Ellagitannins and Oligomeric Proanthocyanidins of Three Polygonaceous Plants

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.


Polyphenols from Persicaria Chinensis
Similar separation of an aqueous acetone extract of the fresh aerial part of Persicaria chinensis collected in Nagasaki, Japan, afforded 1-O- [19] and 1,2,6-tri-O-galloyl-β-D-glucoses [20], geraniin (2) [21], quercetin 3-O-(2"-α-rhamnopyranosyl)-β-glucuronop yranoside [22], and a new ellagitannin called persicarianin. Persicarianin (3) was obtained as a brown amorphous powder, and high-resolution electrospray ionization time of flight (HRESITOF) MS indicated a molecular formula of C 27 1 H NMR spectrum showed signals arising from αand β-hexopyranoses, and it is apparent that the sugar is a 4 C 1 -glucopyranose based on the large coupling constants of the pyranose ring proton signals (J 2,3 , J 3,4 , J 4,5 = 8-10 Hz). The low field shifts of H- ] indicates acylation of these positions. The acyl groups were shown to be a galloyl and a dehydrohexahydroxydiphenoyl (DHHDP) group by 13 C 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 1 H 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) correlations 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 concluded 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 [25,26]. Based on this evidence, persicarianin was characterized to be 3-O-galloyl-4,6-(S)-DHHDP-D-glucose (3). HPLC analysis of the aforementioned hydrolysis products, before condensation with cysteine, revealed production of gallic acid (4), ellagic acid (5), and brevifolin carboxylic acid (6) ( Figure S2, Supplementary Materials). Ellagic acid is a bislactone form of the HHDP group, and therefore, a reduction product of the DHHDP ester of 3. Similar production of 5 from DHHDP groups on acid hydrolysis have been also observed for 2 and related ellagitannins, and the reaction was deduced to be a redox disproportionation [27,28]. Ellagitannins were originally defined as hydrolyzable tannins which afford 5 upon hydrolysis, and 5 is usually considered to be originated from HHDP groups. In this context, despite the absence of a HHDP group, 3 is also an ellagitannin.

Polyphenols from Persicaria Chinensis
Similar separation of an aqueous acetone extract of the fresh aerial part of Persicaria chinensis collected in Nagasaki, Japan, afforded 1-O- [19] and 1,2,6-tri-O-galloyl-β-D-glucoses [20], geraniin (2) [21], quercetin 3-O-(2''-α-rhamnopyranosyl)-β-glucuronopyranoside [22], and a new ellagitannin called persicarianin. Persicarianin (3) was obtained 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 1 H NMR spectrum showed signals arising from α-and β-hexopyranoses, and it is apparent that the sugar is a 4 C1-glucopyranose based on the large coupling constants of the pyranose ring proton signals ( The large chemical shift differences of the glucose H-6 methylene proton signals in the 1 H 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) correlations 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 concluded 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 [25,26]. Based on this evidence, persicarianin was characterized to be 3-O-galloyl-4,6-(S)-DHHDP-D-glucose (3). HPLC analysis of the aforementioned hydrolysis products, before condensation with cysteine, revealed production of gallic acid (4), ellagic acid (5), and brevifolin carboxylic acid (6) ( Figure S2, Supplementary Materials). Ellagic acid is a bislactone form of the HHDP group, and therefore, a reduction product of the DHHDP ester of 3. Similar production of 5 from DHHDP groups on acid hydrolysis have been also observed for 2 and related ellagitannins, and the reaction was deduced to be a redox disproportionation [27,28]. Ellagitannins were originally defined as hydrolyzable tannins which afford 5 upon hydrolysis, and 5 is usually considered to be originated from HHDP groups. In this context, despite the absence of a HHDP group, 3 is also an ellagitannin.

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)

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) by 1D and 2D NMR spectroscopic analysis [29]. Furthermore, the acetonyl derivative 7a was prepared by treatment of 7 with aqueous acetone containing HCO 2 NH 4 , and spectroscopic comparison of 7a with those of an authentic sample provided further evidence for the structure of 7 [30]. In the purification process of 7 (Figure 4), four minor ellagitannins were isolated by a combination of column chromatography using Chromatorex ODS and Toyopearl Butyl-650M to give helioscopinin A (8) [31], davicratinic acids B (9) and C (10) [32], and a new ellagitannin called polygonanin A.
Molecules 2020, 25, x 4 of 11 by 1D and 2D NMR spectroscopic analysis [29]. Furthermore, the acetonyl derivative 7a was prepared by treatment of 7 with aqueous acetone containing HCO2NH4, and spectroscopic comparison of 7a with those of an authentic sample provided further evidence for the structure of 7 [30]. In the purification process of 7 (Figure 4), four minor ellagitannins were isolated by a combination of column chromatography using Chromatorex ODS and Toyopearl Butyl-650M to give helioscopinin A (8) [31], davicratinic acids B (9) and C (10) [32], and a new ellagitannin called polygonanin A.  The 1 H and 13 C 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 1, 7, 9, and 10. Coupling constants of the pyranose ring protons (J1,2, J2,3, 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 7, 9, 10, and 11 adopt a 1 C4 conformation, whereas the glucopyranose core of 1 with a 2,3,4-trigalloyl structure adopts a boat conformation (Figure 1). The NMR 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 8 is generated by addition of H2O to the double bond of 9 or 10 and rearrangements of lactone formation. This was supported by 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 (  The 1 H and 13 C 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 (δ H 5.95, s) and H-6 [δ H 4.99 (t, J = 11.1 Hz), 4.08 (dd, J = 5.3, 11.1 Hz)] with the HHDP ester carbonyl carbons (δ C 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 1, 7, 9, and 10. Coupling constants of the pyranose ring protons (J 1,2 , J 2,3 , J 3,4 , and J 4,5 ) were <2 Hz. This was similar to those of 7, 9, and 10, but different to those of 1 (J 1,2 = 3.2 Hz, J 2,3 = 8.5 Hz, J 3,4 = 7.7 Hz, and J 4,5 = 2.9 Hz), suggesting that the glucopyranoses of 7, 9, 10, and 11 adopt a 1 C 4 conformation, whereas the glucopyranose core of 1 with a 2,3,4-trigalloyl structure adopts a boat conformation (Figure 1). The NMR signals indicated that the 2,4-acyl group of 11 was composed of 3 carboxyl carbons (δ C 172.3 (C-1'), 172.0 (C-6'), 166.1 (C-7')), an oxygenated tertiary carbon (δ C 76.3 (C-4')), an oxygenated methine (δ C 66.3 (C-2')), a methylene (δ C 41.6 (C-5')), and a benzylic methine (δ C 49.2 (C-3')), along with a trihydroxy benzoyl moiety. Taking the molecular formula C 34 H 26 O 24 indicated by HR-fast atom bombardment (FAB) MS into account, these building blocks of the 2,4-acyl group suggested that 8 is generated by addition of H 2 O to the double bond of 9 or 10 and rearrangements of lactone formation. This was supported by 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  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 7. Interestingly, the glucose H-3 of 11 (δ H 5.14) resonated at a much lower field compared to those of 7a (δ H 4.58), 9 (δ H 4.40) and 10 (δ H 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.

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, indicating that the polyphenols are not proanthocyanidins. The 13 C NMR spectrum suggested that the polyphenols are oligomeric hydrolyzable tannins. Further investigations are now underway.

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, indicating that the polyphenols are not proanthocyanidins. The 13

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. ECD spectra were measured with a JASCO J-725N spectrophotometer. 1 H-and 13 C-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 1 H and 13 C nuclei, respectively. 1 H-and 13 C-NMR spectra were also recorded on a JEOL JNM-AL400 spectrometer (JEOL Ltd., Tokyo, Japan) operating at 400 and 100 MHz for the 1 H and 13 C 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-

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. ECD spectra were measured with a JASCO J-725N spectrophotometer. 1 H-and 13 C-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 1 H and 13 C nuclei, respectively. 1 H-and 13 C-NMR spectra were also recorded on a JEOL JNM-AL400 spectrometer (JEOL 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).

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

Acid Hydrolysis of 3
Persicarianin (3) (10 mg) was hydrolyzed by heating in 5% H 2 SO 4 (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 t R 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 BaSO 4 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).

Computational Calculation of 8
A conformational search was performed using the Monte Carlo method and the MMFF94 force field with Spartan 14 (Wavefunction, Irvine, CA). The low-energy conformers 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 confirm 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][37][38].

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].

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 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.