Identification of Unstable Ellagitannin Metabolites in the Leaves of Quercus dentata by Chemical Derivatization

The identification of unstable metabolites of ellagitannins having ortho-quinone structures or reactive carbonyl groups is important to clarify the biosynthesis and degradation of ellagitannins. Our previous studies on the degradation of vescalagin, a major ellagitannin of oak young leaves, suggested that the initial step of the degradation is regioselective oxidation to generate a putative quinone intermediate. However, this intermediate has not been identified yet. In this study, young leaves of Quercus dentata were extracted with 80% acetonitrile containing 1,2-phenylenediamine to trap unstable ortho-quinone metabolites, and subsequent chromatographic separation afforded a phenazine derivative of the elusive quinone intermediate of vescalagin. In addition, phenylenediamine adducts of liquidambin and dehydroascorbic acid were obtained, which is significant because liquidambin is a possible biogenetic precursor of C-glycosidic ellagitannins and ascorbic acid participates in the production of another C-glycosidic ellagitannin in matured oak leaves.


Introduction
Ellagitannins are a group of hydrolyzable tannins having hexahydroxydiphenoyl (HHDP) esters connected mainly to glucose. Owing to their large structural diversity and rich chemistry stemming from the different location of the ester groups on the glucose core, oxidation of the HHDP aromatic rings, coupling with other metabolites, and oligomerization, ellagitannins attract considerable interest in organic and natural product chemistry [1][2][3][4][5]. Among the known ellagitannins, C-glycosidic ellagitannins form a distinct group represented by vescalagin (1) and its C-1 epimer castalagin (2) (Figure 1). These tannins are the major constituents of oak wood, used in barrel making [6][7][8]. During the aging of wine and whisky in oak barrels, tannins 1 and 2 degrade via autoxidation and react with coexisting compounds through chemical reactions that are important in the sensory evaluation of alcoholic beverages [9][10][11][12]. In this context, we previously investigated and proposed the chemical mechanisms involved in the autoxidation of oak ellagitannins and the subsequent addition of ethanol during whisky aging [12]. The related oxidative degradation of oak ellagitannin 1 is also observed in the growing young leaves of Q. glauca [13]. The degradation of 1 by a wood-rotting fungi (Shiitake mushroom, Lentinula edodes) in Japanese oak wood also proceeds according to the same mechanism [13]. These oxidative degradations, that is, autoxidation during the aging of spirits, the metabolism in oak young leaves, and degradation during mushroom cultivation, proceed via a common chemical mechanism, which is depicted in Scheme 1. This mechanism was proposed on the basis of the isolation of intermediate 3 [13] or ethanol adduct 4 [12] having a cyclopentenone structure. The production of 3 in the young leaves of Q. glauca was confirmed by detection 2 of 11 of an adduct 3a after treatment with 1,2-phenylenediamine. In addition, when 1 was treated with mycelia of Lentinula edodes, 3 is accumulated in the mixture [13], and the structure of 3 was determined based on the spectroscopic data of 3a obtained by treatment with 1,2phenylenediamine. These results strongly suggested that the initial step of the degradation of 1 is the selective oxidation of the pyrogallol rings connected to the glucose C-1 position, in order to generate intermediate 1a possessing a cyclohexenetrione structure. However, no direct chemical evidence has been provided to date for the generation of 1a as the initial intermediate of the oxidation of 1. Therefore, the aim of this study was to confirm the presence of 1a and related unstable quinone metabolites in young oak leaves via the conversion of ortho-quinones to stable derivatives. In this study, 1,2-phenylenediamine was used as the derivatization reagent because it is conventionally used to trap diketone compounds in tannin chemistry. The leaves of Q. dentata, a deciduous tree widely distributed in Japan and East Asia, were selected because 1 and 2 are the major constituents of young leaves and their content decreases as the leaves grow. In addition, these leaves are very popular in Japan as a rice cake wrapper.
Molecules 2023, 28, x FOR PEER REVIEW 2 of 11 cyclopentenone structure. The production of 3 in the young leaves of Q. glauca was confirmed by detection of an adduct 3a after treatment with 1,2-phenylenediamine. In addition, when 1 was treated with mycelia of Lentinula edodes, 3 is accumulated in the mixture [13], and the structure of 3 was determined based on the spectroscopic data of 3a obtained by treatment with 1,2-phenylenediamine. These results strongly suggested that the initial step of the degradation of 1 is the selective oxidation of the pyrogallol rings connected to the glucose C-1 position, in order to generate intermediate 1a possessing a cyclohexenetrione structure. However, no direct chemical evidence has been provided to date for the generation of 1a as the initial intermediate of the oxidation of 1. Therefore, the aim of this study was to confirm the presence of 1a and related unstable quinone metabolites in young oak leaves via the conversion of ortho-quinones to stable derivatives. In this study, 1,2-phenylenediamine was used as the derivatization reagent because it is conventionally used to trap diketone compounds in tannin chemistry. The leaves of Q. dentata, a deciduous tree widely distributed in Japan and East Asia, were selected because 1 and 2 are the major constituents of young leaves and their content decreases as the leaves grow. In addition, these leaves are very popular in Japan as a rice cake wrapper.

Comparison of Tannin Compositions in Spring and Summer Leaves
First, the tannin compositions in spring and summer leaves of Q. dentata were compared by high-performance liquid chromatography (HPLC) (Figure 2). Leaves collected in April and July were extracted with 80% acetonitrile (CH3CN) (samples A and C) and 80% CH3CN containing 1,2-phenylenediamine and trifluoroacetic acid (TFA) (samples B Molecules 2023, 28, x FOR PEER REVIEW 2 of 11 cyclopentenone structure. The production of 3 in the young leaves of Q. glauca was confirmed by detection of an adduct 3a after treatment with 1,2-phenylenediamine. In addition, when 1 was treated with mycelia of Lentinula edodes, 3 is accumulated in the mixture [13], and the structure of 3 was determined based on the spectroscopic data of 3a obtained by treatment with 1,2-phenylenediamine. These results strongly suggested that the initial step of the degradation of 1 is the selective oxidation of the pyrogallol rings connected to the glucose C-1 position, in order to generate intermediate 1a possessing a cyclohexenetrione structure. However, no direct chemical evidence has been provided to date for the generation of 1a as the initial intermediate of the oxidation of 1. Therefore, the aim of this study was to confirm the presence of 1a and related unstable quinone metabolites in young oak leaves via the conversion of ortho-quinones to stable derivatives. In this study, 1,2-phenylenediamine was used as the derivatization reagent because it is conventionally used to trap diketone compounds in tannin chemistry. The leaves of Q. dentata, a deciduous tree widely distributed in Japan and East Asia, were selected because 1 and 2 are the major constituents of young leaves and their content decreases as the leaves grow. In addition, these leaves are very popular in Japan as a rice cake wrapper.

Comparison of Tannin Compositions in Spring and Summer Leaves
First, the tannin compositions in spring and summer leaves of Q. dentata were compared by high-performance liquid chromatography (HPLC) (Figure 2). Leaves collected in April and July were extracted with 80% acetonitrile (CH3CN) (samples A and C) and 80% CH3CN containing 1,2-phenylenediamine and trifluoroacetic acid (TFA) (samples B Scheme 1. Proposed chemical mechanism of the oxidative degradation of 1.

Comparison of Tannin Compositions in Spring and Summer Leaves
First, the tannin compositions in spring and summer leaves of Q. dentata were compared by high-performance liquid chromatography (HPLC) (Figure 2). Leaves collected in April and July were extracted with 80% acetonitrile (CH 3 CN) (samples A and C) and 80% CH 3 CN containing 1,2-phenylenediamine and trifluoroacetic acid (TFA) (samples B and D). The phenylenediamine used in the latter extraction conditions reacts with 1,2diketone structures quantitatively to produce quinoxaline moieties under acidic conditions. As shown in Figure 2a, the major ellagitannins of the spring young leaves were 1 and 2, whereas the content of both compounds decreased in the summer leaves ( Figure 2c). In the HPLC of the summer leaves, broad peaks attributable to dimers and oligomers of 1 and 2 and grandinin (9) and related compounds [14][15][16], which have polyalcohol moieties at the C-1 position of 1, were observed in the 6-12 min range, indicating that the content of oligomers and 9 and its analogues increases as the leaves grow. The HPLC of the extracts obtained with 1,2-phenylenediamine (Figure 2b, d) showed additional peaks attributable to derivatives produced via the reaction of 1,2-phenylenediamine, probably with metabolites having 1,2-diketone structures. Next, these derivatives were isolated in a larger-scale experiment.
Molecules 2023, 28, x FOR PEER REVIEW 3 of 11 and D). The phenylenediamine used in the latter extraction conditions reacts with 1,2diketone structures quantitatively to produce quinoxaline moieties under acidic conditions. As shown in Figure 2a, the major ellagitannins of the spring young leaves were 1 and 2, whereas the content of both compounds decreased in the summer leaves ( Figure  2c). In the HPLC of the summer leaves, broad peaks attributable to dimers and oligomers of 1 and 2 and grandinin (9) and related compounds [14][15][16], which have polyalcohol moieties at the C-1 position of 1, were observed in the 6-12 min range, indicating that the content of oligomers and 9 and its analogues increases as the leaves grow. The HPLC of the extracts obtained with 1,2-phenylenediamine ( Figure 2b, d) showed additional peaks attributable to derivatives produced via the reaction of 1,2-phenylenediamine, probably with metabolites having 1,2-diketone structures. Next, these derivatives were isolated in a larger-scale experiment.

Separation and Structure Determination
The fresh leaves collected at the end of April were extracted with 80% CH3CN containing 1,2-phenylenediamine and TFA, and separated by column chromatography using Sephadex LH-20, Diaion HP20SS, Chromatorex ODS, and Wakosil C18 columns to yield six compounds, including 1, 2, and three reaction products, i.e., 6, 7, and 8. The unidentified peak 5 in the HPLC chromatograms shown in Figure 2 was identified as a dimeric ellagitannin cocciferin D2 (5) (Figure 3) [17] on the basis of the comparison of spectroscopic data, including two-dimensional (2D) nuclear magnetic resonance (NMR) experiments.

Separation and Structure Determination
The fresh leaves collected at the end of April were extracted with 80% CH 3 CN containing 1,2-phenylenediamine and TFA, and separated by column chromatography using Sephadex LH-20, Diaion HP20SS, Chromatorex ODS, and Wakosil C18 columns to yield six compounds, including 1, 2, and three reaction products, i.e., 6, 7, and 8. The unidentified peak 5 in the HPLC chromatograms shown in Figure 2 was identified as a dimeric ellagitannin cocciferin D 2 (5) (Figure 3) [17] on the basis of the comparison of spectroscopic data, including two-dimensional (2D) nuclear magnetic resonance (NMR) experiments. Molecules 2023, 28, x FOR PEER REVIEW 4 of 11  showed signals ascribable to oxygenated aliphatic methine and methylene units, whose chemical shifts and coupling patterns were related to those of the glucose moiety of 1. The 1 H-1 H correlation spectroscopy (COSY) confirmed the assignments of the glucose proton signals of 6. The observation of two aromatic singlets at δH 6.61 (1H, s) and 6.88 (1H, s) and aromatic and carboxyl carbon signals showing heteronuclear multiple bond correlations (HMBC) (Figure 4) with the two aromatic protons revealed the presence of an HHDP group in the molecule. In addition, the HMBC correlations of the aromatic singlet signal at δH 6.76 (HC-6) and the similarities of the aromatic carbon signals with those of 1 indicated the presence of a nonahydroxytriphenoyl (NHTP) unit [6]. The location of the ester carboxyl carbons on the glucose core was determined according to the HMBC correlations. Specifically, the correlation of glc-H-6 (δH 4.04, 5.12) and HHDP-HE-6′ (δH 6.61) with HHDP-CE-COO (δc 168. 19) and that of glc-H-4 (δH 5.23) and HHDP-HD-6 (δH 6.88) with HHDP-CD-COO (δC 165.51) indicated that the HHDP group was connected to the 4-and 6-positions of glucose. The correlations of glc-H-5 (δH 5.72) and HC-6 (δH 6.76) to CC-COO (δC 165.92) suggested the presence of an ester linkage between glc-5 and the NHTP terminal galloyl unit. Furthermore, HMBC correlations between glc-H-2 (δH 5.47) and CB-COO (δc 163.57) and CA-2 (δc 116.13) and between glc-H-1 (δH 5.35) and CA-1, 2, 3 (δc 135.0, 116.1, 151.8) were indicative of a C-glycosidic linkage of glc-1 to the NHTP ring A. These spectroscopic features are closely related to those of 1 and confirmed that 6 is derived from 1. Meanwhile, the 1 H and 13 C NMR spectra of both compounds differ in the appearance of signals due to a phenylenediamine unit in the spectra of 6 (δH 8 [13]. A comparison of 13 C NMR chemical shifts of the aromatic carbons of 6 with those of 1 indicated that the ring A reacted with the phenylenediamine. The HMBC spectrum showed a correlation between glc-H-1 (δH 5.35) and the oxygen-bearing aromatic carbon signal at δc 151.3 (NHTP-CA-3), indicating that the quinoxaline unit is located at the CA-4 and CA-5 positions of the NHTP moiety. The atropisomerism of the biphenyl linkages was the same as that of 1, as was extracted from the comparison of the electronic circular dichroism (ECD) data with that of 1 [6]. Consequently, the structure of 6 was determined to be that shown in Scheme 1, according to which the quinoxaline derivative 6 was generated from the missing intermediate 1a, thus supporting the degradation mechanism of 1, which was previously speculated from the structure of the degradation products [12,13].  showed signals ascribable to oxygenated aliphatic methine and methylene units, whose chemical shifts and coupling patterns were related to those of the glucose moiety of 1. The 1 H-1 H correlation spectroscopy (COSY) confirmed the assignments of the glucose proton signals of 6. The observation of two aromatic singlets at δ H 6.61 (1H, s) and 6.88 (1H, s) and aromatic and carboxyl carbon signals showing heteronuclear multiple bond correlations (HMBC) (Figure 4) with the two aromatic protons revealed the presence of an HHDP group in the molecule. In addition, the HMBC correlations of the aromatic singlet signal at δ H 6.76 (H C -6) and the similarities of the aromatic carbon signals with those of 1 indicated the presence of a nonahydroxytriphenoyl (NHTP) unit [6]. The location of the ester carboxyl carbons on the glucose core was determined according to the HMBC correlations. Specifically, the correlation of glc-H-6 (δ H 4.04, 5.12) and HHDP-H E -6 (δ H 6.61) with HHDP-C E -COO (δc 168. 19) and that of glc-H-4 (δ H 5.23) and HHDP-H D -6 (δ H 6.88) with HHDP-C D -COO (δ C 165.51) indicated that the HHDP group was connected to the 4-and 6-positions of glucose. The correlations of glc-H-5 (δ H 5.72) and H C -6 (δ H 6.76) to C C -COO (δ C 165.92) suggested the presence of an ester linkage between glc-5 and the NHTP terminal galloyl unit. Furthermore, HMBC correlations between glc-H-2 (δ H 5.47) and C B -COO (δc 163.57) and C A -2 (δc 116.13) and between glc-H-1 (δ H 5.35) and C A -1, 2, 3 (δc 135.0, 116.1, 151.8) were indicative of a C-glycosidic linkage of glc-1 to the NHTP ring A. These spectroscopic features are closely related to those of 1 and confirmed that 6 is derived from 1. Meanwhile, the 1 H and 13 C NMR spectra of both compounds differ in the appearance of signals due to a phenylenediamine unit in the spectra of 6 (δ H 8.  [13]. A comparison of 13 C NMR chemical shifts of the aromatic carbons of 6 with those of 1 indicated that the ring A reacted with the phenylenediamine. The HMBC spectrum showed a correlation between glc-H-1 (δ H 5.35) and the oxygen-bearing aromatic carbon signal at δc 151.3 (NHTP-C A -3), indicating that the quinoxaline unit is located at the C A -4 and C A -5 positions of the NHTP moiety. The atropisomerism of the biphenyl linkages was the same as that of 1, as was extracted from the comparison of the electronic circular dichroism (ECD) data with that of 1 [6]. Consequently, the structure of 6 was determined to be that shown in Scheme 1, according to which the quinoxaline derivative 6 was generated from the missing intermediate 1a, thus supporting the degradation mechanism of 1, which was previously speculated from the structure of the degradation products [12,13].  a-e : Assignment may be interchanged.  Table 2, Supplementary Materials) suggested the presence of a galloyl (δH 7.27 (2H, s, HC-2, 6)) and two HHDP (δH 6.84 (1H, s, HB-6), 6.73 (1H, s, HD-6), 6.44 (1H, s, HE-6), 6.40 (1H, s, HA-6)) groups in the molecule, which was confirmed by the corresponding 13 C NMR, heteronuclear single-quantum correlation (HSQC), and HMBC spectroscopic analyses (Figure 4). The remaining aromatic proton signals at δH 7.69 (2H, dd, J = 6.0, 3.2 Hz, HF-4, 5) and 7.29 (2H, m, HF-3, 6)] were attributable to a benzene ring of the phenylenediamine unit. In addition, the 1 H NMR spectrum exhibited six aliphatic proton signals due to four methines and one methylene of a polyalcohol moiety (   Figure 4) and the coupling constants of the signals were related to those observed for 1, suggesting the presence of a glucose moiety. The lack of a glucose C-1 methine proton was explained by the HMBC correlation of glucose H-2 with a sp 2 quaternary carbon resonating at δ C 146.7 (glc-C-1), suggesting that the C-1 atom of the polyalcohol is connected to the 1,2-phenylenediamine unit to form a benzimidazole unit. This is consistent with the molecular formula suggested by HR-FAB-MS. Two HHDP groups were determined to be connected at the polyalcohol 2,3-and 4,6-positions according to the HMBC correlations of the HHDP ester carbonyl carbons with the HHDP aromatic and polyalcohol aliphatic protons (Figure 4). Although the location of the galloyl group could not be determined on the basis of the usual HMBC experiment with J = 10 Hz, an HMBC spectrum measured with J = 5 Hz exhibited correlations between an ester carbonyl carbon (δ C 165.1) and δ H 5.69 (glc-H-5) and a two-proton singlet at 7.27 (galloyl H-2,6), confirming the location of the galloyl group at the C-5 position of the polyalcohol. These results suggested that the plane structure of 7 is that shown in Figure 4. This structure is related to liquidambin (7a), which has an open-chain form of the glucose core with an aldehyde group at the C-1 position (δ C 194.6) [18]. Furthermore, the coupling constants of the polyalcohol moiety of 7 were very similar to those of the open-chain glucose unit of 7a (J 1,2 = 0 Hz, J 2,3 = 9.5 Hz, J 3,4 = 1 Hz, J 4,5 = 9 Hz, J 5,6 = 3.5 Hz, J 6,6 = 13 Hz). The atropisomerism of the HHDP groups was also presumed to be the same as that of 7a, because the close similarity of the coupling constants strongly suggests that 7 and 7a adopted similar conformations including HHDP and galloyl esters. In addition, the HHDP unit located at the glucose 2,3-and 4,6-positions is known to adopt an S-configuration except for very limited examples [19,20]. This was confirmed by the ECD spectrum [21]. Consequently, 7 was concluded to be a reaction product of 7a with 1,2-phenylenediamine, indicating the presence of 7a in the young leaves of Q. dentata. Moreover, the structural similarity of 7a with 1 and 2 suggests that 7a is a biosynthetic precursor of the C-glycosidic ellagitannins of this plant [22]. So far, compound 7a has been only isolated from Liquidambar formosana; therefore, the present study demonstrates the second identification of 7a in plants. The molecular formula of 8 was determined to be C 12 H 10 N 2 O 4 on the basis of the FAB-MS (m/z 247 [M + H] + ) and 13 C NMR signals. The chemical shifts of the aromatic protons and carbons indicated the presence of a benzene ring of 1,2-phenylenediamine. The remaining six carbons are three sp 2 carbons (δc 165.9 (C-1), 140.4 (C-2), and 159.1 (C-3)), two methines (δc 79.7 (C-4), 71.6 (C-5)), and a methylene (δc 62.3 (C-6)). The HMBC correlations ( Figure 5) between H-4 and the three sp 2 carbons, including the lactone carbonyl carbon, suggested that 8 contained an unsaturated γ-lactone structure. In addition, the 4 J HMBC correlation of H-4 with one of the aromatic carbon signals (δc 143.7) indicated the condensation of 1,2-phenylenediamine with C-2 and C-3 to form a quinoxaline moiety. This is in agreement with the unsaturation index of 8 indicated by the molecular formula. These spectroscopic observations suggested that 8 is a reaction product of dehydroascorbic acid. This was confirmed by preparing 8 via the autoxidation of L-ascorbic acid in a neutral aqueous solution and subsequent condensation with 1,2-phenylenediamine. Thus, the structure of 8 was determined to be 3-(1,2-dihydroxyethyl)furo [3,4-b]quinoxaline-1one [23]. The mechanism of formation of 8 is shown in Scheme 2. A comparison of the young and matured leaves of Q. dentata by reversed phase HPLC (Figure 2) indicated an increase in the content of highly hydrophilic ellagitannins, which were detected as broad peaks with shorter retention times (t R , 6-12 min) compared with those of 1. Previous studies of ellagitannins of Quercus species showed that these highly hydrophilic ellagitannins contain grandinin (9) and related oligomers, and ascorbic acid is suggested to participate in the biosynthesis of 9 [15].
The molecular formula of 8 was determined to be C12H10N2O4 on the basis of the FAB-MS (m/z 247 [M + H] + ) and 13 C NMR signals. The chemical shifts of the aromatic protons and carbons indicated the presence of a benzene ring of 1,2-phenylenediamine. The remaining six carbons are three sp 2 carbons (δc 165.9 (C-1), 140.4 (C-2), and 159.1 (C-3)), two methines (δc 79.7 (C-4), 71.6 (C-5)), and a methylene (δc 62.3 (C-6)). The HMBC correlations ( Figure 5) between H-4 and the three sp 2 carbons, including the lactone carbonyl carbon, suggested that 8 contained an unsaturated γ-lactone structure. In addition, the 4 J HMBC correlation of H-4 with one of the aromatic carbon signals (δc 143.7) indicated the condensation of 1,2-phenylenediamine with C-2 and C-3 to form a quinoxaline moiety. This is in agreement with the unsaturation index of 8 indicated by the molecular formula. These spectroscopic observations suggested that 8 is a reaction product of dehydroascorbic acid. This was confirmed by preparing 8 via the autoxidation of L-ascorbic acid in a neutral aqueous solution and subsequent condensation with 1,2-phenylenediamine. Thus, the structure of 8 was determined to be 3-(1,2-dihydroxyethyl)furo [3,4-b] quinoxaline-1one [23]. The mechanism of formation of 8 is shown in Scheme 2. A comparison of the young and matured leaves of Q. dentata by reversed phase HPLC (Figure 2) indicated an increase in the content of highly hydrophilic ellagitannins, which were detected as broad peaks with shorter retention times (tR, 6-12 min) compared with those of 1. Previous studies of ellagitannins of Quercus species showed that these highly hydrophilic ellagitannins contain grandinin (9) and related oligomers, and ascorbic acid is suggested to participate in the biosynthesis of 9 [15].   The molecular formula of 8 was determined to be C12H10N2O4 on the basis of the FAB-MS (m/z 247 [M + H] + ) and 13 C NMR signals. The chemical shifts of the aromatic protons and carbons indicated the presence of a benzene ring of 1,2-phenylenediamine. The remaining six carbons are three sp 2 carbons (δc 165.9 (C-1), 140.4 (C-2), and 159.1 (C-3)), two methines (δc 79.7 (C-4), 71.6 (C-5)), and a methylene (δc 62.3 (C-6)). The HMBC correlations ( Figure 5) between H-4 and the three sp 2 carbons, including the lactone carbonyl carbon, suggested that 8 contained an unsaturated γ-lactone structure. In addition, the 4 J HMBC correlation of H-4 with one of the aromatic carbon signals (δc 143.7) indicated the condensation of 1,2-phenylenediamine with C-2 and C-3 to form a quinoxaline moiety. This is in agreement with the unsaturation index of 8 indicated by the molecular formula. These spectroscopic observations suggested that 8 is a reaction product of dehydroascorbic acid. This was confirmed by preparing 8 via the autoxidation of L-ascorbic acid in a neutral aqueous solution and subsequent condensation with 1,2-phenylenediamine. Thus, the structure of 8 was determined to be 3-(1,2-dihydroxyethyl)furo [3,4-b] quinoxaline-1one [23]. The mechanism of formation of 8 is shown in Scheme 2. A comparison of the young and matured leaves of Q. dentata by reversed phase HPLC (Figure 2) indicated an increase in the content of highly hydrophilic ellagitannins, which were detected as broad peaks with shorter retention times (tR, 6-12 min) compared with those of 1. Previous studies of ellagitannins of Quercus species showed that these highly hydrophilic ellagitannins contain grandinin (9) and related oligomers, and ascorbic acid is suggested to participate in the biosynthesis of 9 [15].

General Information
Optical rotations were measured on a JASCO DIP-370 digital polarimeter (JASCO, Tokyo, Japan). Ultraviolet (UV) spectra were obtained on a JASCO V-560 UV/Vis spectrophotometer. Infrared (IR) spectra were measured on a JASCO FT/IR-410K IR spectrometer. ECD spectra were obtained using a JASCO J-725N spectrophotometer. 1 H, 13 C, and 2D

Conclusions
In this study, biosynthetic precursors 1a and 7a were isolated as derivatives 6 and 7, respectively, via the reaction with 1,2-phenylenediamine. The isolation of 6 provides evidence for the presence of 1a, which is a missing intermediate in the degradation of C-glycosidic ellagitannin. The presence of 7a was also demonstrated by identifying 7. A previous study suggested that 7a is a precursor of C-glycosidic ellagitannins of Liquidambar formosana [21], and the structural similarity suggested that 7a is also involved in the biosynthesis of 1 and 2 in the leaves of Q. dentata. Since diketone compound 3 exists mainly in its hydrated forms, triketone 1a probably exists as a complex equilibrium mixture of hydrated forms in aqueous solutions [13], and 7a was detected as a broad peak due to the equilibrium between the aldehyde and hydrated forms [18,21]. Therefore, the detection and isolation of intact 1a and 7a is difficult. Furthermore, a quinoxaline derivative of dehydroascorbic acid (8) was obtained. Since ascorbic acid is known to participate in the production of 9 from 1 in mature leaves [15], the observation of a large peak of 8 in the HPLC analysis of mature leaves (Figure 2) may be significant for the elucidation of the ellagitannin biosynthesis. To understand the biosynthesis and degradation of tannins and polyphenols, the characterization of ortho-quinones and related unstable intermediates is important; thus, this study demonstrated that extraction with 1,2-phenylenediamine and characterization of the resulting reaction products is a powerful method in polyphenol chemistry.