molecules

Article and Oligomeric Proanthocyanidins of Three Polygonaceous

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; [email protected] (Y.-Q.L.); [email protected] (Y.M.); [email protected] (Y.S.) 2 College of Medical Laboratory Science, Guilin Medical University, 109, Huancheng North 2 Road, Guilin 541004, China 3 Department of Natural Product Chemistry, School of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan; [email protected] (M.K.); [email protected] (J.T.) 4 Guangxi Key Laboratory of Functional Phytochemicals Research and Utilization, Guangxi Institute of Botany, Guilin 541006, China; [email protected] (Y.-F.W.); [email protected] (Y.-L.H.); [email protected] (D.-P.L.) 5 Usaien Pharmaceutical Company, Ltd., 1-4-6 Zaimoku, Saga 840-0055, Japan; [email protected] 6 Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau 999078, China; [email protected] * Correspondence: [email protected]; 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 chinensis, a   new hydrolyzable tannin called persicarianin was isolated and characterized to be 3-O-galloyl-4,6-(S)- dehydrohexahydroxydiphenoyl-D-. Interestingly, acid hydrolysis of this compound afforded Citation: Li, Y.-Q.; Kitaoka, M.; , despite the absence of a hexahydroxydiphenoyl group. From the rhizome of Takayoshi, J.; Wang, Y.-F.; Matsuo, Y.; Saito, Y.; Huang, Y.-L.; Li, D.-P.; runcinatum var. sinense, a large amount of , along with minor ellagitannins, helioscpoinin Nonaka, G.-i.; Jiang, Z.-H.; et al. A, davicratinic acids B and C, and a new called polygonanin A, were isolated. Based Ellagitannins and Oligomeric on 2D nuclear magnetic resonance (NMR) spectroscopic examination, the structure of polygonanin Proanthocyanidins of Three A was determined to be 1,6-(S)-hexahydroxydiphenoyl-2,4-hydroxychebuloyl-β-D-glucopyranose. Polygonaceous Plants. Molecules 2021, These are the second and third hydrolyzable tannins isolated from Polygonaceous plants. In addition, 26, 337. https://doi.org/10.3390/ oligomeric proanthocyanidins of Persicaria capitatum and P. chinensis were characterized by thiol molecules26020337 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 Keywords: ellagitannin; hydrolyzable tannin; proanthocyanidin; Persicaria; Polygonum; Polygo- Accepted: 7 January 2021 naceae Published: 11 January 2021

Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional clai- 1. Introduction ms in published maps and institutio- nal affiliations. 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 Copyright: © 2021 by the authors. Li- in the kingdom is relatively limited. Among Polygonaceous plants, only 2,3,4-tri-O- censee MDPI, Basel, Switzerland. galloyl-1,6-(S)-HHDP-β-D-glucose (davidiin, 1) has been isolated from Persicaria capitatum This article is an open access article (syn. Polygonum capitatum)[6]; its biological activity and metabolism have also been distributed under the terms and con- reported [7–9]. More recently, the presence of and in the aerial parts ditions of the Creative Commons At- of Persicaria chinense var. hispidum was suggested by the use of high-performance liquid tribution (CC BY) license (https:// chromatography (HPLC)-mass spectroscopy (MS) [10]. In the course of chemical studies creativecommons.org/licenses/by/ on ellagitannins, we analyzed aqueous CH CN extracts of eight Polygonaceous plants, 4.0/). 3

Molecules 2021, 26, 337. https://doi.org/10.3390/molecules26020337 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 337 2 of 11

Molecules 2020, 25, x , Persicaria perfoliata, P. longiseta, P. lapathifolia, P. capitata, P. chinensis2 of 11, Molecules 2020, 25, x 2 of 11 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 (Figure S1, Supplementary(Figure S1, Supplementary Materials). In Materials the study,). Inonly the the study, extracts only of the P. extractscapitata ofand P. P. capitata and P. showed peaks with UV absorption characteristic to ellagitannins (Figure1). The results chinensis showedchinensis peaks showed with UV peaks absorption with UV characteristic absorption characteristic to ellagitannins to ellagitannins(Figure 1). The (Figure 1). The were consistent with the above-mentioned studies; the major ellagitannin of P. capitata results were consistentresults were with consistent the above with-mentioned the above studies-mentioned; the ma studiesjor ellagitannin; the major of ellagitannin P. cap- of P. cap- was davidiin (1), and P. chinensis contains geraniin (2) (Figure2). In addition, we found itata was davidiinitata was (1), davidiinand P. chinensis (1), and contains P. chinensis geraniin contains (2) (Figuregeraniin 2) (.2 )In (Figure addition, 2). weIn addition, we that the rhizome of Polygonum runcinatum var. sinense, a Chinese perennial plant used found that thefound rhizome that theof Polygonum rhizome of runcinatum Polygonum var. runcinatum sinense, avar. Chinese sinense perennial, a Chinese plant perennial plant as a folk medicine for detoxification and hemostasis, contains ellagitannins as the major used as a folkused medicine as a folk for medicinedetoxification for detoxification and hemostasis, and containshemostasis, ellagitannins contains ellagitanninsas the as the constituents. This paper describes the isolation of phenolic substances from these three major constituents.major constituents.This paper describes This paper the describes isolation theof phenolic isolation substance of phenolics from substance these s from these plants and structural determination of the two new ellagitannins obtained from Persicaria three plants andthree structur plantsal and determination structural determination of the two new of ellagitanninsthe two new ellagitanninsobtained from obtained Per- from Per- sicariachinensis chinensisand Polygonum and Polygonum runcinatum runcinatumvar. sinense. var. sinense.In addition, In addition, proanthocyanidin proanthocyanidin oligomers oli- sicaria chinensisfrom andPersicaria Polygonum capitata runcinatumand Persicaria var. sinense. chinensis In addition,were chemically proanthocyanidin characterized. oli- gomers from gomersPersicaria from capitata Persicaria and Persicaria capitata and chinensis Persicaria were chinensis chemically were characterized. chemically characterized.

(a) 1 CH-09 [MaxABS (200.0--550.0 nm)] (b) tsurusoba - CH5 (c) 7 Euphorbia - CH5 (a) 1800000 1 CH-09 [MaxABS (200.0--550.0 nm)] (b) tsurusoba - CH5 (c) 7 Euphorbia - CH5 1800000 1600000 1600000 600000 1400000 600000 1400000 Fl 1200000 Fl 1200000 Fl 2 1000000 Fl 4000002 1000000 1000000 400000 Fl 1000000 800000

Intensity[µAU] Fl Intensity[µV] 3 800000 Fl Intensity[µV]

Intensity[µAU] Intensity[µV] 3 600000 Fl Intensity[µV] 600000 200000 400000 200000 400000 200000

200000 abs) (max absorption UV -0 UV absorption (max abs) (max absorption UV 0 0 -0 0 0 0.0 10.0 20.0 30.0 40.0 50.0 60.0 0.0 10.0 20.0 30.0 40.0 50.0 60.0 0 10 20 Retention 30 Time [min] 40 50 min 0 10 20 30 40 50 min 0.0 10.0 20.0 30.0 40.0 50.0 60.0 0.0 10.0 20.0 30.0 40.0 50.0 60.0 0.0 10.0 20.0 30.0 40.0 50.0 60.0 0 10 20 Retention 30 Time [min] 40 50 min 0 10 20 Retention 30 Time [min] 40 50 min 0 10 20 30 40 50 min 0 0.0 10.010 2020.0 30.030 40.040 50.050 min 60.0 Retention Time [min] Figure 1.1. HPLC profilesprofiles ofof 60%60% CH CH33CNCNextracts extracts of of ( a()a)Persicaria Persicaria capitata capitata,(,b ()bPersicaria) Persicaria chinensis chinensisand and (c) (Polygonumc) Polygonum runcinatum runcina- Figure 1. HPLC profiles of 60% CH3CN extracts of (a) Persicaria capitata, (b) Persicaria chinensis and (c) Polygonum runcina- tum var. sinense. 1: davidiin, 2: geraniin, 3: persicarianin, 7: granatin A and Fl: flavonoid glycosides. tum var. sinense.var. 1: sinense.davidiin,1: 2 davidiin,: geraniin,2: 3 geraniin,: persicarianin,3: persicarianin, 7: granatin7 A: granatin and Fl: flavonoid A and Fl: glycosides. flavonoid glycosides.

HO HO OH OH HO HO OH OH HO HO OH OH HO HO OH OH R HO OH R HO OH HO OH S HO OH S (S)-HHDP OH (S)-HHDP (R)-HHDP OOH O O (R)-HHDP O O O O O O O O O OH O O O O OH HO O O O HO O OH O O O OH O OH HO O OH HO O OH O O O O OHO O O O O O O O H O O O HOO O OO O HO O OH H O H OH galloyl HO H galloyl HO O OH HO O OHO OH HO O OH HO HO O HO OH HO OH HO O OH HO HO O OH HO OH (R)-DHHDP HOOHHO O OH (R)-DHHDP OH 1 2 1 2 Figure 2. Structures of davidiin (1) from Persicaria capitata and geraniin (2) from Persicaria chinensis. FigureFigure 2. 2.Structures Structures of of davidiin davidiin (1 )(1 from) fromPersicaria Persicaria capitata capitataand and geraniin geraniin (2 )(2 from) fromPersicaria Persicaria chinensis. chinensis.

2. Results and2. DiscussionResults and Discussion 2.1. Polyphenols2.1. of Polyphenols Persicaria capitata of Persicaria capitata The EtOH extractThe EtOH of Persicaria extract capitata of Persicaria whole capitata plant, collectedwhole plant plant, in Shui, collected Cheng, in Guizhou,Shui Cheng, Guizhou, Guizhou, China, was fractionatedChina, was was byfractionated fractionated Diaion HP20SS by by Diaion Diaion and furtherHP20SS HP20SS separated and and further further using separated separated Sephadex using using LH Sephadex-20 Sephadex LH LH--20 and Chromatorexand20 and Chromatorex ODS Chromatorex to give davidiinODS ODS to give (1 to) as davidiin give the davidiinmajor (1) constituentas (the1) asmajor the (7% constituent major from constituent the (7%extract), from (7% the from extract), the extract), along with minor compounds, which were identified as 2,3-di- [11], 2,4-di- [12], along with minoralong compounds, with minor compounds,which were identifiewhich wered as identifie2,3-di- [11d ]as, 2,4 2,3-di-di- -[ 12[11],] ,2,3,4 2,4--ditri- -[ 12], 2,3,4-tri- 2,3,4-tri- [13], and 2,3,6-tri-O-galloyl [14], 6’-O-galloyl arbutin [15], 4-hydroxy-3- [13], and 2,3,6[13-tri],- Oand-galloyl 2,3,6- triglucoses-O-galloyl [14] glucoses, 6’-O-galloyl [14], arbutin6’-O-galloyl [15], arbutin4-hydroxy [15-]3,- 4methox--hydroxy-3-methox- yphenolmethoxyphenol 1-O-β-D 1--(6O-’-βO-D-galloyl)-(6’-O-galloyl)-glucopyranoside-glucopyranoside [16], 4 [-16hydroxy], 4-hydroxy-2-methoxyphenol-2-methoxyphenol 1-O- yphenol 1-O-β-D-(6’-O-galloyl)-glucopyranoside [16], 4-hydroxy-2-methoxyphenol 1-O- 1-β-OD-(6β-’D-O-(6’--galloyl)O-galloyl)-glucopyranoside-glucopyranoside [16], [quercetin16], quercetin 3-O-β 3--DO--glucosideβ-D-glucoside [17], [quercetin17], quercetin 3-O- β-D-(6’-O-galloyl)-glucopyranoside [16], quercetin 3-O-β-D-glucoside [17], quercetin 3-O- α3--OL--rhamnopyranosideα-L-rhamnopyranoside [17], [ quercetin17], quercetin 3-O- 3-αO-L--α(3-L-(3”-’’-O-galloyl)O-galloyl)-rhamnopyranoside-rhamnopyranoside [18] [,18 el-], α-L-rhamnopyranoside [17], quercetin 3-O-α-L-(3’’-O-galloyl)-rhamnopyranoside [18], el- ellagic acid, and quercetin. lagic acid, andlagic quercetin. acid, and quercetin.

Molecules 2021, 26, 337 3 of 11 Molecules 2020, 25, x 3 of 11

2.2. Polyphenols from Persicaria Chinensis 2.2. Polyphenols from Persicaria Chinensis Similar separation of an aqueous acetone extract of the fresh aerial part of Persicaria Similar separation of an aqueous acetone extract of the fresh aerial part of Persi- chinensis collected in Nagasaki, Japan, afforded 1-O- [19] and 1,2,6-tri-O-galloyl-β-D-glu- caria chinensis collected in Nagasaki, Japan, afforded 1-O-[19] and 1,2,6-tri-O-galloyl-β- coses [20], geraniin (2) [21], quercetin 3-O-(2’’-α-rhamnopyranosyl)-β-glucuronopyra- D-glucoses [20], geraniin (2)[21], quercetin 3-O-(2”-α-rhamnopyranosyl)-β-glucuronop noside [22], and a new ellagitannin called persicarianin. Persicarianin (3) was obtained as yranoside [22], and a new ellagitannin called persicarianin. Persicarianin (3) was obtained aas brown a brown amorphous amorphous powder, powder, and and high high-resolution-resolution electrospray electrospray ionization ionization time time of of flight flight (HRESITOF) MS indicated a molecular formula of C27H22O19 (m/z: 673.0689, calculated for (HRESITOF) MS indicated a molecular formula of C27H22O19 (m/z: 673.0689, calculated C27H22O19Na: 673.0653). The 1H NMR1 spectrum showed signals arising from α- and β-hex- for C27H22O19Na: 673.0653). The H NMR spectrum showed signals arising from α- and opyranoses, and it is apparent that the sugar is a 4C1-glucopyranose4 based on the large β-hexopyranoses, and it is apparent that the sugar is a C1-glucopyranose based on the coupling constants of the pyranose ring proton signals (J2,3, J3,4, J4,5 = 8–10 Hz). The low large coupling constants of the pyranose ring proton signals (J2,3, J3,4, J4,5 = 8–10 Hz). The fieldlow fieldshifts shifts of H of-3 H-3[δ 5.63 [δ 5.63(α-H (-α3),-H-3), 5.40 5.40(β-H (-β3)],-H-3)], H-4 H-4[5.40 [5.40 (α-H (-α4),-H-4), 3.94 3.94(β-H (-β4)],-H-4)], and andH-6 [4H-6.94, [4.94, 3.95 3.95 (α-H (α-6),-H-6), 5.40, 5.40, 3.92 3.92 (β- (Hβ--H-6)]6)] indicate indicatess acylation acylation of of these these positions. positions. The The acyl groupsgroups were were shown shown to to be be a galloyl a galloyl and and a dehydrohexahydroxydiphenoyl a dehydrohexahydroxydiphenoyl (DHHDP) (DHH groupDP) groupby 13C by NMR 13C NMR spectroscopy, spectroscopy which, which exhibited exhibited signals signals attributable attributable to an to aliphatic an aliphatic methine me- thine(δ 43.0), (δ 43.0), a double a double bond bond (δ 151.7 (δ 151.7 and and 130.8), 130.8), a ketone a ketone (δ 192.0), (δ 192.0), and and two two acetal acetal carbons carbons (δ (δ91.5 91.5 and and 96.5), 96.5), constructing constructing a hydrated a hydrated cyclohexenetrione cyclohexenetrione ring ring of the of DHHDPthe DHHDP group. group. The Thelarge large chemical chemical shift shift differences differences of the of glucose the glucose H-6 methylene H-6 methylene proton proton signals signals in the in1H the NMR 1H NMRspectrum spectrum suggested suggested that the that DHHDP the DHHDP esters esters bridges bridges between between the C-4 the and C-4 C-6and hydroxy C-6 hy- droxygroups groups [23]. This [23]. was This confirmed was confirmed by heteronuclear by heteronuclear multiple multiple bond bond coherence coherence (HMBC) (HMBC cor-) correlationsrelations of theof the glucose glucose H-6 H- and6 and H-4 H- to4 to the the DHHDP DHHDP C-7’ C-7 and’ and C-7 C-7 ester ester carbonylcarbonyl carbons,carbons, respectivelyrespectively (Figure (Figure 33).). TheThe configurationconfiguration of thethe DHHDPDHHDP methine carbon (C-1’)(C-1’) was con- cludedcluded to to be be SS,, based on on the the appearance appearance of negative and positive Cotton effects at 213 nm andand 234 234 nm, respectively [24] [24].. The l locationocation of the galloyl group was determined to be at thethe glucose glucose C C-3-3 hydroxy hydroxy group group by observation observation of HMBC correlations between the glucose HH-3-3 and galloyl C C-7.-7. The D configuration configuration of of the the glucosy glucosyll moiety was determined via acid hydrolysishydrolysis followed byby HPLCHPLC analysisanalysis of of the the thiazolidine thiazolidine derivatives derivatives prepared prepared by by reaction reac- tionwith with L-cysteine L-cysteine and o -tolylisothiocyanateand o-tolylisothiocyanate [25,26 ].[25,26 Based]. onBased this evidence,on this evidence, persicarianin persicar- was ianincharacterized was characterized to be 3-O-galloyl-4,6-( to be 3-O-galloylS)-DHHDP--4,6-(S)-DDHHDP-glucose-D (3-glucose). HPLC (3 analysis). HPLC of analysis the afore- of thementioned aforementioned hydrolysis hydrolysis products, products before condensation, before condensation with cysteine, with cysteine revealed, productionrevealed pro- of ductiongallic acid of (gallic4), ellagic acid acid (4), (ellagic5), and acid brevifolin (5), and carboxylic brevifolin acid carboxylic (6) (Figure acid S2, Supplementary(6) (Figure S2, SupplementaryMaterials). Ellagic Materials) acid is a. Ellagic bislactone acid form is a bislactone of the HHDP form group, of the and HHDP therefore, group, a and reduction there- fore,product a reduction of the DHHDP product ester of the of DHHDP3. Similar ester production of 3. Similar of 5 productionfrom DHHDP of 5 groups from DHHDP on acid groupshydrolysis on acid have hydrolysis been also observedhave been for also2 and observed related for ellagitannins, 2 and related and ellagitannins the reaction, wasand thededuced reaction tobe was a redox deduced disproportionation to be a redox [disproportionation27,28]. Ellagitannins [2 were7,28]. originally Ellagitannins defined were as originallyhydrolyzable defined tannins as hydrolyzable which afford tannins5 upon which hydrolysis, afford and 5 upon5 is usuallyhydrolysis considered, and 5 is tousu- be allyoriginated considered from to HHDP be originated groups. Infrom this HHDP context, groups. despite In the this absence context, of despite a HHDP the group, absence3 is ofalso a HHDP an ellagitannin. group, 3 is also an ellagitannin.

O 3' HO O 7" OH OH O 6' HO O O OH HO H1' 6 1 CO H 6 4 O HO OH 2 5% H2SO4 O O HO 7 O O O 3 + + OH + D-glucose OH OH O O 3 O 7 OH 105 °C, 5 h O HO O CO2H OH (S)-DHHDP OH 2 OH OH OH OH 3 4 5 6 FigureFigure 3. 3.Selected Selected heteronuclear heteronuclear multiple multiple bond bond coherence coherence (HMBC) (HMBC correlations) correlations and and acid acid hydrolysis hydrolysis of of 3- 3O-O-galloyl-4,6-(-galloyl-4,6S- )- DHHDP-D-glucose (3). (S)-DHHDP-D-glucose (3).

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 (7)

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 Molecules 2020, 25, x showed a prominent peak arising from a principal phenolic component (Figure1c), which4 of 11 was isolated by Diaion HP20SS column chromatography and identified as granatin A (7) by 1Dby 1D and and 2D NMR2D NMR spectroscopic spectroscopic analysis analysis [29]. [ Furthermore,29]. Furthermore, theacetonyl the acetonyl derivative derivative7a was 7a 7 preparedwas prepared by treatment by treatment of with of 7 aqueouswith aqueous acetone acetone containing containing HCO2 HCONH4,2NH and4 spectroscopic, and spectro- comparisonscopic comparison of 7a with of 7a those with those of an of authentic an authentic sample sample provided provided further further evidence evidence for thefor structurethe structure of 7 of[30 7 ].[30 In]. In the the purification purification process process of of7 7(Figure (Figure4), 4), four four minor minor ellagitannins ellagitannins werewere isolatedisolated byby aa combination ofof columncolumn chromatography usingusing Chromatorex ODSODS andand Toyopearl Butyl-650MButyl-650M to to give give helioscopinin helioscopinin A A (8 ()8[)[31],31 davicratinic], davicratinic acids acids B (9 B) and (9) andC (10 C) 10 ([32)[], 32and], anda new a new ellagitannin ellagitannin called called polygonanin polygonanin A. A.

HO HO OH OH HO HO OH OH HO HO OH OH

HO OH OH HO OH HO OH

O O galloyl = OH O O O O O O O O-R O OH O O O OH OH O O O

O O O O O O O H O O O O O O O 5' H H OH H HO O HO HO O HO 3' O HO O 2' OH H O OH 1' HO O HO O OH HO O OH OH HO OH CO2H OH O 7 : R = H 7a 9 : 2' -H 10: 2' -H 8 : R = galloyl FigureFigure 4.4. StructuresStructures ofof 7–107–10 andand acetonylacetonyl derivativederivative 7a7a..

1 13 The 1H and 13CC NMR NMR spectra spectra of of polygonanin polygonanin A A ( (1111)) was related to those of 9 and 10, indicatingindicating 1,2,4,6-acylated1,2,4,6-acylated glucopyranoseglucopyranose withwith aa HHDPHHDP group.group. TheThe locationlocation ofof thethe HHDPHHDP group atat thethe 1,6-positions1,6-positions ofof glucoseglucose waswas confirmedconfirmed by by HMBC HMBC correlations correlations of of glucose glucose H-1 H- δ δ J J (1 H(δ5.95,H 5.95, s) s) and and H-6 H [-6H [δ4.99H 4.99 (t, (t,= J 11.1 = 11.1 Hz), Hz), 4.08 4.08 (dd, (dd,=5.3, J = 11.15.3, 11.1 Hz)] Hz)] with with the HHDP the HHDP ester carbonyl carbons (δ 166.4, 168.2). The atropisomerism of the HHDP was shown to be an S ester carbonyl carbonsC (δC 166.4, 168.2). The atropisomerism of the HHDP was shown to configurationbe an S configuration by appearance by appearance of positive of positive and negative and negative Cotton effects Cotton at effects 242 nm at and242 262nm nm,and respectively,262 nm, respectively, in the electronic in the electronic circular circular dichroism dichroism (ECD) spectrum.(ECD) spectrum. The configuration The configura- is thetion same is the as same that as of that1, 7 ,of9, 1and, 7, 910, and. Coupling 10. Coupling constants constant of thes of pyranose the pyranose ring protonsring protons (J1,2, J , J , and J ) were <2 Hz. This was similar to those of 7, 9, and 10, but different to (2,3J1,2, J3,42,3, J3,4, and4,5 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 those of 1 (J1,2=3.2 Hz, J2,3=8.5 Hz, J3,4=7.7 Hz,1 and J4,5=2.9 Hz), suggesting that the glucopy- glucopyranoses of 7, 9, 10, and 11 adopt a C4 conformation, whereas the glucopyranose ranoses of 7, 9, 10, and 11 adopt a 1C4 conformation, whereas the glucopyranose core of 1 core of 1 with a 2,3,4-trigalloyl structure adopts a boat conformation (Figure1). The NMR with a 2,3,4-trigalloyl structure adopts a boat conformation (Figure 1). The NMR signals signals indicated that the 2,4-acyl group of 11 was composed of 3 carboxyl carbons (δC indicated that the 2,4-acyl group of 11 was composed of 3 carboxyl carbons (δC 172.3 (C- 172.3 (C-1’), 172.0 (C-6’), 166.1 (C-7’)), an oxygenated tertiary carbon (δC 76.3 (C-4’)), an 1’), 172.0 (C-6’), 166.1 (C-7’)), an oxygenated tertiary carbon (δC 76.3 (C-4’)), an oxygenated oxygenated methine (δC 66.3 (C-2’)), a methylene (δC 41.6 (C-5’)), and a benzylic methine methine (δC 66.3 (C-2’)), a methylene (δC 41.6 (C-5’)), and a benzylic methine (δC 49.2 (C- (δC 49.2 (C-3’)), along with a trihydroxy benzoyl moiety. Taking the molecular formula 3’)), along with a trihydroxy benzoyl moiety. Taking the molecular formula C34H26O24 in- C H O indicated by HR-fast atom bombardment (FAB) MS into account, these building dicated34 26 by24 HR-fast atom bombardment (FAB) MS into account, these building blocks of blocks of the 2,4-acyl group suggested that 8 is generated by addition of H2O to the the 2,4-acyl group suggested that 8 is generated by addition of H2O to the double bond of double bond of 9 or 10 and rearrangements of lactone formation. This was supported by 9 or 10 and rearrangements of lactone formation. This was supported by HMBC correla- HMBC correlations, as illustrated in Figure5. The planar structure of this acyl group is the sametions, asas theillustrated 4’-hydroxychebuloyl in Figure 5. The group plan ofar an structure ellagitannin of this isolated acyl group from ais Euphorbiaceous the same as the plant4’-hydroxychebuloyl [33], and the chemical group shifts of an of ellagitannin the aliphatic isolated proton from and carbon a Euphorbiaceous signals in the plant literature [33], and the chemical shifts of the aliphatic proton and carbon signals in the literature (δH 4.84 (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’); (d, J = 5Hz, 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- 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,2’), HChe 47.3 (C H-2’)-3’), and77.6 (C 4.88-4’ (d,), 42.5J = (C 2.8-5’ Hz,)) are HChe similar H-3’), to those 3.35 of and 11 2.78(δH 4.83 (each (br d, s,J HChe= 15.9 H Hz,-2’) and 4.88 (d, J = 2.8 Hz, HChe H-3’), 3.35 and 2.78 (each d, J = 15.9 Hz, 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 (NO- ESY) 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 Fig- ure 5(b). 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

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 Figure5b. The NOESY correlation between H-2’ and one of the H-5’ methylene Molecules 2020, 25, x 5 of 11 protons confirmed the relative configurations of C-2’ – C-5’. From the biogenesis illustrated SchemeScheme 1,1 the, the configuration configuration of the of the benzylic benzylic methines methines of the of acyl the acylgroups groups of 11 ofwa11s deducedwas deduced to be the same as that of the DHHDP group of 7. Interestingly, the glucose H-3 of 11 (δ to be the same as that of the DHHDP group of 7. Interestingly, the glucose H-3 of 11 (δH H 5.14) resonated at a much lower field compared to those of 7a (δ 4.58), 9 (δ 4.40) and 10 5.14) resonated at a much lower field compared to those of 7a (δH 4.58)H , 9 (δH 4.40)H and 10 δ (δ(H H4.44).4.44). This This could could be bedue due to the to thedeshielding deshielding effect effect of the of ester the ester carbonyl carbonyl group group attached attached toto the the glucose glucose C- C-22 hydroxy hydroxy group group (Figure (Figure 5b),5 b),suggesting suggesting that that hydroxylation hydroxylation at C- at4’ C-4’of the of the 2,42,4-acyl-acyl group group affect affectss the the conformation conformation of the of themacrocyclic macrocyclic ester ester structure. structure.

HO HO OH OH

HO OH

O O O O OH O

O O

O O CO2H H HO OH H HO O OH HMBC (H to C) O (a) (b)

FigureFigure 5. 5.(a)( aStructure) Structure and and selected selected HMBC HMBC correlations correlations for 11. for (b)11 A.( partialb) A partialstructure structure of the most of the most stablestable conformer conformer of 11 of and11 and nuclear nuclear Overhauser Overhauser effect spectroscopy effect spectroscopy (NOESY (NOESY)) correlations correlations of the of the glucose and 2,4-acyl group. glucose and 2,4-acyl group.

2.4. Proanthocyanidins of Persicaria Capitata and Persicaria Chinensis O O In additionH to hydrolyzable tannins, oligomeric proanthocyanidins are also important HconstituentsO of PersicariaO capitata and Persicaria chinensis. The oligomers were detected as broad humps on theOH HPLC baseline; thiol degradation using 2-mercaproethanol was used HO O OH O to characterizeH+ the structural components [34] (Figure S3, Supplementary Materials). HPLC H analysis of4 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 proanthocyanidinsO O of P. chinensis yieldedO 2-hydroxyethylsulfanylO derivativesO of epicatechin,O H epigallocatechin,H epicatechin-3-+ H2O O-gallate and epigallocatechin-3- H2O O-gallate originatingH from HextensionO units, alongO with the freeHO form of epicatechin-3-CO2H O-gallateHO originating fromO the H HO OH HO H O terminal unit. The results indicates that proanthocyanidinsOH in these two plants belonging HO HO H HO CO2H HO OH CO H the same genusCO2PersicariaH are composed of different flavan-3-ol units (Figure6). In contrast,2 high-molecular weight polyphenols obtained from the rhizome of Polygonum9, 10 runcinatum + H2O 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 O O CO2H O O CO2H underway. H H2O H HO OH HO OH HO H HO H HO OH CO2H HO O O 11 Scheme 1. Plausible production mechanism of 9–11 from 4.

Molecules 2020, 25, x 5 of 11

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 7. Interestingly, the glucose H-3 of 11 (δH 5.14) resonated at a much lower field compared to those of 7a (δH 4.58), 9 (δH 4.40) and 10 (δH 4.44). This could be due to the deshielding effect of the ester carbonyl group attached 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.

HO HO OH OH

HO OH

O O O O OH O

O O

O O CO2H H HO OH H HO O OH HMBC (H to C) O (a) (b) Molecules 2021, 26Figure, 337 5. (a) Structure and selected HMBC correlations for 11. (b) A partial structure of the most 6 of 11 stable conformer of 11 and nuclear Overhauser effect spectroscopy (NOESY) correlations of the glucose and 2,4-acyl group.

O O H HO O OH HO O OH O H+ H Molecules 2020, 25, x 4 6 of 11 2.4. Proanthocyanidins of Persicaria Capitata and Persicaria Chinensis In addition to hydrolyzable tannins, oligomeric proanthocyanidins are also im-

O Oportant constituents of PersicariaO capitataO and Persicaria chinensisO . TheO oligomers were de- tected as broad humps on the HHPLC baseline; thiol degradation using 2-mercaproethanol H + H2O H2O H HO wasO used to characterizHO e the structuralCO components2H [H3O4] (Figure S3, SupplementaryO Mate- H HO rials).O HPLCH analysis of the HdegradationO products obtained fromH proanthocyanidinsO of OH HO HO H HO CO2H HO OH CO H CO2PersicariaH capitata exhibited peaks attributable to 4β-(2-hydroxyethylsulfanyl)2 derivatives of epicatechin and epicatechin-3-O-gallate originating from extension9, 10 units, accompanied by small peaks of catechin, epicatechin,+ H2O 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 O O originating from extension units, alongCO 2withH the free formO of epicatechinO CO-23H-O-gallate orig- H inating from the terminal unit. The results indicates H2O that proanthocyanidinsH in these two HO OH plants belonging the same genus Persicaria are composedHO of differentOH flavan-3-ol units HO H HO H (Figure 6). In contrast, high-molecular weight polyphenols obtained from the rhizome of HO OH CO2H HO O Polygonum runcinatum var. sinense did not yield 2-hydroxyethylsulfanylO derivatives on thiol degradation, indicating that the polyphenols are not proanthocyanidins.11 The 13C Scheme 1. PlausibleNMR productionScheme spectrum 1. mechanismPlausible suggested production of 9 –that11 from the mechanism 4polyphenols. of 9–11 arefrom oligomeric4. hydrolyzable tannins. Fur- ther investigations are now underway.

R1 OH

HO O OH

O-R2 R1 OH OH

extension units HO O OH n O-R 2 Persicaria capitata OH OH R1 = H HO O R2 = H or galloyl OH Persicaria chinensis terminal units O-R2 R1 = H or OH OH R2 = H or galloyl FigureFigure 6. 6.Structures Structures of of oligomeric oligomeric proanthocyanidins proanthocyanidins of ofPersicaria Persicaria capitata capitataand andP. P chinensis. chinensissuggested sug- bygested thiol by degradation. thiol degradation.

3.3. MaterialsMaterials andand MethodsMethods 3.1.3.1. GeneralGeneral InformationInformation OpticalOptical rotationsrotations were measured measured on on a aJASCO JASCO P- P-10201020 digital digital polarimeter polarimeter (JASCO (JASCO,, To- Tokyo,kyo, Japan). Japan). IR spectra IR spectra were were measured measured on a on JASCO a JASCO FT/IR FT/IR 410 spectrophotometer. 410 spectrophotometer. Ultra- Ultravioletviolet (UV) (UV) spectra spectra were were obtained obtained on a onJASCO a JASCO V-560 V-560 UV/VIS UV/VIS spectrophotometer. spectrophotometer. ECD 1 13 ECDspectra spectra were were measured measured with with a JASCO a JASCO J-725N J-725N spectrophotometer. spectrophotometer. 1H- andH- and13C-NMRC-NMR spec- spectratra were were recorded recorded on ona Varian a Varian Unity Unity plus plus 500 500 spectrometer spectrometer (Agilent (Agilent Technologies,Santa Santa 1 13 Clara,Clara, CA, CA, USA) USA) operating operating at at 500 500 MHz MHz and and 126 126 MHz MHz for for the the H1H and and 13CC nuclei,nuclei, respectively.respectively. 1 13 1H-H- andand 13C-NMRC-NMR spectraspectra werewere alsoalso recordedrecorded onon aa JEOLJEOL JNM-AL400JNM-AL400 spectrometerspectrometer (JEOL (JEOL 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., To- kyo, 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 Chem- ical Co., Tokyo, Japan), Chromatorex ODS (Fuji Silysia Chemical Ltd., Kasugai, Japan),

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 0 (79 mg), 4-hydroxy-3-methoxyphenol 1-O-β-D-(6 -O-galloyl)-glucopyranoside (37 mg), 0 4-hydroxy-2-methoxyphenol 1-O-β-D-(6 -O-galloyl)-glucopyranoside (46 mg). Fr. 6 was purified by precipitation from water to give davidiin (1) (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-α-rhamnoside (2.5 g), quercetin (719 mg), quercetin 3-O-β-D-glucopyranoside (182 mg), quercetin 3-O-α-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 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. × 50 cm) with 0–100% MeOH (10% stepwise gradient elution, each 500 mL) to give 11 fractions. Crystallization of fr. 3 from H2O yielded 1-O-galloyl-β-D-glucopyranose (301 mg). Separation of fr. 7 (4.61 g) by Sephadex LH-20 (0–100% MeOH) gave polymeric proanthocyanidins (1.37 g), 3 (1.37 g), and 1,2,6-tri-O-galloyl-β-D-glucopyranose (76 mg). Fr. 8 (11.2 g) was subjected to Sephadex LH-20 (0–100% MeOH and then MeOH–H2O–acetone, 8:1:1 and 3:1:1) to give quercetin 3-O-(2”-α-rhamnopyranosyl)-β-glucuronopyranoside (481 mg) and 2 (553 mg).

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. × 35 cm) with 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 7 as the major component. Fr. 3 (29.7 g) was separated by Sephadex LH-20 column chromatography (6 cm i.d. × 31 cm) with 0–100% MeOH to give 7 (6.68 g), 10 (1.44 g), and a subfraction containing 9 and 11. Further chromatography of the subfraction using Chromatorex ODS (0–50% MeOH) and Toyopearl butyl-650M (0–50% MeOH) to yield 9 (490 mg) and 11 (19 mg). Fr. 11 (40.2 g) 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) −1 Brown amorphous powder, [α]D +43.8 (c 0.1, MeOH), IR νmax cm : 3412, 1728, 1708, 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): −25.16 (213), +6.68 (234), 1 −5.47 (283), +2.36 (374). H-NMR (acetone-d6, 500 MHz) δH: 7.19 (s, α-galloyl-2,6), 7.18 (s, β-galloyl-2,6), 6.76 (s, α,β-DHHDP-6”), 6.38 (s, α-DHHDP-3), 6.37 (s, β-DHHDP-3), 5.63 (dd, J = 9.8, 9.5 Hz, α-glc H-3), 5.42 (t, J = 9.4 Hz, β-glc H-3), 5.40 (t, J = 9.4, α,β-glc H-4), 5.32 (d, J = 3.4 Hz, α-glc H-1), 5.01 (dd, J = 4.8, 10.7 Hz, α-glc H-6), 4.94 (dd, J = 4.8, 10.7 Hz, α-glc H-6), 4.88 (d, J = 7.8 Hz, β-glc H-1), 4.65 (s, α-DHHDP-1’), 4.63 (s, β-DHHDP-1’), 4.25 (m, α-glc H-5), 3.95 (m, α-glc H-5, α-glc H-6, β-glc H-6), 3.89 (dd, J = 9.8, 3.4 Hz, α-glc H-2), 3.66 13 (dd, J = 9.4, 7.8 Hz, β-glc H-2), C NMR (acetone-d6, 125 MHz) δC: 192.0 (α,β-DHHDP-4’), 168.1 (α,β-DHHDP-7), 166.9 (galloyl-7), 164.6 (α,β-DHHDP-7’), 151.7 (α,β-DHHDP-2’), 146.0 (α,β-DHHDP-4), 145.9 (galloyl-3,5), 142.3 (α,β-DHHDP-6), 138.8 (galloyl-4), 135.4 (α,β-DHHDP-5), 130.8 (α,β-DHHDP-3’), 124.1 (α,β-DHHDP-2), 121.7 (galloyl-1), 112.7 (α,β-DHHDP-1), 110.1 (galloyl-2,6), 107.7 (β-DHHDP-3), 107.6 (α-DHHDP-3), 97.8 (β-glc H-1), 96.5 (α,β-DHHDP-5’), 93.6 (α-glc C-1), 91.5 (α,β-DHHDP-6’), 75.6 (β-glc C-3), 74.1 (α-glc C-4), 74.0 (β-glc C-4), 73.7 (β-glc C-2), 73.3 (α-glc C-3), 71.2 (α-glc C-2), 68.3 (β-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’). Molecules 2021, 26, 337 9 of 11

3.5.2. Polygonanin A (11) ◦ + Tan amorphous powder, [α]D −51.3 (c 0.1, MeOH), FABMS m/z: 841 (M + Na) , 819 + + -1 (M + H) , HRFABMS m/z: 819.0891 (M + H) (Calcd for C34H27O24: 819.0887), IR νmax cm (neat): 3412, 1719, 1616, 1445, 1313, 1223, 1190. UV λmax (MeOH) nm (log ε): 266 (4.40), 230 (4.70). ECD (MeOH) λmax (∆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, 13 d, J = 15.9 Hz, HChe H-5’). C 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 (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 MMFF94 force field with Spartan 014 (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 10 of 11

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 and 2D NMR spectra of 11. Figures S15 and S16: 1H 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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