
max
(MeOH) nm (log
ε
): 266 (4.40), 230
(4.70). ECD (MeOH)
λ
max
(
∆ε): 316 (
−
2.4), 283 (+12.6), 261 (
−
8.9), 241 (+17.9), 227 (+0.6),
1
217 (+5.5). H NMR (500 MHz, acetone-d6) δH: 7.46 (1H, s, hydroxychebuloyl (HChe) H-3),
6.83 (1H, s, HHDP-3), 6.79 (1H, s, HHDP-3’), 5.95 (1H, s, glc H-1), 5.14 (1H, br s, glc H-3),
5.10 (1H, br s, glc H-4), 4.99 (1H, t, J = 11.1 Hz, glc H-6), 4.88 (1H, d, J = 2.8 Hz, HChe
H-3’), 4.83 (1H, br s, HChe H-2’), 4.72 (1H, br s, glc H-2), 4.48 (1H, dd, J = 5.3, 11.1 Hz, glc
H-5), 4.08 (1H, dd, J = 5.3, 11.1 Hz, glc H-6), 3.35 (1H, d, J = 15.9 Hz, HChe H-5’), 2.78 (1H,
d, J = 15.9 Hz, HChe H-5’). 13C NMR (125 MHz, acetone-d6) δC: 172.3 (HChe-1’), 172.0
(C-6’), 168.2 (HHDP-7’), 166.7 (HChe-7), 166.4 (HHDP-7), 166.1 (HChe-7’), 146.0 (HChe-4),
145.3 (HHDP-4’), 144.5, 144.9 (HHDP-6, 6’), 143.0 (HChe-6), 144.7 (HHDP-4), 138.5 (HChe-
5), 136.9 (HHDP-5), 136.3 (HHDP-5’), 125.9 (HHDP-2), 125.7 (HHDP-2’), 121.0 (HChe-2),
116.7 (HHDP-1), 116.2 (HChe-3), 115.4 (HHDP-1’), 114.5 (HChe-1), 109.8 (HHDP-3), 108.7
(HHDP-3’), 90.4 (glc C-1), 76.3 (HChe-4’), 71.7 (glc C-2), 70.9 (glc C-5), 69.2 (glc C-4), 66.3
(HChe-2’), 64.2 (glc C-6), 59.1 (glc C-3), 49.2 (HChe-3’), 41.6 (HChe-5’).
3.6. Acid Hydrolysis of 3
◦
Persicarianin (3) (10 mg) was hydrolyzed by heating in 5% H2SO4 (1 mL) at 105 C for
5 h in a screw capped vial. The precipitates (2.7 mg) formed in the mixture were collected
by filtration. HPLC analysis of the filtrate showed peaks assignable to gallic acid (8.94 min),
brevifolin carboxylic acid (21.36 min), and ellagic acid (30.66 min), which were identified by
comparison of tR and UV absorption with those of authentic samples. HPLC analysis of the
precipitates showed only a peak for ellagic acid. The filtrate was neutralized with saturated
Ba(OH)2 and resulting precipitate of BaSO4 was removed by filtration. The filtrate was
concentrated and the residue was dissolved in 0.5 mL of pyridine containing L-cysteine
HCl (5.0 mg), and heated at 60 ◦C for 1 ◦h. To the mixture was added o-tolylisothiocyanate
(20 µL) and this mixture heated at 60 C for 1 h. The final mixture was then cooled to
ambient temperature and directly analyzed using HPLC. The retention time of the peak at
34.8 min coincided with that of the thiazolidine derivatives of D-glucose (L-glucose: 35.
6 min).
3.7. Computational Calculation of 8
A conformational search was performed using the Monte Carlo method and the
0
MMFF94 force field with Spartan 14 (Wavefunction, Irvine, CA). The low-energy con-
formers within a 6 kcal/mol window were optimized at the B3LYP/6-31G(d,p) level in
acetone (SMD). The vibrational frequencies were also calculated at the same level to con-
firm their stability, and no imaginary frequencies were found. The magnetic shielding
constants (σ) of the low-energy conformers with Boltzmann populations greater than
1% were calculated using the gauge-independent atomic orbital (GIAO) method at the
mPW1PW91/6-311+G(2d,p) level in acetone (PCM) and were weight-averaged [36–38].
3.8. Thiol Degradation
Polymeric polyphenols obtained from the three plants (5 mg) were dissolved in a
solution containing 4% 2-mercaptoethanol and 0.1% HCl in 60% EtOH (1 mL). The mixture
◦
was heated at 70 C for 7 h and analyzed by HPLC. The standard thiol degradation products
were obtained from persimmon proanthocyanidins [34].
4. Conclusions
Distribution of hydrolyzable tannins in the plant kingdom is much more limited
compared with that of proanthocyanidins. In Polygonaceous plants, a hydrolyzable tannin
had only been isolated from Persicaria capitatum. In this study, we reinvestigated P. capitatum
and showed the presence of minor gallotannins and proanthocyanidin oligomers comprised

Molecules 2021, 26, 337
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of epicatechin and epicatechin-3-O-gallate. From Persicaria chinensis, a new hydrolyzable
tannin called persicarianin (3) was isolated together with an ellagitannin geraniin and
proanthocyanidin oligomers mainly comprising epicatechin, epigallocatechin and their
galloyl esters. The rhizome of Polygonum runcinatum var. sinense contained granatin A
(7) as the major constituent along with minor ellagitannins, including a new ellagitannin
polygonanin A (11). These results prompted us to continue investigations into hydrolyzable
tannins in this plant family.
Supplementary Materials: The following are available online, Figure S1: HPLC of extracts of Polyg-
onaceous plants, Figure S2: HPLC analysis of acid hydrolysis products of 3, Figure S3: Thiol degrada-
tion of proanthocyanidin oligomers, Figures S4–S8: 1D and 2D NMR spectra of
3
, Figures S9–S14: 1D
1
and 2D NMR spectra of 11. Figures S15 and S16: H and 13C NMR spectra of
7, Figures S17 and S18:
1H and 13C NMR spectra of 7a.
Author Contributions: Y.-Q.L., M.K., J.T. performed experiments including extraction, chromato-
graphic separation, and spectroscopic analyses. T.T., Y.-L.H., D.-P.L., Z.-H.J. and G.-i.N. conceived
and designed the experiments. Z.-H.J., Y.-F.W., Y.-L.H., T.T. and G.-i.N. collected plant materials. Y.M.
performed the computational calculation. Y.-Q.L., M.K., Y.S. and Y.M. analyzed the data. Y.M. and
T.T. wrote the paper. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by JSPS KAKENHI Grant Numbers 20K07102, and 17K08338.
Acknowledgments: The authors are grateful to N. Tsuda, K. Chifuku and H. Iwata, Center for
Industry, University and Government Cooperation, Nagasaki University, for recording NMR and MS
data. Some computations were carried out using the computer facilities at the Research Institute for
Information Technology, Kyushu University.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples are not available from the authors.
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