Production of ellagitannin hexahydroxydiphenoyl ester by spontaneous reduction of dehydrohexa-hydroxydiphenoyl ester

Article abstract of DOI:10.3390/molecules25051051

Amariin is an ellagitannin with two dehydrohexahydroxydiphenoyl (DHHDP) moieties connecting glucose 2,4- and 3,6-hydroxy groups. This tannin is predominant in the young leaves of Triadica sebifera and Carpinus japonica. However, as the leaves grow, the 3,6-DHHDP is converted to its reduced form, the hexahydroxydiphenoyl (HHDP) group, to generate geraniin, a predominant ellagitannin of the matured leaves. The purified amariin is unstable in aqueous solution, and the 3,6-(R)-DHHDP is spontaneously degraded to give HHDP, whereas 2,4-(R)-DHHDP is stable. The driving force of the selective reduction of the 3,6-DHHDP of amariin is shown to be the conformational change of glucose from ^O,3B to ¹C₄. Heating geraniin with pyridine affords 2,4-(R)-DHHDP reduction products. Furthermore, the acid hydrolysis of geraniin yields two equivalents of ellagic acid. Although the reaction mechanism is still ambiguous, these results propose an alternative biosynthetic route of the ellagitannin HHDP groups.

Full text of DOI:10.3390/molecules25051051

Article
Production of Ellagitannin Hexahydroxydiphenoyl
Ester by Spontaneous Reduction of Dehydrohexa-
hydroxydiphenoyl Ester
Manami Era, Yosuke Matsuo, Yoshinori Saito and Takashi Tanaka *
Department of Natural Product Chemistry, Graduate School of Biomedical Sciences, Nagasaki University,
1-14 Bunkyo-machi, Nagasaki 852-8521, Japan; manamiso912@gmail.com (M.E.);
y-matsuo@nagasaki-u.ac.jp (Y.M.); saiyoshi@nagasaki-u.ac.jp (Y.S.)
* Correspondence: t-tanaka@nagasaki-u.ac.jp; Tel.: +81-95-819-2432
Academic Editor: Teresa Escribano-Bailón; Ignacio García-Estévez
Received: 31 January 2020; Accepted: 24 February 2020; Published: 26 February 2020
Abstract: Amariin is an ellagitannin with two dehydrohexahydroxydiphenoyl (DHHDP) moieties
connecting glucose 2,4- and 3,6-hydroxy groups. This tannin is predominant in the young leaves of
Triadica sebifera and Carpinus japonica. However, as the leaves grow, the 3,6-DHHDP is converted to
its reduced form, the hexahydroxydiphenoyl (HHDP) group, to generate geraniin, a predominant
ellagitannin of the matured leaves. The purified amariin is unstable in aqueous solution, and the
3,6-(R)-DHHDP is spontaneously degraded to give HHDP, whereas 2,4-(R)-DHHDP is stable. The
driving force of the selective reduction of the 3,6-DHHDP of amariin is shown to be the
O,3
1
conformational change of glucose from B to C4. Heating geraniin with pyridine affords 2,4-(R)-
DHHDP reduction products. Furthermore, the acid hydrolysis of geraniin yields two equivalents of
ellagic acid. Although the reaction mechanism is still ambiguous, these results propose an
alternative biosynthetic route of the ellagitannin HHDP groups.
Keywords: dehydrohexahydroxydiphenic acid; hexahydroxydiphenic acid; amariin; geraniin;
ellagitannin
1. Introduction
Ellagitannin is a group of polyphenols having hexahydroxydiphenoyl (HHDP) ester or its
metabolites connected to mostly glucose [1,2]. Due to its high structural diversity and wide
distribution in the plant kingdom, ellagitannins continue to attract intense interest within the natural
product and organic chemistry communities [3,4]. The accumulation of ellagitannins in some plants
suggests that these tannins play a significant role in plant defense systems [5–7], while the
antiproliferative and antioxidative activities of ellagitannins in fruits and medicinal plants are well
studied [8–14]. However, the biosynthesis of ellagitannins including the production mechanism of
the HHDP group remains obscure. The HHDP group was supposed to be formed by oxidative
coupling between two galloyl esters on glucopyranose, and this mechanism has been evidenced by
the enzymatic preparation of ellagitannin from a gallotannin [15–17]. In addition, many successful
total ellagitannin syntheses are also based on similar biomimetic strategies [18,19]. However, in 1993,
Foo reported an important finding [20]. More specifically, that amariin, 1-O-galloyl-2,4;3,6-bis-(R)-
dehydrohexahydroxydiphenoyl (DHHDP)-β-D-glucose (1), degraded spontaneously in aqueous
ethanol to give the reduction product geraniin, 1-O-galloyl-2,4-(R)-DHHDP-3,6-(R)-HHDP-β-D-
glucopyranose (2) as a major product [21,22]. Based on the implicit assumption that polyphenols are
susceptible to oxidation, most natural product chemists supposed that DHHDP is generated by the
Molecules 2020, 25, 1051; doi:10.3390/molecules25051051
www.mdpi.com/journal/molecules

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oxidation of HHDP. However, Foo’s finding indicated that HHDP is possibly generated by the
reduction of DHHDP. In this study, we investigated the reductive conversion of the DHHDP esters
to HHDP esters.
2. Results and Discussion
2.1. Composition of Ellagitannins in Young and Matured Leaves.
In our continuous chemical study on ellagitannins, we found that similar reductive production
of HHDP from DHHDP occurs in the fresh leaves of Triadica sebifera (syn. Sapium sebiferum). Amariin
(1) was predominant in the small young leaves at the top of the twig collected in June; however, 1
was not detected in the larger and harder leaves of the same twig (Figure 1 and Supplementary Figure
S1). Leaves of different sizes, ranging between the smallest leaf at the top and the large matured leaf
were lyophilized, and the 60% acetonitrile extracts were analyzed by HPLC. The concentration of
geraniin (2) per leaf increased proportionally with the leaf size and reached a plateau (Figure 2). In
contrast, the concentration of 1 per leaf first increased as the leaves grew and then decreased as the
leaves matured. These observations suggest that 2 is biosynthesized via 1. Similarly, 1 was
predominant in the young leaves of Carpinus laxiflora (collected in April) (Figure 3) and decreased as
the leaves grew; however, here 2 become predominant instead of 1 in the matured leaves in October
[23]. A significant amount of elaeocarpsin, an ascorbic acid adduct of 2, was also detected in the
matured leaves [24]. Furthermore, the young soft leaves of Elaeocarpus sylvestris var. ellipticus contain
1 as the major ellagitannin accompanied by less 2 content, and the matured, hard leaves contain 2
(Figure S2). These findings also suggest that 1 is a biosynthetic precursor of 2.
2200000
2000000
2200000
2000000
2200000
2000000
2200000
2000000
2
A
B
C
D
2
2
1000000
1000000
1000000
1000000
2
1
1
1
1
0
0
0
0
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
0
10
20
30
40 (min)
0
10
20
30
40 (min)
0
10
20
30
40 (min)
0
10
20
30
40 (min)
Figure 1. HPLC profiles (310 nm) of extracts of the leaves of Triadica sebifera.
A: young leaf at the top of a twig (leaf size about 1 cm in diameter), D: matured leaf of the same
twig (about 8.5 cm in diameter), B (about 2 cm) and C (about 3.5 cm) are the leaves between A and D
(50 mg of lyophilized leaf was extracted with 1.5 mL of 60% CH3CN, 0.1% TFA).

Molecules 2020, 25, 1051
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12
10
8
geraniin
amariin
6
4
2
0
1
A
1.5
2
B
2.5
3
C
3.5
4
D
Figure 2. Concentration of amariin (1) and geraniin (2) per leaf at different growing stages. A–D: see
Figure 1.
Aka427_Fr36Cryst - CH5
1600000
1500000
1600000
1500000
amariin (1)
A
B
C
geraniin (2)
geraniin (2)
1000000
500000
0
chlorogenic
acid
isoamariin (3)
chlorogenic
acid
1000000
500000
0
1000000
500000
0
elaeocarpusin
davidiin
amariin (1)
corilagin
furosin
flavonol glycosides
pentagalloyl
glucose
0.0
0
5.0
10.0
10
15.0
20.0
20
25.0
30.0
30
35.0
40.0
40
45.0
50.0
55.0
60.0
0.0
0
5.0
10.0
10
15.0
20.0
20
25.0
30.0
30
35.0
45.0
50.0
55.0
440.00
50 (mi6n0.)0
0
10
220.00
30
404.00
50 (m6in0.)0
50.0
0.0
10.0
30.0
50 (min)
Figure 3. HPLC profiles of extracts of Carpinus laxiflora fresh leaves collected in April (A) and collected
in October (B), and amariin (1) isolated from the April leaves (C).
2.2. Structure of Isoamariin
In the young leaves of C. laxiflora, 1 was accompanied by a minor isomer named isoamariin (3).
The molecular formula C41H28O28 was confirmed by high resolution fast atom bombardment mass
+
1
13
spectrometry (HRFABMS) (m/z: 991.0656 [M + Na] , calcd for C41H28O28Na, 991.0665). The H and C
NMR spectra are closely related to 1. They indicated that the molecular parts of 3 are the same as
those of 1. The NMR spectra exhibited duplicated signals caused by an equilibrium between the 6-
and 5-membered ring hemiacetal structures of DHHDP group connected to the glucose 2,4-positions.
In contrast to the 3,6-DHHDP of 1, the 3,6-DHHDP of 3 was almost fixed in the 6-membered ring
form. In addition, the HMBC correlations (Figure 4) revealed that 3 is an isomer of 1, differing in the
orientation of the 3,6-DHHDP on glucopyranose. The configuration of the DHHDP group was
concluded to be R based on large negative and positive Cotton effects at 236 and 209 nm, respectively,
in the electronic circular dichroism (ECD) spectrum. The structure of 3 is the same as that previously
1
reported for didehydrogeraniin [25]; however, the H NMR chemical shifts of the didehydrogeraniin
phenazine derivative in the literature coincided with those of the phenazine derivative of 1 and did
not match the data for the phenazine derivative of 3 (Supplementary Table S1).

Molecules 2020, 25, 1051
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OH
HO
O
OH
O
HO
OH
O
O
O
O
OH
OH
3
O
O
1
4
O
OH
2
O
O
O
O
HO
O
HO
OH
O
OH
OH
Figure 4. Selected HMBC correlations of major component of 3.
As reported by Foo [20], in aqueous solution 1 decomposed to give reduction product 2 as a
major product (Scheme 1, Supplementary Figure S3). Similarly, isoamariin (3) was also decomposed
under the same conditions to yield 2 (Supplementary Figure S4). Considering that reduction of 1 to 2
proceeded without any reductant, the reaction was presumed to be reduction-oxidation
disproportionation. The isolation yield of 2 from 1 in citrate-phosphate buffer at pH 6 was 47%. Other
products were identified to be 1-O-galloyl-2,4-(R)-DHHDP-β-D-glucopyranose (furosin) [25], 1-O-
galloyl-3,6-(R)-HHDP-β-D-glucopyranose (corilagin) [21], gallic acid, ellagic acid, and a new
compound, 1-O-galloyl-3,6-(S)-HHDP-D-glucose (Supplementary Figure S5). The structure of the
1
13
new compound was determined based on electrospray ionization (ESI)MS, HRFABMS, H, C NMR,
1
1
H- H correlation spectroscopy (COSY), heteronuclear single quantum coherence (HSQC),
heteronuclear multiple bond correlation (HMBC) and ECD spectra as shown in the Supplementary
Materials (Figure S6). The 3,6-(S)-HHDP glucose is rare in nature; however, 1,2,4-tri-O-galloyl-3,6-
(S)-HHDP-α-D-glucose is one of the major ellagitannin of Nuphar japonicum [26]. At the present time,
we have not succeeded in identifying the oxidation products that are expected to be generated
together with the reduction products.
DHHDP
HHDP
OH
OH
OH
HO
O
OH
O
HO
O
HO HO OH OH
HO
O
O
OH
OH
O
HO
O
R
OH
R
HO
OH
HO
OH
OH
H
HO
OH
OH
O
H
O
H
O
O
R
R
O
O
O
O
O
pH 6
O
pH 6
OH
OH
OH
O
O
O
OH
O
O
O
O
3
4
O
6
O
O
OH
OH
O
O
O
O
O
2
O
1
O
OH
O
O
O
O
O
O
H
O
O
O
O
O
H
R
O
O
H
H
O
H
H
O
HO
HO
HO
OH
O
OH
OH
HO
HO
HO
HO
HO
HO
O
OH
OH
O
OH
OH
OH
O
O
OH
O
OH
O
R
O
O
HO
OH
HO
HO
OH
OH
O
OH
OH
OH
OH
OH
1
2
3
Scheme 1. Production of geraniin (2) from amariin (1) and isoamariin (3) in citrate-phosphate buffer
(pH 6) at room temperature.
Only the 3,6-DHHDP of the two DHHDP groups of 1 selectively undergo the reduction;
however, computational calculation indicated that the structures of the 2,4- and 3,6-DHHDP moieties
are both ridged and essentially the same. The difference of the reactivity is thought to be caused by a
difference in the strain from glucose to the DHHDP groups. The coupling constants of glucopyranose
(J1,2 = 5.1 Hz, J2,3 = 1.3 Hz, J3,4 = 3.7 Hz, and J4,5 = 1.5 Hz) suggest that conformation of the glucopyranose
O,3
of 1 is of the B form. In contrast, X-ray crystallography confirmed that the glucopyranose core of 2
1
adopts the C4 conformation [27]. Due to the difficulty in analyzing the NMR signals caused by the
equilibrium of the DHHDP groups, a bisacetonyl derivative 1a was prepared to fix the DHHDP
groups in the 5-membered ring hemiacetal form [28], and DFT calculation was applied to 1a. The
coupling constants observed for glucopyranose of 1a were essentially the same as those of 1, and they
were also in agreement with those of 1a obtained by DFT calculation. The experimental NOEs

Molecules 2020, 25, 1051
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observed for 1a agreed with the calculation results (Figure 5). Calculation also indicated that a
1
O,3
conformer with C4-glucopyranose is less stable than in the B form (ΔG = + 4.5 kcal/mol). In contrast,
1
2 prefers the C4 conformation, and 1-O-galloyl-2,4-(R)-DHHDP-β-D-glucose is also shown to be
adapted to C4 conformation based on the coupling constants and DFT calculation. These
1
observations suggested that the difference in the reactivity of 3,6- and 2,4-DHHDP of 1 was due to
the difference of strain present from the glucose to ester carbonyl groups.
OH
O
H
OH
HO
O
O
OH
3.5 Å
O
O
O
O
6
4
OH
OH
O
1
3
O
H
OH
O
2.9 Å
H
H
O
O
O
O
H
O
OH
OH
O
O
NOE
OH
HO
Figure 5. Selected NOE correlations and stereostructure obtained by DFT calculation of 1a.
DHHDP is regarded as a hydrated quinone dimer of galloyl groups. Similar pyrogallol-quinone
dimers of epigallocatechin (4), that is, dehydrotheasinensins (5) are produced during the tea
fermentation process in black tea manufacturing (Scheme 2) [29,30]. The catechin dimers are unstable
and decompose during the drying process of black tea production to generate reduction products,
theasinensins (6), as the major products. Its hexahydroxybiphenyl structure is structurally related to
the HHDP group of ellagitannins. Experiments using pure dehydrotheasinensins showed that the
reaction is reduction-oxidation disproportionation. However, only limited oxidation products, such
as oolongtheanins (7) [31], were identified due to the complexity of the oxidation products.
Theasinensins are catechin oxidation products characteristic of black tea, and the dimerization
process seems to be related to the 3,6-HHDP formation of 2. The aforementioned production of the
3,6-(S)- and 3,6-(R)-HHDP glucoses from the (R)-DHHDP of 1 was also related to the production of
theasinensins with R and S biphenyl bond, respectively, from (S)-dehydrotheasinensins.
OH
O
OH
R-O
O
OH
OH
HO
HO
HO
O
HO
O
O
R or
S
OH
OH
OH
OH
[O]
OH
O-R
O-R
OH
OH
OH
4a
HO
O
4
OH
4
[O]
O-R
OH
OH
R = H or
OH
OH
O
6
O-R
HO
R-O
reduction-oxidation
disproportionation
OH
OH
OH
OH
HO
O
HO
O
H
S
H
O
OH
HO
O
O
O
HO
HO
O
H
OH
O-R
OH
O
O-R
OH
OH
7
5
Scheme 2. Reduction-oxidation disproportionation of catechin quinone dimer during black tea
production.

Molecules 2020, 25, 1051
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Previously, we reported that heating of 2 with pyridine in CH3CN yielded reduction products
mallotusinin (8) with a characteristic dibenzofuran structure, 1-O-galloyl-2,4;3,6-bis-(R)-HHDP-β-D-
glucose (10), and a decarboxylation product (11) [32]. The major product was 8 (30%, recovery of 2
1
13
was 15%)(Figure 6). In this study, an additional product 9 was isolated. The H and C NMR spectra
indicated that 9 is a pyridine adduct of 8. The binding site of the pyridine was confirmed to be at the
glucose C-4 side of the dibenzofuran moiety by HMBC correlations of pyridinium H-2,6 with the
benzofuran 3’ carbon (Figure 7). This was supported by a large up field shift of the glucose H-4 (Δδ
0.67) as compared to that of 8. The production mechanism of 9 from 2 is proposed as shown in Scheme
3, and this may be related to the reduction of 2 to 8. The attack of hydride instead of pyridine produces
8.
Geraniin - CH5
Geraniin_pyridine - CH5
A
B
2
pyridine
2000000
1000000
0
8
1500000
1000000
500000
ellagic acid
2
10
11
1-O-galloyl-3,6-(R)-
HHDP-β-D-glucose
(corilagin)
9
oligomeric polyphenols
0
10.0
20.0
40.0
50.0
60.0
00.0
10
20
330.00
40
50 (min)
0.0
10.0
20.0
30.0
40.0
50.0
50 (m6i0n.0)
0
10
20
30
40
Retention Time [min]
Retention Time [min]
Figure 6. HPLC profiles of geraniin (2) (A) and reaction mixture after treatment with 4% pyridine in
CH3CN (80 ˚C, 90 min) (B).
HO HO OH OH
HO
HO HO OH OH
HO HO OH OH
HO HO OH OH
HO
OH
HO
OH
HO
OH
OH
OH
OH
OH
OH
O
O
O
O
O
O
O
O
O
O
O
O
OH
OH
OH
OH
OH
O
O
O
O
OH
O
O
OH
O
O
O
O
O
O
O
O
OH
O
O
OH
HO
O
O
O
O
O
O
O
O
OH
O
O
O
O
O
O
O
N+
HO
OH
OH
-O
HO
OH
OH
HO
HO
OH
OH
O
O
HO
OH OH OH
O
O
10
11
8
9
Figure 7. Products obtained by treatment of 2 with pyridine in CH3CN (80 °C, 90 min) and HMBC
correlations of 9.
N
O
H
O
O
O
O
N
O
- 2H2O
9
HO
OH
O
OH
OH
HO
OH
OH
HO
O
2
Scheme 3. Plausible production mechanism of 9 from 2.
In this experiment, we could not identify the expected oxidation products; however, oligomeric
products (isolation yield 9.4%) may be candidates. The mixture of oligomers was detected at the
origin during thin layer chromatography (TLC) and as a broad hump on the baseline of the HPLC
13
(Figures 6 and S7). The C NMR spectrum showed signals attributable to galloyl and HHDP esters
together with glucose (Figure S8). Methylation and alkaline degradation of the oligomers confirmed

Molecules 2020, 25, 1051
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the presence of galloyl, HHDP, and tetrahydroxy dibenzofuran moieties; however, we could not
obtain evidence that the oligomers are oxidation products of 2.
Similar but contradictory results were obtained during acid hydrolysis. Foo reported that acid
hydrolysis of amariin (1) gave ellagic acid [20], and Okuda et al. also indicated the production of
excess ellagic acid upon acid hydrolysis of 2 [22]. These results suggested that reduction of the
DHHDP esters also occurs during hydrolysis. Reexamined the acid hydrolysis of 2 with 2% H2SO4 at
100 ˚C afforded ellagic acid in the isolation yield of 180 mol%. Furthermore, the acid hydrolysis of 1-
O-galloyl-2,4-(R)-DHHDP-β-D-glucopyranose with 2% H2SO4 also yielded gallic acid and ellagic acid
in the molar ratio of 1:0.73 (Supplementary Figure S9). These results suggested that the reduction of
DHHDP to HHDP during the hydrolysis is not reduction-oxidation disproportionation. In this
reaction, a possible electron donor candidate was glucose simultaneously generated by the
13
hydrolysis; however, the C NMR spectrum and TLC of the sugar fraction obtained by hydrolysis
confirmed the recovery of glucose.
3. Materials and Methods
3.1. General
Ultraviolet (UV) spectra were obtained on a JASCO V-560 UV/VIS spectrophotometer (JASCO,
Tokyo, Japan). Optical rotations were measured with a JASCO DIP-370 digital polarimeter. The ECD
1
13
spectra were measured with a JASCO J-725N spectrophotometer. H and C NMR spectra were
recorded on a Varian Unity Plus 500 spectrometer (Agilent Technologies, Santa Clara, CA, USA)
1
13
operating at 500 and 125 MHz for the H and C nuclei, respectively. NMR spectra were also recorded
on a JEOL JNM-AL 400 spectrometer (JEOL Ltd., Tokyo, Japan) operating at 400 and 100 MHz for the
1
13
H and C nuclei, respectively. ESIMS were obtained using a JEOL JMS-T100TD spectrometer.
FABMS were recorded on a JMS700N spectrometer (JEOL Ltd.) using m-nitrobenzyl alcohol or
glycerol as the matrix. Column chromatography was performed using Sephadex LH-20 (25–100 mm,
GE Healthcare UK Ltd., Little Chalfont, UK), MCI-gel CHP20P (75–150 mm, Mitsubishi Chemical
Co., Tokyo, Japan), Diaion HP20SS (Mitsubishi Chemical Co.), Toyopearl Butyl-650C (Tosoh
Bioscience Japan, Tokyo, Japan), Cosmosil 75C18OPN (Nacalai Tesque Inc., Kyoto, Japan), and
Chromatorex ODS (Fuji Silysia Chemical Ltd., Kasugai, Japan) columns. TLC was performed on
precoated Kieselgel 60 F254 plates (0.2-mm thickness, Merck, Darmstadt, Germany), using toluene–
ethyl formate–formic acid (1:7:1, v/v) and CHCl3–MeOH–H2O (7:3:0.5, v/v) mixtures as the eluents.
The spots were detected using ultraviolet illumination and by spraying with 2% ethanolic FeCl3
solution (for phenolic compounds), 5% anisaldehyde in ethanolic 5% H2SO4 solution (for
proanthocyanidins), or 5% H2SO4 solution followed by heating. Analytical HPLC was performed on
a Cosmosil 5C18-ARII (Nacalai Tesque Inc., Kyoto, Japan) column (250 × 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-2018 plus). Geraniin was obtained as
yellow crystalline powder from previous our studies [33].
3.2. Plant Material
Fresh leaves of Triadica sebifera and Elaeocarpus sylvestris var. ellipticus were collected in Bunkyo
campus of Nagasaki University. Fresh leaves of Carpinus laxiflora were collected Mt. Gokahara,
Isahaya, Nagasaki prefecture.
3.3. Quantification of Ellagitannins in the Leaves of T. Sebifera
Fresh leaves of T. sebifera were lyophilized and powdered by grinding in a mortar. Aliquots (50
mg) of the powder (each 4 samples) were extracted with 60% CH3CN containing 0.1% trifluoroacetic
acid (1.5 mL) at room temperature for 7 h. The extracts were analyzed by HPLC. Calibration curves
were built using geraniin (320 nm) isolated in our previous study [24] and amariin (275 nm) isolated
in this study. Average dried weights of the leaves in the Figure 1 were as follows: A, 0.007 g/leaf

Molecules 2020, 25, 1051
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(fresh: 0.022 g/leaf); B, 0.029 g/leaf (fresh: 0.089 g/leaf); C, 0.065g/leaf (fresh: 0.20 g/leaf), and D, 0.195
g/leaf (fresh: 0.63 g/leaf).
3.4. Isolation of Amariin (1) and Isoamariin (3)
Fresh young leaves of Carpinus laxiflora (750 g) collected in April 30, 2014 were homogenized
with 80% acetone containing 0.005% trifluoroacetic acid (TFA) (3 L) at room temperature 2 times. The
extract was filtered, and the filtrate was concentrated using rotary evaporator. Resulting precipitates
formed in the aqueous solution were removed by filtration. The filtrate (about 800 mL) was applied
to Diaion HP20SS (8 cm i.d. × 37 cm) column with 0.005% TFA containing increasing proportions of
MeOH [0% (500 mL), 20% (1 L), 30% (1 mL), 40% (1 L), 50% (1 L), 60% (1 L), 80% (1 L), and 100% (2
L)] to give 5 fractions. Fraction (Fr.) 3 (16.8 g) mainly containing 1 was subjected to Sephadex LH-20
column chromatography (5 cm i.d. × 22 cm) with 0%–100% MeOH in 0.005% TFA (20% stepwise, each
300 mL) to yield a crude sample of 1 (10.9 g). A portion (7.5 g) of the crude sample was purified by
Diaion HP20SS column chromatography (4 cm i.d. × 27 cm, 0%–60% MeOH in 0.005% TFA, 5%
stepwise, each 200 mL). Tubes containing 1 were combined and concentrated by rotary evaporator
(40 ˚C) to give yellow precipitates of 1, which were collected by filtration (3.7 g). The filtrate (0.76 g)
was separated by Chromatorex ODS (3 cm i.d. × 25 cm, 0%–50% MeOH in 0.005% TFA, 5% stepwise,
each 100 mL) to yield 3 (121 mg).
1
Amariin (1): Yellow powder, H NMR (500 MHz, acetone-d6) δ: galloyl: 7.23, 7.22 (each s, galloyl-2,6);
DHHDP (5- and 6-membered-ring hemiacetal structure): 7.26, 7.238, 7.236, 7.235, 7.233 (each s, H-3’),
6.71, 6.67, 6.60, 6.58 [each s, H(6)-3], 6.31, 6.26 [each d, J = 1.5 Hz, H(5)-3], 5.31. 5.29, 5.27, 5.26 [each s,
H(6)-1], 4.90, 4.88, 4.86, 4.85 [each d, J = 1,5 Hz, H(5)-1]; glucose (two major isomers among 4 isomers),
isomer A: 6.40 (d, J = 5.4 Hz, glc-H-1), 5.79 (dd, J = 1.5, 3.2 Hz, glc-3), 5.50 (br d, J = 3.2 Hz, glc-4), 5.47
(dt, J = 1.5, 5.4 Hz, glc-2), 5.23 (dd, J = 2.7, 13.3 Hz, glc-6), 4.77 (br s, glc-5), 4.39 (br d, J = 13.3 Hz, glc -
6); isomer B: 6.35 (d, J = 5.1 Hz, glc-1), 5.65 (dt, J = 1.3, 3.7 Hz, glc-4), 5.62 (dt, J = 1.5, 5.1 Hz, glc-2), 5.53
(dd, J = 1.5, 3.7 Hz, glc-3), 5.33 (dd, J = 2.6, 13.3 Hz, glc-6), 4.83 (br s, glc-5), 4.39 (br d, J = 13.3 Hz, glc
13
-6); C NMR (125 MHz, acetone-d6) δ: only signals of major isomers COO: 168.4, 168.1, 166.2, 165.1,
165.0, 164.8, 164.7, 164.6, 164.4; galloyl: 146.21, 146.18 (C-3,5), 139.9, 139.2 (C-4), 110.40, 110.39 (C-2,6);
DHHDP: 194.2 [C(5)-4], 191.6, 191.2 [C(6)-4], 153.6, 152.5, 152.1 [C(6)-2], 148.6, 147.8. 147.3 [C(5)-6’],
143.5, 143.0, 142.9 [C(6)-6’], 129.2, 128.6 [C(6)-3], 125.7 [C(5)-3], 96.6, 96.2 [C(6)-5], 92.31, 92.27, 92.0
2
[C(5)-5, C(6)-6], 52.2, 51.1 [C(5)-1], 46.4, 45.0, 44.8 [C(6)-1]; other aromatic and sp carbons: 145.93,
145.86, 145.83, 139.2, 138.5, 138.4, 137.6, 120.3, 120.0, 119.9, 119.8, 119.7, 118.7, 117.0, 116.0, 115.0, 114.9,
113.9, 113.6, 113.1; glucose (two major isomers among 4 isomers): 95.7, 94.4 (glc-1), 78.1, 77.7 (glc-5),
73.7, 72.7 (glc-2), 70.4, 69.7 (glc-4), 67.6,66.1 (glc-3), 66.0, 65.8 (glc-6).
Isoamariin (3): Yellow powder, [α]1D7 −136.8 (c 0.1, MeOH); FAB-MS m/z: 991 [M + Na] ; HR-FAB-MS
+
+
−1
m/z: 991.0656 [M + Na] (calcd. for C41H28O28Na, 991.0665); IR νmax cm : 3413, 1705, 1613, 1528, 1447,
1325, 1213; UV (MeOH) λmax (log ε): 279 (4.43), 221 (4.89); ECD (MeOH) λmax (Δε): 369 (−3.9), 326 (−1.4),
1
293 (−8.6), 270 (−2.1), 262 (−2.4), 257 (−2.1), 236 (−14.7), 228 (0), 209 (+36.4). H NMR (500 MHz, acetone-
d6) δ: galloyl: 7.21, 7.20 (each s, galloyl-2,6); 2,4-DHHDP 5-membered-ring hemiacetal structure: 7.29
(s, H-3’), 6.26 (d, J = 1.4 Hz, H-3), 4.95 (d, J = 1.4 Hz, H-1); 2,4-DHHDP 6-membered-ring hemiacetal
structure: 7.27 (s, H-3’), 6.60 (s, H-3), 5.35 (s, H-1); glucose with 5-membered-ring hemiacetal 2,4-
DHHDP: 6.42 (d, J = 5.2 Hz, glc-1), 5.67 (dt, J = 1.3, 5.2 Hz, glc-2), 5.51 (dd, J = 1.3, 3.7 Hz, glc-3), 6.17
(dt, J = 1.3, 3.7 Hz, glc-4), 4.72 (br s, glc-5), 4.60 (dd, J = 3.1, 13.3 Hz, glc-6), 4.90 (dd, J = 1.7, 13.3 Hz, glc
-6); glucose with 6-membered-ring hemiacetal 2,4-DHHDP: 6.36 (d, J = 4.7 Hz, glc-1), 5.78 (dt, J = 1.4,
4.7 Hz, glc-2), 5.36 (dd, J = 1.4, 3.7 Hz, glc-3), 6.28 (dt, J = 1.3, 3.5 Hz, glc-4), 4.76 (br s, glc-5), 4.62 (dd,
13
J = 3.1, 12.3 Hz, glc-6), 4.93 (dd, J = 1.8, 12.3 Hz, glc-6); C NMR (125 MHz, acetone-d6) δ: galloyl: 119.7,
119.9 (galloyl-1), 110.3 (galloyl-2,6), 146.2 (galloyl-3,5), 139.8, 139.9 (galloyl-4), 164.8, 164.9 (galloyl-7);
2,4-DHHDP 5-membered-ring hemiacetal structure: 52.1 (C-1), 148.9 (C-2), 125.8 (C-3), 194.3 (C-4),
92.4 (C-5), 109.3 (C-6), 117.1 (C-1’), 120.3 (C-2’), 113.1 (C-3’), 147.8 (C-4’), 137.6 (C-5’), 147.3 (C-6’); 2,4-
DHHDP 6-membered-ring hemiacetal structure: 46.4 (C-1), 154.0 (C-2), 129.3 (C-3), 191.7 (C-4), 96.2
(C-5), 92.4 (C-6); 116.1 (C-1’), 119.0 (C-2’), 113.6 (C-3’), 145.9 (C-4’), 139.2 (C-5’), 144.0 (C-6’); glucose

Molecules 2020, 25, 1051
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with 5-membered-ring hemiacetal 2,4-DHHDP: 94.3 (glc-1), 74.2 (glc-2), 67.5 (glc-3), 69.6 (glc-4), 77.1
(glc-5), 68.0 (glc-6); glucose with 6-membered-ring hemiacetal 2,4-DHHDP: 95.4 (glc-1), 73.6 (glc-2),
69.0 (glc-3), 68.7 (glc-4), 76.5 (glc-5), 67.9 (glc-6).
3.5. Preparation of Acetonyl Derivative
Amariin (1) (130 mg) was dissolved in 80% acetone (20 mL) containing HCO2NH4 (130 mg) and
stirred at r.t. for 13h. After removal of acetone by evaporation, the aqueous solution was subjected to
Diaion HP20SS column chromatography (2 cm i.d. × 15 cm, 0%–60% MeOH in H2O, 5% stepwise,
each 50 mL) to yield 1a (57.2 mg).
Bisacetonyl derivative (1a): off-white amorphous powder, [α]1D7 −55.2 (c 0.1, MeOH); IRνmax cm : 3406,
−1
1
1715, 1620, 1531, 1441; UV (MeOH) λmax (log ε): 282 (4.56), 221 (4.91). H NMR (500 MHz, acetone-d6)
1
δ: H-NMR (acetone-d6, 500 MHz) δ: 2.19 (3H, s, 2,4-acyl-CH3), 2.23 (3H, s, 3,6-acyl-CH3), 2.96 (1H, d,
J = 15.3 Hz, 3,6-acyl-CH2), 3.06 (1H, d, J = 15.6 Hz, 2,4-acyl-CH2), 3.46 (1H, d, J = 15.6 Hz, 2,4-acyl-CH2),
3.48 (1H, d, J = 15.3 Hz, 3,6-acyl-CH2), 4.21 (1H, dd, J = 1.2, 13.1 Hz, glc-6a), 4.83 (1H, d, J = 1.5 Hz, 3,6-
acyl-1), 4.88 (1H, br. s, glc-5), 4.98 (1H, d, J = 1.5 Hz, 2,4-acyl-1’), 5.39 (1H, dt, J = 1.4, 3.8 Hz, glc-4),
5.47 (1H, dt, J = 1.5, 4.9 Hz, glc-2), 5.57 (1H, dd, J = 3.2, 13.1 Hz, glc-6b), 5.70 (1H, dd, J = 1.4, 3.8 Hz,
glc-3), 6.35 (1H, d, J = 1.5 Hz, 2,4-acyl-3’), 6.44 (1H, d, J = 4.9 Hz, glc-1), 6.46 (1H, d, J = 1.5 Hz, 3,6-acyl-
13
3), 7.20 (1H, s, 3,6-acyl-3’), 7.22 (1H, s, 2,4-acyl-3), 7.27 (2H, s, galloyl-H); C NMR (acetone-d6, 125
MHz) δ: 32.0 (2,4-acyl-CH3), 32.2 (3,6-acyl-CH3), 49.2 (3,6-acyl-CH2), 49.9 (2,4-acyl-CH2), 50.8 (3,6-acyl-
1), 51.9 (2,4-acyl-1’), 64.5 (glc-6), 66.0 (glc-3), 69.7 (glc-4), 73.8 (glc-2), 78.1 (glc-5), 80.2 (3,6-acyl-5), 80.6
(2,4-acyl-5’), 94.3 (glc-1), 110.0 (2,4-acyl-6’), 110.4 (galloyl-2, 6), 110.6 (3,6-acyl-6), 113.0 (2,4-acyl-3, 3,6-
acyl-3’), 116.9 (2,4-acyl-1), 117.9 (3,6-acyl-1’), 119.9 (3,6-acyl-2’), 120.1 (galloyl-1), 120.5 (2,4-acyl-2),
127.1 (3,6-acyl-3), 127.5 (2,4-acyl-3’), 136.5 (3,6-acyl-5’), 137.3 (2,4-acyl-5), 139.6 (galloyl-4), 144.6 (3,6-
acyl-2), 144.9 (2,4-acyl-2’), 146.1 (galloyl-3, 5), 146.7 (3,6-acyl-6’), 147.0 (2,4-acyl-6), 147.4 (2,4-acyl-4,
3,6-acyl-4’), 164.3 (2,4-acyl-7), 164.9 (galloyl-7, 3,6-acyl-7), 166.1 (2,4-acyl-7’), 166.9 (3,6-acyl-7’), 196.9
(3,6-acyl-4), 197.2 (2,4-acyl-4’), 205.7 (2,4-acyl-CO), 205.9 (3,6-acyl-CO).
3.6. Preparation of Phenazine Derivative
Amariin (1) (50 mg) and O-phenylenediamine (10 mg) was dissolved in 10% AcOH in EtOH (2
mL) and heated at 50˚C for 2 h. The mixture was applied to Sephadex LH-20 (2 cm i.d. × 15 cm, 0-20%
H2O in EtOH, 10% stepwise, each 100 mL) to yield 1b (30.2 mg). Phenazine derivative 3a was
prepared from 3 in the same way.
Phenazinde derivative (1b): UV (MeOH) λmax (log ε) 445 (3.56), 378 (4.13), 281 (4.98), 205 (4.87); ECD
1
(MeOH) Δε (nm) 377 (−1.48), 325 (+3.86), 284 (−67.5), 250 (+72.1), 221 (−42.0). H NMR (acetone-d6, 500
MHz) δ: 4.11 (1H, dd, J = 3.3, 12.0 Hz, glc-6a), 4.98 (1H, dd, J = 8.1, 12.0, glc-6b), 5.04 (1H, dd, J = 3.3,
8.1, glc-5), 5.40 (1H, d, J = 4.1, glc-4), 5.80 (1H, d, J = 4.1, glc-3), 5.86 (1H, d, J = 5.5, glc-2), 6.23 (1H, d, J
= 5.5, glc-1), 7.01 (2H, s, galloyl-H), 7.03 (1H, s, 3,6-acyl-3’), 7.52 (1H, s, 2,4-acyl-3), 8.24 (1H, s, 3,6-acyl-
13
3), 8.31 (1H, s, 2,4-acyl-3’); C-NMR (acetone-d6, 125 MHz) δ: 64.7 (glc-6), 67.7 (glc-3), 68.3 (glc-4), 76.2
(glc-2), 77.0 (glc-5), 91.7 (glc-1), 109.7 (2,4-acyl-3), 110.1 (galloyl-2, 6), 113.2 (3,6-acyl-3’), 115.2 (3,6-
acyl-1’), 116.4 (2,4-acyl-1), 116.7 (3,6-acyl-1), 116.8 (2,4-acyl-1’), 119.8 (galloyl-1), 120.1 (2,4-acyl-3’),
120.6 (2,4-acyl-2), 120.7 (3,6-acyl-3), 123.1 (3,6-acyl-2’), 130.0, 130.4, 130.5 (2,4-acyl-4”, 5”, 3,6-acyl-4”,
5”), 132.2, 132.3 (2,4-acyl-3”, 6”, 3,6-acyl-3”, 6”), 136.0 (2,4-acyl-2’), 136.3 (3,6-acyl-2), 137.4 (3.6-acyl-
5’), 138.0, 139.0 (2,4-acyl-5’, 3,6-acyl-5), 139.3 (2,4-acyl-5), 139.6 (galloyl-4), 142.8, 143.0, 143.1, 145.1,
145.3, 145.4, 145.6, 145.9 (2,4-acyl-6, 4’, 1”, 2”, 3,6-acyl-4, 4’, 6’, 1”, 2”), 145.3 (2,4-acyl-4), 146.0 (galloyl-
3, 5), 152.1 (3,6-acyl-6), 152.3 (2,4-acyl-6’), 164.7 (galloyl-7), 165.9 (3,6-acyl-7), 166.6 (2,4-acyl-7’), 167.8
(3,6-acyl-7’), 167.9 (2,4-acyl-7).
Phenazine derivative (3a): Brown amorphous powder, [α]1D7 −323.9 (c 0.06, MeOH); FAB-MS m/z: 1077
+
+
+
[M + H] , 1099 [M + Na] ; HR-FAB-MS m/z: 1099.1403 [M + Na] , (calcd. for C53H32O22N4Na, 1099.1406);
−1
IR vmax cm : 3336, 1732, 1613, 1509, 1463, 1355, 1335, 1308, 1189; UV (MeOH) λmax (log ε): 446 sh (3.51),
375 (4.15), 279 (5.00), 223 sh (4.86), 207 (4.89); ECD (MeOH) λmax (Δε): 381 (+3.8), 359 (+1.3), 304 (+16.9),

Molecules 2020, 25, 1051
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1
292 (0), 274 (−79.0), 257 (0), 247 (+29.4), 230 (0), 220 (−24.8), 212 (0); H NMR (acetone-d6, 500 MHz) δ:
4.12 (1H, t, J = 5.1, 11.6 Hz, glc-6a), 4.84 (1H, dd, J = 8.5, 11.6, glc-6b), 5.11 (1H, dd, J = 5.1, 8.5, glc-5),
5.48 (1H, d, J = 4.1, glc-3), 5.63 (1H, dd, J = 0.7, 4.1, glc-4), 5.71 (1H, d, J = 5.7, glc-2), 6.22 (1H, d, J = 5.7,
glc-1), 6.99 (2H, s, galloyl-H), 7.16, 7.46, 7.86, 8.27 (each 1H, each s, aromatic) 7.93–8.01, 8.15–8.36 (each
13
m, phenazine-H); C NMR (acetone-d6, 125 MHz) δ: 66.7, 68.2, 68.7, 76.2, 76.8 (glc-2~6), 91.7 (glc-1),
110.2 (galloyl-2,6), 113.5 (acyl-3), 115.7, 116.0, 116.6, 117.0, 118.5 (galloyl-1, acyl-1, 1’), 120.1, 120.2,
120.5, 122.7 (acyl-2, 3’), 130.2, 130.6, 130.7 (acyl-4”, 5”), 132.2, 132.4, 132.5 (acyl-3”, 6”), 136.1, 136.2
(acyl-2’), 138.2, 139.2, 139.5, 139.8, 139.9, 142.8, 142.9, 143.3, 143.5, 145.2, 145.3, 145.5, 145.7, 145.9
(galloyl-4, acyl-4, 5, 6, 4’, 5’, 1”, 2”), 146.2 (galloyl-3, 5), 152.3, 152.4 (acyl-6’), 164.9, 166.2, 166.6, 167.5,
167.9 (esters).
3.7. Treatment of 1 at pH 6
Amariin (1) (1.0 g) was dissolved in citrate-phosphate buffer (0.05 M, pH 6.0) and stirred at r.t.
for 20 h. The mixture was acidified by addition of 5% TFA to pH 2 and applied to Sephadex LH-20
column chromatography (3 cm i.d. × 30 cm, 0%–100% MeOH in H2O, 20% stepwise, each 200 mL,
followed by MeOH-acetone-H2O, 90:6:4, 80:12:8, 70:18:12, each 200 mL) to yield 11 fractions. Fr. 3
(23.5 mg) was identified as gallic acid. Fr. 4 (60.6 mg) was subjected to Chromatorex ODS column
chromatography (2 cm i.d. × 17 cm, 0–70% MeOH in H2O, 5% stepwise, each 100 mL) to give 1-O-
galloyl-3,6-(S)-HHDP-β-D-glucose (17.6 mg). Fr. 5 (43.7 mg) was purified by MCI gel CHP 20P
column chromatography (2 cm i.d. × 17 cm, 0-50% MeOH in H2O, 5% stepwise, each 100 mL) to afford
1-O-galloyl-3,6-(R)-HHDP-β-D-glucose (corilagin, 24.0 mg). Similar separation of Fr. 7 (63.9 mg)
yielded 1-O-galloyl-2,3-(R)-DHHDP-β-D-glucose (furosin, 34.4 mg). Fractions 9 and 10 were
identified to be geraniin (2)(463.9 mg) and ellagic acid (48.0 mg), respectively.
1-O-Galloyl-3,6-(S)-HHDP-β-D-glucose: Brown amorphous powder; [α]2D9 +20.7° (c 0.10, MeOH); UV
−1
(MeOH) λmax (log ε): 293 (4.36), 273 (4.49), 227 (4.80), 219 (4.88); IR νmax cm : 3378, 1709, 16213; ESI-
−
MS (negative) m/z: 633 [M − H] ; HR-FAB-MS m/z: 657.0705 (calcd for C27H22O18Na, 657.0703); ECD
1
(MeOH) Δε (nm): +3.38 (229), −2.16 (267), +1.45 (293); H NMR (acetone-d6, 500 MHz) δ: 7.21 (2H, s,
galloyl-H-2,6), 7.23 (1H, s, HHDP-H-3), 7.01 (1H, s, HHDP-H-3’), 6.29 (1H, d, J = 9.0 Hz, glc-1), 5.10
(1H, dt, J = 1.1, 5.7 Hz, glc-3), 4.88 (1H, d, J = 12.3 Hz, glc-6), 4.25 (1H, br d, J = 2.4 Hz, glc-5), 3.96 (1H,
13
dt, J = 5.7, 9.0 Hz, glc-2), 3.84 (1H, dd, J = 3.4, 12.3 Hz, glc-6), 3.59 (1H, br s, glc-4); C NMR (acetone-
d6, 125 MHz) δ: 168.0 (HHDP-7), 167.7 (HHDP-7’), 166.1 (galloyl-7), 145.8 (galloyl-3,5), 144.6, 144.5
(HHDP-4,4’,6,6’), 139.1 (galloyl-4), 138.1 (HHDP-5), 136.4 (HHDP-5’), 126.4 (HHDP-2, 2’), 121.2
(galloyl-1), 117.5 (HHDP-1), 116.7 (HHDP-1’), 112.4 (HHDP-3’), 110.3 (galloyl-2,6), 108.1 (HHDP-3’),
92.3 (glc-1), 81.2 (glc-3), 80.1 (glc-5), 72.7 (glc-4), 70.2 (glc-2), 63.3 (glc-6).
3.8. Heating of 2 with Pyridine in CH3CN
A solution of 2 (2.0 g) in 4% pyridine in CH3CN (50 mL) was heated at 80 ˚C for 90 min. After
cooling, the solution was mixed with 2% aqueous TFA (25 mL) and concentrated. Resulting aqueous
solution was applied to Sephadex LH-20 column chromatography (3 cm i.d. × 25 cm, 60%–100%
MeOH in H2O, 10% stepwise, each 100 mL, followed by MeOH-acetone-H2O, 80:10:10, 200 mL) to
give 7 fractions. Fr. 3 (123.8 mg) was mainly composed of 1-O-galloyl-3,6-(R)-HHDP-β-D-glucose
(corilagin) and product 9. Fr. 4 (78.7 mg) contained corilagin, 9, and ellagic acid. Fr. 5 (473.5 mg) was
separated by Chromatorex ODS (4 cm i.d. × 25 cm, 0%–55% MeOH in H2O, 5% stepwise, each 100
mL) to give 2 (290.6 mg) and a mixture of 9 and 11, which were separated by Diaion HP20SS (3 cm
i.d. × 17 cm, 10–60% MeOH in H2O, 5% stepwise, each 50 mL) followed by purification using
Chromatorex ODS chromatography (3 cm i.d. × 28 cm) to yield 9 (23.2 mg) and 11 (39.3 mg). Fr. 6 was
separated H2O soluble and insoluble parts, and the soluble fraction was purified by Avicel cellulose
column chromatography (2 cm i.d. × 15 cm) with 2% AcOH to give 1-O-galoyl-2,3;4,6-bis-(R)-HHDP-
β-D-glucose (10) (30.9 mg). The H2O insoluble precipitates were combined with Fr. 7 and subjected to
size-exclusion column chromatography using Sephadex LH-20 (2 cm i.d. × 55 cm) with 7M
urea:acetone (2:3, v/v, containing conc. HCl at 5 mL/L)[34] to give fractions containing oligomeric

Molecules 2020, 25, 1051
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polyphenols and 8. The removal of urea by Diaion HP20SS column chromatography afforded 8 (595.8
mg) and oligomeric polyphenols (188.3 mg).
Pyridine adduct (9): A tan amorphous powder, [α]1D7 −47.5 (c 0.1, MeOH); IR νmax cm : 3383, 1730, 1616,
−1
+
1344; UV (MeOH) λmax (log ε): 349 (3.88), 280 (4.48), 217 (4.78). ESI-MS m/z: 996 [M + H] , m/z: 995 [M
−
+
1
− H] ; HRFABMS: m/z 996.1108 [M + H] (calcd for C46H30NO25, 996.1107); H NMR (acetone-d6, 500
MHz) δ: 9.53, 9.08 [each 1H, br d, J = 6.4 Hz, pyridine (py)-2,6], 8.62, 8.45 (each 1H, br t, J = 6.4 Hz, py-
3,5), 7.37 [1H, s, benzofuran (BF)-3], 7.09 (2H, s, galloyl-2,6), 7.02 (1H, s, HHDP-3), 6.67 (1H, s, HHDP-
3’), 6.39 (1H, br s, glc-3), 6.20 (1H, d, J = 3.1 Hz, glc-1), 5.39 (1H, br s, glc-2), 4.70 (1H, d, J = 3.8 Hz, glc-
13
4), 4.41 (2H, m, glc-5, 6), 4.18 (1H, dd, J = 5.1, 10.6 Hz, glc-6); C NMR (acetone-d6, 125 MHz) δ: 168.3
(HHDP-7’), 166.7 (BF-7), 166.6 (HHDP-7), 165.2 (BF-7’), 165.1 (galloyl-7), 149.7, 149.2 (py-2,6), 147.9,
147.7, 147.5 (py-4, BF-4’,6’), 146.0 (galloyl-3,5), 145.9 (BF-6), 145.13, 145.06, 145.0, 144.6 (HHDP-
4,4’,6,6’), 140.8 (BF-5’), 139.9 (galloyl-4), 137.3 (HHDP-5), 136.7 (HHDP-5’), 135.8 (BF-5), 134.6 (BF-2’),
129.8, 129.0 (py-3,5), 125.9 (BF-3’), 124.4, 123.8 (HHDP-2,2’), 119.6 (galloyl-1), 117.0 (BF-3), 116.7
(HHDP-1), 115.8 (BF-2), 115.7 (HHDP-1’), 115.1 (BF-1’), 114.3 (BF-1), 110.3 (galloyl-2,6), 109.5 (HHDP-
3), 108.8 (HHDP-3’), 91.8 (glc-1), 74.7 (glc-5), 73.6 (glc-2), 69.3 (glc-4), 64.2 (glc-6), 62.1 (glc-3).
3.9. Methylation and Alkaline Degradation of Oligomeric Product
Oligomeric products (100 mg) in methanol (2 mL) was treated with etherial diazomethane for 2
days. The mixture was heated with 5% NaOH at 80 °C for 12 h, acidified with diluted HCl, and then
partitioned with diethyl ether. The ether layer was concentrated and treated with diaomethane, and
the products were separated by silica gel column chromatography (30%–100% acetone in hexane,
10% stepwise, each 50 mL) to yield methyl trimethoxybenzoate (5 mg), dimethyl (R)-
hexamethoxydiphenate (2.0 mg) and dimethoxycarbonyl tetramethoxydibenzofuran (1.7 mg).
3.10. Acid Hydrolysis of DHHDP Esters
A portion (0.50 mL) of an acetone solution of 2 (10.0 mg/mL), 1-O-galloyl-2,4-(R)-DHHDP-β-D-
glucopyranose (6.0 mg/mL), and 1-O-galloyl-3,6-(R)-HHDP-β-D-glucopyranose (6.0 mg/mL) was
dispensed into 5 mL screw-capped vials, and acetone was removed in vacuo. To each vial, 0.50 mL
of 2% H2SO4 was added and heated at 100 °C for 7 h. The reaction mixture was mixed with DMSO
(4.5 mL) to dissolve ellagic acid and analyzed by HPLC (Figure S9). Yield of the products was
estimated based on the peak area and calibration curve obtained by using standard compounds: 4.76
μmol of gallic acid and 10.09 μmol of ellagic acid was produced from 5.25 μmol of 2. Similarly, 4.16
μmol of gallic acid and 3.05 μmol of ellagic acid was produced from 4.61 μmol of 1-O-galloyl-2,4-(R)-
DHHDP-β-D-glucopyranose, and 4.84 μmol of gallic acid and 5.59 μmol of ellagic acid was produced
from 4.72 μmol of 1-O-galloyl-3,6-(R)-HHDP-β-D-glucopyranose.
3.11. Acid Hydrolysis of 2
Geraniin (2) (1.00 g, 1.05 mmol, dried over P4O10) was hydrolyzed with 2% H2SO4 (50 mL) under
reflux for 2 days. Insoluble precipitates were collected by filtration and washed with MeOH to give
ellagic acid (504.5 mg, 1.67 mmol). The filtrate was directly subjected to Diaion HP20SS column
chromatography (3 cm i.d. × 25 cm, 0%–80% MeOH in H2O, 10% stepwise, each 100 mL) to yield gallic
acid (206.2 mg, 1.21 mmol), flavogallonic acid (9.8 mg, 0.02 mmol), and ellagic acid (66.0 mg, 0.22
mol). Total isolation yield of ellagic acid was 1.89 mmol.
3.12. Computational Methods
A conformational search was performed using the Monte Carlo method and the MMFF94 force
field with Spartan ′14 (Wavefunction, Irvine, CA, USA). The obtained low-energy conformers within
6 kcal/mol were optimized at the B3LYP/6-31G(d,p) level in acetone (PCM). The vibrational
frequencies were also calculated at the same level to confirm their stability, and no imaginary
1
frequencies were found. H NMR coupling constants of the low-energy conformers with Boltzmann
populations greater than 1% were calculated at the B3LYP/6-31G(d,p)u + 1s (using only the Fermi

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contact term) level in acetone (PCM) and scaled by using the slope parameter 0.94 [35]. The calculated
data for each conformer were averaged according to the Boltzmann distribution theory at 298 K based
on their relative Gibbs free energies. All DFT calculations were performed using Gaussian 09 [36].
GaussView was used to draw the three-dimensional molecular structures [37].
4. Conclusion
The results presented and described in this paper strongly suggest that HHDP is produced by
the reduction of DHHDP, at least in the case of 3,6-HHDP of geraniin (2) from amariin (1) and
isoamariin (3). This may be the key mechanism in the ellagitannin biosynthesis, which otherwise
remains unclear. The similarity to the reaction mechanism for the epigallocatechin B-B’ ring biphenyl
bond formation during black tea production also supports our hypothesis. The difference in reactivity
between 3,6- and 2,4-DHHDP is probably explained by tensile forces from glucose, which depend on
the conformation of the pyranose ring. In our investigation on the production mechanism of HHDP
from DHHDP, understanding the missing electron donor is the most critical issue. However,
presently, we can only introduce the experimental results as described herein.
Supplementary Materials: The following are available online. Figure S1. The leaves of Triadica sebifera used for
the HPLC analysis, Figure S2. HPLC profiles of 60% CH3CN extracts of Elaeocarpus sylvestris var. ellipticus fresh
leaves, Table S11H NMR data for phenazine derivatives. Figure S3. HPLC profiles of aqueous solution of amariin
(1) in pH 6 citrate-phosphate buffer, Figure S4. HPLC profiles of isoamariin (3) in pH 6 citrate-phosphate buffer,
Figure S5. Structures of minor products produced by degradation of 1, Figure S6. HMBC correlations and ECD
spectra of 1-O-galloyl-3,6-(S)-HHDP-β-D-glucose. Figure S7. HPLC profile (A) and UV spectrum (B) of
oligomeric polyphenol fraction obtained from 2, Figure S8. 13C NMR spectrum of oligomeric polyphenol fraction
measured in acetone-d6. Figure S9. HPLC profiles of acid hydrolysis of 2, 1-O-galloyl-2,4-(R)-DHHDP-β-D-
glucopyranose, and 1-O-galloyl-3,6-(R)-HHDP-β-D-glucopyranose. Figures S10–S31, 1D, 2D NMR spectra of
compounds 1, 1a, 3, 3a, and 9, and computational methods.
Author Contributions: T.T. and Y.M. conceived and designed the experiments, and analyzed the data; M.E.
performed the experiments and analyzed the data. Y.M. and M.E. performed the DFT calculation. M.E., Y.S. and
T.T. discussed the conclusion and wrote the paper. All authors have read and agreed to the published version
of the manuscript.
Funding: This work was supported by the Japan Society for the Promotion of Science KAKENHI (Grant Nos.
17K08338 and 16K07741).
Acknowledgments: The authors are grateful to N. Tsuda, K. Chifuku, and H. Iwata at the Center for Industry,
University and Government Cooperation, Nagasaki University, for recording the NMR and MS data, and Dr.
M. Tanaka (Graduate School of Biomedical Sciences, Nagasaki University) for providing access to the ECD
spectrometer. The computation was partly carried out using the computer facilities at the Research Institute for
Information Technology, Kyushu University. This work was the result of using research equipment shared in
MEXT Project for promoting public utilization of advanced research infrastructure (Program for supporting
introduction of the new sharing system) Grant Number JPMXS0422500320.
Conflicts of Interest: The authors declare no conflict of interest.
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