Highly Oxidized Ellagitannins of Carpinus japonica and Their Oxidation-Reduction Disproportionation

Article abstract of DOI:10.1021/acs.jnatprod.0c00893

In the research on ellagitannin metabolism, two unique dehydroellagitannins, carpinins E (1) and F (2), bearing dehydrohexahydroxydiphenoyl (DHHDP) and hydrated biscyclohexenetrione dicarboxyl ester (HBCHT) groups, were isolated from young leaves of Carpinus japonica. Upon heating in H2O or treatment with pH 6 buffer at room temperature, 1 and 2 afforded the reduction product 3, isocarpinin A, with an (R)-hexahydroxydiphenoyl (HHDP) group, suggesting the occurrence of redox disproportionation of the (S)-DHHDP group. This was supported by the increase in production of 3 in the pH 6 buffer solution by coexistence of epigallocatechin-3-O-gallate (15), accompanied by oxidation of 15. In contrast, treatment of 1 and 2 with ascorbic acid yielded 4, carpinin A, with an (S)-HHDP group. Upon heating with ascorbic acid, the HBCHT group was also reduced to an (S)-HHDP group, and 2 was converted to 2,3;4,6-bis(S)-HHDP glucose. In leaves of C. japonica, the tannins 1 and 2 are dominant in young spring leaves, but compounds 3 and 4 become the major components of tannins in mature leaves. These results suggest that, in ellagitannin biosynthesis, oxidative coupling of the two galloyl groups first generates a DHHDP group, and subsequent reduction of DHHDP esters produces HHDP esters.

Full text of DOI:10.1021/acs.jnatprod.0c00893

pubs.acs.org/jnp
Article
Highly Oxidized Ellagitannins of Carpinus japonica and Their
Oxidation−Reduction Disproportionation
Daisetsu Kojima,# Kengo Shimizu,# Kosuke Aritake,# Manami Era, Yosuke Matsuo, Yoshinori Saito,
Takashi Tanaka,* and Gen-ichiro Nonaka
Cite This: https://dx.doi.org/10.1021/acs.jnatprod.0c00893
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: In the research on ellagitannin metabolism, two unique dehydroellagitannins, carpinins E (1) and F (2), bearing
dehydrohexahydroxydiphenoyl (DHHDP) and hydrated biscyclohexenetrione dicarboxyl ester (HBCHT) groups, were isolated
from young leaves of Carpinus japonica. Upon heating in H2O or treatment with pH 6 buffer at room temperature, 1 and 2 afforded
the reduction product 3, isocarpinin A, with an (R)-hexahydroxydiphenoyl (HHDP) group, suggesting the occurrence of redox
disproportionation of the (S)-DHHDP group. This was supported by the increase in production of 3 in the pH 6 buffer solution by
coexistence of epigallocatechin-3-O-gallate (15), accompanied by oxidation of 15. In contrast, treatment of 1 and 2 with ascorbic
acid yielded 4, carpinin A, with an (S)-HHDP group. Upon heating with ascorbic acid, the HBCHT group was also reduced to an
(S)-HHDP group, and 2 was converted to 2,3;4,6-bis(S)-HHDP glucose. In leaves of C. japonica, the tannins 1 and 2 are dominant
in young spring leaves, but compounds 3 and 4 become the major components of tannins in mature leaves. These results suggest
that, in ellagitannin biosynthesis, oxidative coupling of the two galloyl groups first generates a DHHDP group, and subsequent
reduction of DHHDP esters produces HHDP esters.
llagitannins are a group of hydrolyzable tannins
Ebiosynthesized by oxidative metabolism of galloyl
glucoses.1−6 The most common and diagnostic acyl group is
the hexahydroxydiphenoyl (HHDP) group, which is a simple
galloyl dimeric moiety. Another common acyl group is the
dehydrohexahydroxydiphenoyl (DHHDP) group, which is an
oxidized form of the HHDP group with a hydrated tricarbonyl
structure (Scheme 1).1,2,7 Because polyphenols are susceptible
to oxidation, it is thought that HHDP esters are produced by
oxidative coupling of two galloyl groups. Subsequent oxidation
of the HHDP group affords DHHDP esters (Scheme 1 route
A).1,8 However, in 1993, Foo reported the reductive
production of an HHDP ester from a DHHDP ester by a
nonenzymatic spontaneous reaction under mild conditions.9
Heating of amariin, 1-O-galloyl-2,4;3,6-bis(R)-DHHDP glu-
cose, in aqueous EtOH yielded geraniin, 1-O-galloyl-2,4-(R)-
DHHDP-3,6-(R)-HHDP glucose. This result demonstrated
the spontaneous reduction of a DHHDP group to an HHDP
group in a nonenzymatic reaction. It was noted that this
reaction of amariin was similar to that of the partially
characterized ellagitannin, isogeraniin, reported by Haddock
et al. in 1982.2,10 Geraniin is the most widely distributed
DHHDP-bearing ellagitannin (so-called dehydroellagitan-
nin).1,2,7 Haddock et al. demonstrated that isogeraniin coexists
with geraniin in various geraniin-rich plants belonging to the
Geraniaceae, Aceraceae, Simaroubaceae, and Cercidiphylla-
ceae.2 If isogeraniin is identical to amariin, the coexistence of
geraniin and isogeraniin in those plants suggests that the
reduction of DHHDP esters may be related to the biosynthesis
of the 3,6-HHDP ester of geraniin (Scheme 1, route B). Our
Received: August 12, 2020
© XXXX American Chemical Society and
American Society of Pharmacognosy
https://dx.doi.org/10.1021/acs.jnatprod.0c00893
J. Nat. Prod. XXXX, XXX, XXX−XXX
A

Journal of Natural Products
pubs.acs.org/jnp
Article
ellagitannins, carpinins A−D (4−7), with a highly oxidized
form of the HHDP group named hydrated biscyclo-
hexenetrione dicarboxyl (HBCHT) (Figure 1).13 In this
study, we show that ellagitannin composition changes
markedly in the leaves of C. japonica as they mature, and we
isolated unstable dehydroellagitannins, carpinins E (1) and F
(2), from the young leaves. Furthermore, the unusual reactivity
of these tannins provides further evidence for the putative
ellagitannin metabolism route B shown in Scheme 1.
Scheme 1. Conventional (Black) and Alternative (Red)
Routes for Production of HHDP and DHHDP Esters
RESULTS AND DISCUSSION
■
HPLC Analyses of Compounds in Spring and Summer
Leaves. In our previous study, carpinins A (4), B (5), C (6),
and D (7) were isolated from leaves of C. japonica collected in
June (Figure 2).13 However, HPLC analyses of the fresh young
leaves collected in April revealed that tannins 1 and 2 were the
major tannins (Figure 1). In mature leaves collected in August,
tannins 1 and 2 had almost disappeared and 3, 4, 5, and the
uncharacterized tannin 3 were the major components.
The concentrations of 1 and 2 in extracts of leaves collected
in April gradually decreased during extraction with 80%
CH3CN at room temperature for 15 h. However, these
compounds were much more stable in solvent containing small
amounts (0.05−0.1%) of trifluoroacetic acid (TFA) (Figures 2
and 3A). When the young leaves collected in April were
extracted with 80% CH3CN at 70 °C for 1 h, 1 and 2 were
absent, and instead, a peak attributable to unidentified tannin 3
was observed (Figure 3B). Dramatic changes in the HPLC
profile were also observed when ascorbic acid was added to the
extraction solvent as a reducing reagent (40 °C for 2 h), and
the chromatogram exhibited large peaks arising from 4 instead
of peaks of 1 and 2 (Figure 3C). These results suggested that 1
and 2 in the young leaves are the precursors to 3 and 4.
Structure Determination of 1−3. To isolate the
uncharacterized unstable tannins 1−3, fresh leaves collected
recent studies on seasonal changes in ellagitannin composition
demonstrated that amariin is a major ellagitannin in the young
spring leaves of Carpinus laxiflora, Triadica sebifera, and
Elaeocarpus sylvestris var. ellipticus and that amariin content
decreases as the leaves mature.11 A similar decrease of
DHHDP esters has also been detected in the young spring
leaves of Castanopsis sieboldii.12 Furthermore, detailed analyses
of the reactions of purified amariin in aqueous solution at room
temperature and spontaneous reduction of the DHHDP group
of geraniin on heating with pyridine suggested that the
conversion of a DHHDP ester to an HHDP ester comprises a
redox disproportionation.11 The results suggested that the
HHDP group is produced from the DHHDP group (Scheme
1, route B). These findings prompted us to study seasonal
changes in ellagitannin composition in the leaves of Carpinus
japonica, because these leaves contain several characteristic
Figure 1. Structures of carpinins E (1), F (2), A (4), B (5), C (6), and D (7) and isocarpinin A (3).
B
https://dx.doi.org/10.1021/acs.jnatprod.0c00893
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
Isomers 1 and 2 were separated by Chromatorex ODS column
chromatography, and 3 and 4 were separated by Toyopearl
HW40F column chromatography.
The 13C NMR spectra of carpinins E (1) and F (2) were
related to those of 4−7, showing signals arising from the α-
and β-anomers of 4C1-glucopyranose and HBCHT ester
groups.13 The NMR spectrum of 2 was much simpler than
that of 1, because the proportion of α- and β-anomers of 2 was
7:1, while that of 1 was near 1:1. This may explain the
differences in the HPLC peak areas of the two anomers for
each compound (Figure 2A). The HBCHT group is a highly
oxidized form of the HHDP and DHHDP groups commonly
found in carpinins and is characterized by two sets of
conjugated carbonyls (data for the α-anomer of 2: δ 192.1,
192.3, HBCHT-4,4′), a double bond (δ 129.8, 130.4,
HBCHT-3,3′; 145.90, 149.94, HBCHT-2,2′), two hemiacetal
moieties (δ 91.6, 91.7, HBCHT-5,5′; 103.6, 103.8, HBCHT-
6,6′), a benzylic methine (δ 48.5, 48.6, HBCHT-1,1′), and
ester carbonyls (δ 166.1, 166.5, HBCHT-7,7′). The remaining
component of molecules 1 and 2 was determined to be a
DHHDP group, on the basis of signals of the hydrated
cyclohexenetrione carboxyl moiety (data for the α-anomer of
2: δ 44.2, C-1; 91.5, C-6; 97.0, C-5; 131.4, C-3; 150.1, C-2;
164.6, C-7; 191.5, C-4) and the trihydroxybenzoyloxy moiety
(data for 2: 106.5, C-3′; 112.0, C-1′; 124.6, C-2′; 135.4, C-5′,
142.9, C-6′, 146.3, C-4′, 170.4, C-7′). These spectroscopic
observations were consistent with the same molecular formulas
of 1 and 2 (C34H24O25) indicated by HRFABMS. Usually,
DHHDP groups exist as equilibrium mixtures of six- and five-
membered hemiacetal forms;14 however, the 1H and 13C NMR
spectra of 1 and 2 indicated that the DHHDP esters of these
compounds predominantly adopted six-membered hemiacetal
Figure 2. HPLC chromatograms (220 nm) of fresh leaf extracts of C.
japonica collected on April 28 (A) and August 3, 2016 (B), from the
same tree (extraction: 1.0 g fresh leaf material/15 mL 80% CH3CN
containing 0.1% TFA, at room temperature for 2 h). Tannins 1−7
and 12 were detected as a pair of peaks corresponding to α- and β-
anomers. Chl: chlorogenic acid, PG: pentagalloyl glucose, FG:
flavonol glycosides, EA: ellagic acid. Chl, PG, and EA were identified
by comparison of the retention times and UV absorption with those
of authentic samples.
1
structures (6:1 for 1 and 7:1 for 2, based on H NMR signals
of the DHHDP H-1 methine protons).
The location of the acyl groups in 1 and 2 was determined
by HMBC correlations between ester carbonyls and glucosyl
protons (Figure 4). In the spectra of 1 and 2, glucosyl H-4 and
H-6 showed HMBC correlations with the ester carbonyl
carbons of the HBCHT group, confirming the location of the
HBCHT diester group at C-4 and C-6 of the glucosyl unit. As
for the DHHDP esters, the DHHDP C-7 carboxylic carbon of
1 correlated with the DHHDP C-1 methine proton and
glucosyl H-3. The C-7′ ester carbonyl showed cross-peaks with
the H-3′ aromatic methine and glucosyl H-2. The HMBC
spectrum of 2 showed correlations between the DHHDP C-7
ester carbonyl and the H-1 methine proton and glucosyl H-2
in April were homogenized with 80% acetone at room
temperature and extracted for 5 h. The extract was first
fractionated by Sephadex LH-20 column chromatography, and
the fraction containing 1−4 was further separated by column
chromatography using Diaion HP20SS and Chromatorex ODS
columns with aqueous CH3CN containing 0.05% TFA.
Figure 3. HPLC chromatograms (220 nm) of fresh leaves collected on April 23, 2017 (extraction: 0.20 g fresh leaf material/4.0 mL solvent). (A)
Extracted with 80% CH3CN containing 0.1% TFA, at 40°C for 1 h. (B) Extracted with 80% CH3CN at 70°C for 1 h. (C) Extracted with 80%
CH3CN containing ascorbic acid (80 mg) at 40 °C for 2 h. Chl: chlorogenic acid, GG: galloyl glucose, PG: pentagalloyl glucose, FG: flavonol
glycosides.
C
https://dx.doi.org/10.1021/acs.jnatprod.0c00893
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
Figure 4. Selected HMBC and ROESY correlations for 1 and 2.
and correlations between the C-7′ ester carbonyl and the
aromatic methine H-3′ and glucosyl H-3. These correlations
indicated that 1 and 2 are isomers differing in the connectivity
of the DHHDP ester to the glucopyranosyl moiety.
spectra of 3 also resembled those of 4, indicative of the
presence of α- and β-glucopyranosyl anomers bearing HBCHT
and HHDP ester moieties. The location of the HBCHT ester
units at C-4 and C-6 of the glucosyl moieties was determined
from the HMBC correlations between the C-7 and C-7′ ester
carbonyls and H-4 and H-6 of the glucosyl unit. However, in
the 1H NMR spectrum, severe broadening of the HHDP
aromatic protons of the α-anomer (δH 6.92, 6.98, each brs)
was observed. In addition, the 13C NMR chemical shifts of the
HHDP C-1,1′ (HHDP C-1,1′ of α- and β-anomers: δC 117.0,
117.47, 117.54, 117.6) and C-2,2′ (δC 119.3, 121.1, 121.4,
122.9) were different from those observed for the 2,3-(S)-
HHDP group of 4 (δC 114.10, 114.14, 114.57, 114.61; C-2, 2′:
δC 126.42, 126.43, 126.46, 126.62). These spectroscopic
features were similar to those reported for the 2,3-(R)-
HHDP group of cuspinin [1-O-galloyl-2,3-(R)-4,6-(S)-bis-
HHDP-β-D-glucose] isolated from Castanopsis cuspidata var.
sieboldii.19,20 In addition, the ECD spectrum of 3 showed a
negative Cotton effect at 229 nm and a positive Cotton effect
at 264 nm, opposite to those observed for 4. Thus the structure
of isocarpinins A (3) was defined as shown in Figure 1.
Reactions of 1 and 2. The isomers 1 and 2 showed similar
and unusual reactivities: heating in H2O afforded the reduction
product 3. This reaction was the same as that observed when
the fresh young leaves were heated in 80% CH3CN (Figure
3B). Besides product 3, separation of the reaction products of
2 after heating at 60 °C for 46 h afforded 7, 2,3-(R)-HHDP-
4,6-(S)-DHHDP-D-glucoses (8), 4,6-(S)-DHHDP-D-glucoses
(9),15 brevifolin carboxylic acid (10),21 ellagic acid (11), and a
mixture of oligomeric products (Scheme 2, HPLC profiles in
Figure S2, Supporting Information). The structure of 8 was
determined based on HMBC correlations (Figure S34,
Supporting Information) and the ECD spectrum showing a
negative Cotton effect at 217 nm (S-DHHDP) and a positive
Cotton effect at 261 nm (R-HHDP).15 Product 9 was
confirmed by comparison of its spectroscopic data with those
of the partial hydrolysate of 1,2,3-trigalloyl-4,6-(S)-DHHDP-β-
D-glucose prepared by treatment with tannase.22 Compound 9
was also obtained by heating of 7 in H2O (100 °C, 5 h).
The production of 3 from 2 and 9 from 7 only by heating in
H2O suggested that the reaction is a redox disproportionation,
and the simultaneous production of oxidation products was
expected. However, the products isolated from the reaction
mixture of 2 were reduction products or hydrolysates. The
The configuration of the DHHDP groups of 1 and 2 was
deduced to be S from the negative Cotton effect at 207 nm and
the positive Cotton effect at 238 nm in their ECD spectra.15,16
This was supported by the ROESY correlations between the
DHHDP benzylic methine and glucosyl H-2 for 1 and between
the benzylic methine and glucosyl H-3 for 2 (Figure 4).
To chemically confirm the presence of the acyl groups, 2 was
treated with 1,2-phenylenediamine and the mixture of products
was methylated with CH2N2−ether. The products were
hydrolyzed by heating under alkaline conditions, and the
resulting carboxylic acid was methylated with CH2N2−ether.
The methyl esters were identified as (S)-methyl 4-methoxy-3-
[2,3,4-trimethoxy-6-(methoxycarbonyl)phenyl]-2-phenazine-
carboxylate (2a)15,17,18 and (S)-methyl 4-methoxyphenazine-2-
carboxylate 3,3′-dimer (2b). The configuration of the biphenyl
bonds of 2a and 2b was determined by comparison of their
ECD spectra with literature data and confirmed by computed
ECD data (Figure S1, Supporting Information).15 Based on
these spectroscopic and chemical results, the structures of 1
and 2 were determined as shown in Figure 1.
Another new ellagitannin named isocarpinin A (3) was
isolated as a minor constituent of young leaves collected in
April. Its molecular formula, C34H24O24, was shown to be the
1
same as that of 4 by HRFABMS. The H and 13C NMR
Figure 5. Structures of phenazine derivatives 2a and 2b.
D
https://dx.doi.org/10.1021/acs.jnatprod.0c00893
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
Scheme 2. Reaction of 2 in H2O and 1% H2SO4
Scheme 3. Production of Pedunculagins (12) and Desgalloyl Cuspinins (13) and 4,6-(S)-HHDP-Glucoses (14) from 2
only candidate oxidation products were the oligomeric
products detected at the origin on silica gel TLC plates and
as a broad hump on the HPLC baseline. The 13C NMR
spectrum of the oligomers (Figure S3, Supporting Informa-
tion) exhibited signals attributable to the HBCHT group and
glucosyl moieties; however, the spectrum did not provide
evidence that the oligomers were oxidation products. In
contrast, heating of 2 in 1% H2SO4 (100 °C, 3.5 h) afforded 4
with an (S)-HHDP group as well as 7, 9, 10, and 11 (Scheme
2, HPLC profiles in Figure S2, Supporting Information). The
reason for the (R)-HHDP group being produced upon heating
in H2O and the (S)-HHDP group being formed upon heating
under acidic conditions is unclear.
Information). Furthermore, the reaction of 2 with ascorbic acid
at a higher temperature (100 °C for 1 h) afforded
pedunculagin (12)25 and 4 (Scheme 3). The ellagitannin 12
was detected in mature leaves of C. japonica collected in
August (Figure 2B), suggesting that a similar metabolic
pathway exists in the leaves. The combination of the
aforementioned reactions of 2, that is, heating in H2O (100
°C for 20 min) and subsequent heating with ascorbic acid (100
°C for 1 h), yielded desgalloyl cuspinins [2,3-(R)-HHDP-4,6-
(S)-HHDP-D-glucose, 13] along with its partial hydrolysate,
4,6-(S)-HHDP-glucose (14), and 11 (Scheme 3). Compound
13 was identified as the tannase hydrolysate of cuspinin [1-O-
galloyl-2,3-(R)-HHDP-4,6-(S)-HHDP-D-glucose].19 Owing to
incomplete conversion of 2 to 3 in this experiment, 12 was also
isolated from the reaction mixture.
As observed for fresh young leaves treated with ascorbic acid
(Figure 3C), the reduction of 1 and 2 with ascorbic acid in
H2O at 40 °C for 1 h yielded 4 (HPLC profiles in Figure S5
and possible reaction mechanism in Scheme S2, Supporting
Our previous study showed that reduction of the DHHDP
group also occurred by heating with pyridine in CH3CN (80
E
https://dx.doi.org/10.1021/acs.jnatprod.0c00893
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
°C for 90 min),11,26 but the mechanism of the reaction remains
unclear. Treatment of 4 and 5 under the same conditions
afforded 12 and 2,3-di-O-galloyl-4,6-(S)-HHDP-D-glucose,27,28
respectively, indicating that the HBCHT esters are also
reduced by heating with pyridine (HPLC profiles in Figure
S6, Supporting Information).
Tannins 1 and 2 were gradually converted to 3 in a pH 5−7
buffer solution at room temperature (HPLC profiles in Figure
S4, Supporting Information). This suggested that dissociation
of the phenolic group is important during stereoselective
conversion and that deprotonation at the benzylic methine is
probably involved in the conversion to the (R)-HHDP group.
Interestingly, the color of both 1 and 2 in pH 6 and 7 buffer
solutions was greenish-blue (Figure 6). This suggests that an
and 3 was detected as the major product (Figure 8B). When
15 was present in the solution of 2, the yield of 3 increased to
153% of the yield from 2 alone. The reaction mixture also
contained oxidation products of 15, that is, 15a−d (Figure
8D). These results confirm the electron transfer from the B-
ring of 15 to the DHHDP group of 2 and support our
speculation that the reaction mechanism of the production of 3
from 2 is a redox disproportionation caused by intermolecular
electron transfer.
The results show that the ellagitannin composition of C.
japonica leaves changes markedly as they grow, and extraction
of young leaves and subsequent chromatographic separation
led to the isolation of two unique dehydroellagitannins,
carpinins E (1) and F (2), with DHHDP and HBCHT
groups. The ellagitannins 1 and 2 are unstable and afforded the
reduction product isocarpinin A (3) with an (R)-HHDP group
by treatment with pH 6 buffer at room temperature. This
suggested the occurrence of a redox disproportionation by
intermolecular electron transfer. Supporting evidence was
provided by the increase in the quantity of reduction product
3 in the presence of 15 and the formation of oxidation
products of 15. Interestingly, the results demonstrate stereo-
selective reduction of the (S)-DHHDP ester moieties of 1 and
2 to (S)- and (R)-HHDP groups and suggest that the
dissociation of the phenolic group of the DHHDP pyrogallol
ring participates in the production of the (R)-HHDP group
(Scheme S1, Supporting Information). The HBCHT group
was reduced to form an (S)-DHHDP group on heating in
H2O, and 2 was converted into the bis-HHDP analogues 12
and 13 by heating with ascorbic acid. These results provide
further evidence for the putative pathway of ellagitannin
metabolism represented by route B in Scheme 1. That is,
oxidative coupling of two galloyl groups generates the
DHHDP group, and subsequent reduction produces HHDP
esters. This mechanism is similar to that by which black
polyphenols theasinensins A (15b) and D (15c) are generated
via reduction of dehydrotheasinensin A (15a).28 The
mechanism of that reaction is also thought to be a redox
disproportionation, suggesting that a common reaction
mechanism operates for the oxidative coupling of pyrogallol-
type phenolic compounds. Although the mechanism for
stereoselective production of 3 from 1 and 2 at pH 6 is not
clear, stereoselective reduction of 1 and 2 to 4 with ascorbic
acid is probably related to stereoselective addition of ascorbic
acid to the (R)-DHHDP group,33,34 where ascorbic acid
attacks from the less hindered face of the DHHDP group
(Scheme S2, Supporting Information). Results of this study do
Figure 6. Color of solutions of 1 in citrate−phosphate buffer (rt, 30
min).
intermolecular charge-transfer complex may have formed. A
similar color change was observed upon oxidation of the black
tea pigment theaflavin, where a charge-transfer complex forms
between theaflavin and the o-quinone B-ring moiety of
epicatechin.23,24 Possible reaction mechanisms based on
intermolecular electron transfer from the DHHDP aromatic
ring to the cyclohexenetrione ring are proposed in Scheme S1,
in the Supporting Information.
Reaction of 2 in the Presence of Epigallocatechin
Gallate. To confirm the intermolecular electron transfer
between the DHHDP cyclohexenetrione and pyrogallol rings,
the conversion of 2 to 3 in the presence of electron-rich
pyrogallol-type aromatic rings was examined. Epigallocatechin-
3-O-gallate (15) is the representative tea catechin with an
electron-rich pyrogallol-type B-ring.29,30 Enzymatic oxidation
of 15 affords dehydrotheasinensin A (15a), theasinensins A
(15b) and D (15c),29 and theacitrin C (15d), which are
catechin dimers characteristic of black tea polyphenols (Figure
7).31,32 In pH 6 buffer solution (room temperature, 3 h), 15
alone did not change, whereas 2 alone completely disappeared
Figure 7. Structures of 15 and its oxidation products 15a−d generated by reaction with 2.
F
https://dx.doi.org/10.1021/acs.jnatprod.0c00893
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
Figure 8. HPLC chromatograms of reaction mixtures of 2 and 15 in pH 6 buffer. (A) Aqueous solution of 2. (B) 2 in pH 6 buffer at room
temperature for 3 h. (C) Aqueous solution of a mixture of 2 and 15. (D) 2 and 15 in pH 6 buffer at room temperature for 3 h. Compound 15 alone
did not change under the same conditions.
specimen has been deposited at the Graduate School of Biomedical
Sciences, Nagasaki University.
not provide evidence that the redox disproportionation is the
representative mechanism of HHDP production. Further
studies from the viewpoints of biochemistry and organic
synthesis are necessary to understand ellagitannin biosynthesis.
HPLC Analysis of Leaf Extracts. To examine seasonal changes in
ellagitannin content, the fresh leaves collected on April 28 were cut
with scissors into small pieces and extracted with 80% aqueous
CH3CN containing 0.1% TFA at room temperature for 2 h (1.0 g
fresh weight/15 mL). After filtration through a membrane filter (0.45
μm), the filtrate was analyzed by HPLC (Figure 1). Leaves collected
on August 3 were also analyzed under the same conditions.
Heating of Extract. A small portion of fresh leaves collected on
April 23 (200 mg) was extracted with 80% aqueous CH3CN (4.0 mL)
at 70 °C for 1 h. After filtration through a membrane filter (0.45 μm),
the filtrate was analyzed by HPLC (Figure 3B).
Treatment of Extract with Ascorbic Acid. A small portion of
fresh leaves collected on April 23 (200 mg) was mixed with ascorbic
acid (80 mg) and extracted with 80% aqueous CH3CN (4.0 mL) at
40 °C for 2 h. After filtration through a membrane filter (0.45 μm),
the filtrate was analyzed by HPLC (Figure 3C).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Ultraviolet (UV) spectra
were obtained using a JASCO V-560 UV/VIS spectrophotometer
(JASCO, Tokyo, Japan). Optical rotations were measured with a
JASCO DIP-370 digital polarimeter. The ECD spectra were measured
with a JASCO J-725N spectrophotometer. The H and 13C NMR
1
spectra were recorded on a Varian Unity Plus 500 spectrometer
(Agilent Technologies, Santa Clara, CA, USA) operating at 500 and
1
126 MHz for the H and 13C nuclei, respectively. The NMR spectra
were also recorded on a JEOL JNM-AL 400 spectrometer (JEOL Ltd,
1
Tokyo, Japan) operating at 400 and 100 MHz for the H and 13C
nuclei, respectively. The FABMS data were recorded on a JMS700N
spectrometer (JEOL Ltd.) using m-nitrobenzyl alcohol or glycerol as
matrix. Column chromatography was performed using Sephadex LH-
20 (25−100 mm, GE Healthcare UK Ltd., Little Chalfont, UK),
Diaion HP20SS (Mitsubishi Chemical Co.), Avicel cellulose
(Funakoshi Co., Ltd, Tokyo, Japan), Chromatorex ODS (Fuji Silysia
Chemical Ltd., Kasugai, Japan), Toyopearl HW40F, and Toyopearl
Ether 650M (Tosoh Bioscience Co., Tokyo, Japan). The TLC
analyses were performed on 0.25 mm thick, precoated silica gel 60
F254 plates (Merck, Darmstadt, Germany) with toluene−ethyl
formate−formic acid (1:7:1, v/v/v) and on 0.1 mm thick, precoated
cellulose F plates (Merck, Darmstadt, Germany) with 2% aqueous
HOAc. Spots were detected by illumination under a short UV
wavelength (254 nm), followed by spraying with 2% ethanolic FeCl3
or 5% H2SO4 solution and then heating. Analytical HPLC was
performed on a Cosmosil 5C18-ARII (Nacalai Tesque Inc., Kyoto,
Japan) column (250 × 4.6 mm, i.d.) with 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).
Isolation of Compounds. Fresh young leaves (600 g) of C.
japonica collected on April 26, 2016, were homogenized with 80%
acetone (5.0 L) in a Waring blender, and the homogenate was kept at
room temperature for 5 h with occasional shaking. After filtration, the
extract was concentrated using a rotary evaporator. The insoluble
precipitate that formed in the aqueous solution was removed by
filtration. The filtrate was subjected to chromatographic separation on
a Sephadex LH-20 column (7.0 cm i.d. × 31 cm) with H2O
containing increasing proportions of MeOH (0, 10, 30, 50, 60, 80, and
100% MeOH, each 0.5 L) and finally with 60% acetone (2 L) to give
fraction (Fr.) 1 (34.9 g) containing 1−4 and Fr. 2 (23.0 g) containing
5 and pentagalloyl glucose. Fr. 1 was separated by Diaion HP20SS
column chromatography (5.0 cm i.d. × 32 cm) with 0−40% CH3CN
containing 0.05% TFA (5% stepwise, each 500 mL) to yield Fr. 1-1
(9.6 g, mainly composed of 1, 3, and 4), 1-2 (26 g, mainly composed
of 2), and a mixture of flavonol glycosides (0.95 g). Fr. 1-2 was
separated by chromatography on a Chromatorex ODS column (5.0
cm i.d. × 27 cm) with 0−30% CH3CN containing 0.1% TFA (5%
stepwise, each 500 mL) to give Fr. 1-2-1 (2.9 g, a mixture of 1, 3, 4,
and chlorogenic acid), Fr. 1-2-2 (9.6 g, a mixture of 2 and chlorogenic
acid), and 2 (10.7 g). Fractions 1-1 and 1-2-1 were combined and
separated by Chromatorex ODS column chromatography to give a
Plant Material. Leaves of C. japonica, Betulaceae, were collected
at Mt. Gokahara, Isahaya city, Nagasaki prefecture. A voucher
G
https://dx.doi.org/10.1021/acs.jnatprod.0c00893
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
mixture of 3 and 4 (1.81 g) and 1 (3.51 g). Finally, 3 and 4 were
separated by Toyopearl HW40F column chromatography (3.0 cm i.d.
× 20 cm) with 60−80% MeOH (5% stepwise, each 200 mL) to yield
3 (418 mg) and 4 (32.8 mg). Owing to the instability of the
compounds, especially 1 and 2, the isolated compounds were
lyophilized to remove H2O completely.
(DHHDP-7), 166.1 (HBCHT-7′), 166.5 (HBCHT-7), 170.4
(DHHDP-7′), 191.5 (DHHDP-4), 192.1 (HBCHT-4′), 192.3
(HBCHT-4). Anomer: α:β = 7:1; DHHDP: six-membered-ring:five-
membered-ring = 9:1.
Isocarpinins A (3): pale yellow lyophilized powder; [α]D −75 (c
0.3, MeOH); UV (MeOH) λmax (log ε) 229 (4.59), 275sh (4.13),
322sh (3.69), 353sh (3.07); IR νmax cm−1 3433, 2967, 1713, 1615,
1514, 1443, 1350; FABMS m/z 815 [M − H]−, HRFABMS m/z:
839.0556 [M + Na]+ (calcd for C34H24O24Na, 839.0550); ECD
(MeOH) λmax (Δε) 334 (+0.5), 293 (-9.4), 278 (0), 264 (+7.6), 253
(0), 229 (−30.2), 214 (0), 203 (+24.2); anal. calcd for
C34H24O24H2O C, 48.93, H, 3.14; found C, 49.12, H, 3.20; 1H
NMR (500 MHz, acetone-d6) δ 3.89 (d, J =13.6 Hz, α-HBCHT-1),
3.90 (d, J =13.6 Hz, β-HBCHT-1), 3.94 (d, J =13.6 Hz, α-HBCHT-
1′), 4.00 (d, J =13.6 Hz, β-HBCHT-1′), 4.15 (m, α-glc-6, β-glc-5),
4.16 (brt, J =11.1 Hz,β-glc-6), 4.40 (bt t, J =8.8 Hz, α-glc-5), 4.60 (dd,
J =7.8, 9.7 Hz, β-glc-2), 4.78 (m, α-glc-2, 6), 4.83 (t, J =10.4 Hz, β-
glc6), 5.02 (brt, J =9.2 Hz, β-glc-), 5.09 (t, J =9.8 Hz, β-glc-3), 5.12
(d, J =7.8 Hz, β-glc-1), 5.18 (brs, α-glc-4), 5.30 (t, J =9.7 Hz, α-glc-3),
5.52 (brs, α-glc-1), 6.51 (brs, α-glc-1-OH), 6.60 (s, β-HHDP-3), 6.67,
6.68 (each s, α-HBCHT-3,3′), 6.68 (s, β-HBCHT-3′), 6.74 (s, β-
HBCHT-3), 6.92, 6.98 (each brs, α-HHDP-3,3′), 7.20 (s, β-HHDP-
3′); 13C NMR (126 MHz, acetone-d6) δ 48.4, 48.46, 48.52 (HBCHT-
1,1′), 64.5 (α-glc-5), 65.9 (β-glc-6), 66.6 (α-glc-6), 68.8 (β-glc-5),
74.8 (β-glc-4), 74.9 (α-glc-3), 75.2 (α-glc-4), 76.5 (α-glc-2), 77.5 (β-
glc-3), 79.2 (β-glc-2), 91.3 (α-glc-1), 91.6, 91.65, 91.70 (HBCHT-
5,5′), 95.5 (β-glc-1), 103.54, 103.56, 103.59, 103.62 (HBCHT-6,6′),
107.4 (β-HHDP-3), 109.0, 110.0 (α-HHDP-3,3′), 112.0 (β-HHDP-
3′), 117.0, 117.5 (β-HHDP-1′), 117.5, 117.6 (α,β-HHDP-1,1′), 119.3
(β-HHDP-2′), 121.1, 121.4 (α-HHDP-2,2′), 122.9 (β-HHDP-2),
129.8, 130.0 (β-HBCHT-3,3′), 130.2 (α-HBCHT-3,3′), 136.6 (β-
HHDP-5′), 137.6, 137.9 (α-HHDP-5,5′), 139.1 (β-HHDP-5′), 144.6,
144.7 (α-HHDP-4,4′), 144.8 (β-HHDP-4), 145.0 (β-HHDP-4′),
145.4, 145.45, 145.47, 145.48 (α,β-HHDP-6,6′), 145.8, 145.9, 146.0,
146.2 (α,β-HBCHT-2,2′), 166.0 (α-HBCHT-7′), 166.0 (α-HBCHT-
7), 166.3 (β-HBCHT-7′), 166.4 (β-HBCHT-7), 168.2 (β-HHDP-7′),
168.4 (α-HHDP-7), 168.5 (α-HHDP-7′), 168.9 (β-HHDP-7), 192.1,
192.2 (α-HBCHT-4,4′), 192.3, 192.4 (β-HBCHT-4,4′).
Carpinins E (1): pale yellow lyophilized powder; [α]D +74 (c 0.2,
MeOH); UV (MeOH) λmax (log ε) 208 (4.58), 243sh (4.37), 272sh
(3.93), 335 (3.42), 403sh (3.08); IR νmax cm−1 3448, 2968, 1739,
1717, 1623, 1512, 1453, 1375; FABMS m/z 833 [M + H]+, 855 [M +
Na]+; HR-FABMS m/z 855.0504 [M + Na]+ (calcd for C34H24O25Na,
855.0499); ECD (MeOH) λmax (Δε) 385 (+1.4), 346 (0), 326
(−0.84), 290 (0), 282 (+0.40), 266 (0), 259 (−0.50), 253 (0), 238
(+4.8), 229 (0), 207 (−15.3); anal. calcd for C34H24O25·0.5H2O C,
48.53, H, 2.99; found C, 48.34, H, 3.03; 1H NMR (500 MHz,
acetone-d6) δ 3.92 (m, HBCHT-1′), 3.95 (m, HBCHT-1), 4.17 (dd, J
= 3.3, 11.0 Hz, α-glc-6), 4.25 (m, β-glc-5,6), 4.43 (s, α-DHHDP-1),
4.47 (s, β-DHHDP-1), 4.49 (m, α-glc-5), 4.55 (d, 1.6 Hz, DHHDP-
3(5)), 4.95 (dd, J = 4.9, 11.2 Hz, β-glc-6), 4.98 (dd, J = 3.7, 11.1 Hz,
α-glc-6), 5.19 (t, J = 9.9 Hz, β-glc-3), 5.33 (d, J = 8.1 Hz, β-glc-1),
5.37 (t, J = 9.4 Hz, β-glc-4), 5.37 (dd, J = 7.9, 9.9 Hz, β-glc-2), 5.40 (t,
J = 9.8 Hz, α-glc-3), 5.44 (t, J = 9.5 Hz, α-glc-4), 5.51 (dd, J = 3.2,
10.3 Hz, α-glc-2), 5.72 (d, J = 3.2 Hz, α-glc-1), 6.39 (d, J = 1.6 Hz,
DHHDP-3′), 6.52 (s, α-HBCHT-3), 6.55 (s, DHHDP-3′, β-
HBCHT-3), 6.68 (s, α-HBCHT-3′), 6.70 (s, β-HBCHT-3′), 6.78
(s, α-DHHDP-3), 6.79 (s, β-DHHDP-3); 13C NMR (126 MHz,
acetone-d6) δ 44.0 (β-DHHDP-1), 44.3 (α-DHHDP-1), 48.4 (α,β-
HBCHT-1,1′), 65.7 (α-glc-5), 65.9 (β-glc-6), 66.4 (α-glc-6), 69.4 (β-
glc-5), 74.1 (α-glc-2), 75.0 (β-glc-2), 75.1 (α-glc-3), 75.2 (α-glc-4),
76.2 (β-glc-4), 77.9 (β-glc-3), 91.3 (β-DHHDP-6), 91.5 (α-glc-1),
91.5 (α-DHHDP-6), 91.6 (HBCHT-5), 91.7 (HBCHT-5′), 95.2 (β-
glc-1), 97.0 (α-DHHDP-5), 97.1 (β-DHHDP-5), 103.5, 103.6
(HBCHT-6,6′), 105.7 (β-DHHDP-3′), 106.3 (α-DHHDP-3′),
112.1 (β-DHHDP-1′), 112.3 (α-DHHDP-1′), 124.3 (α-DHHDP-
2′), 124.5 (β-DHHDP-2′), 129.8 (α,β-HBCHT-3), 130.3 (β-
HBCHT-3′), 130.4 (α-HBCHT-3′), 131.4 (α-DHHDP-3), 131.7
(β-DHHDP-3), 135.4 (β-DHHDP-5′), 135.5 (α-DHHDP-5′), 143.0
(β-DHHDP-6′), 143.0 (α-DHHDP-6′), 145.7 (α-HBCHT-2′), 145.7
(β-HBCHT-2′), 145.9 (α-HBCHT-2), 146.0 (β-HBCHT-2), 146.4
(α-DHHDP-4′), 146.5 (β-DHHDP-4′), 150.0 (β-DHHDP-2), 150.4
(α-DHHDP-2), 165.8 (β-DHHDP-7), 166.2 (α-DHHDP-7), 166.2
(α,β-HBCHT-7′), 166.3 (α-HBCHT-7), 166.4 (β-HBCHT-7), 169.9
(α-DHHDP-7′), 169.9 (β-DHHDP-7′), 191.35 (β-DHHDP-4),
191.41 (α-DHHDP-4), 192.2 (α,β-HBCHT4,4′), 192.2 (α,β-
HBCHT4,4′). Anomer: α:β = 1:1; DHHDP: six-membered-ring:-
five-membered-ring = 6:1.
Carpinins A (4): pale yellow lyophilized powder; [α]D −52 (c 0.2,
MeOH); UV (MeOH) λmax (log ε) 231 (4.60), 284sh (4.06), 327sh
(3.35); IR νmax cm−1 3446, 2962, 1743, 1717, 1620, 1513, 1445, 1329;
FABMS m/z 815 [M − H]−; HRFABMS m/z 839.0554 [M + Na]+
(calcd for C34H24O24Na, 839.0550); ECD (MeOH) λmax (Δε) 314
(−0.5), 287 (+12.4), 262 (−11.2), 238 (+31.5), 217 (−5.9), 200
(−14.0); anal. calcd for C34H24O24·H2O C, 48.92, H, 3.14; found C,
1
48.79, H, 3.32; H NMR (500 MHz, acetone-d6) δ 3.95 (d, J = 13.6
Carpinins F (2): pale yellow lyophilized powder; [α]D +82 (c 0.2,
MeOH); UV (MeOH) λmax (log ε) 219 (4.57), 242sh (4.37), 273sh
(3.86), 334 (3.37), 402sh (3.00); IR νmax cm−1 3437, 2961, 1738,
1718, 1624, 1512, 1451, 1374; FABMS m/z 855 [M + Na]+, 833 [M
+ H]+, 815 [M − H2O + H]+; HRFABMS m/z 833.0684 [M + H]+
(calcd for C34H25O25, 833.0679); ECD (MeOH) λmax (Δε) 384
(+1.7), 340 (0), 324 (−0.62), 283 (0), 236 (+6.1), 226 (0), 207
Hz, α-HBCHT-1′), 3.95 (dd, J = 13.6, 0.6 Hz, β-HBCHT-1′), 4.03
(d, J =13.6 Hz, β-HBCHT-1), 4.05 (d, J = 13.6 Hz, α-HBCHT-1),
4.18-4.23 (m, β-glc-5, α glc-6), 4.25 (dd, J = 3.5, 10.9 Hz, β-glc-6),
4.49 (dt, J = 2.9, 9.8 Hz, α-glc-5) 4.87−4.92 (m, β-glc-2, 6, α-glc-6),
5.11 (dd, J = 3.4, 9.5 Hz, α-glc-2), 5.22 (t, J = 7.8 Hz, β-glc-1), 5.34−
5.39 (m, β-glc-3,4), 5.41 (t, J = 9.8 Hz, α-glc-4), 5.54 (brt, J = 3.8 Hz,
α-glc-1), 5.58 (t, J = 9.8 Hz, α-glc-3), 6.44 (s, β-HBCHT-3), 6.471 (s,
β-HHDP-3′), 6.473 (s, α-HHDP-3′), 6.54 (s, α-HBCHT-3), 6.58 (s,
α,β-HHDP-3) 6.64 (brs, α-glc-1-OH), 6.69 (d, J = 0.6 Hz, β-
HBCHT-3′), 6.69 (d, J = 0.7 Hz, α-HBCHT-3′), 6.75 (d, J = 7.1 Hz,
β-glc-1-OH); 13C NMR (126 MHz, acetone-d6) δ 48.4, 48.5
(HBCHT-1,1′), 64.7 (α-glc-5), 66.2 (β-glc-6), 66.7 (α-glc-6), 69.1
(β-glc-5), 73.7 (α-glc-3), 74.2 (β-glc-4), 74.6 (α-glc-4), 74.8 (α-glc-
2), 76.2 (β-glc-3), 77.4 (β-glc-2), 91.6 (α-glc-1), 91.6 (α-HBCHT-
5,5′), 91.6 (β-HBCHT-5,5′), 91.7 (β-HBCHT-5,5′), 95.4 (β-glc-1),
103.61, 103.63, 103.65 (HBCHT-6,6′), 107.28, 107.32 (β-HHDP-
3,3′), 107.4, 107.6 (α-HHDP-3,3′), 114.10, 114.57 (α-HHDP-1,1′),
114.14, 114.61 (β-HHDP-1,1′), 126.42, 126.43 (β-HHDP-2,2′),
126.5, 126.6 (α-HHDP-2,2′), 129.5 (β-HBCHT-3), 129.6 (α-
HBCHT-3), 130.1 (β-HBCHT-3′), 130.2 (α-HBCHT-3′), 136.1
(α-HHDP-5′), 136.2 (β-HHDP-5′), 136.4 (αβ-HHDP-5), 144.30,
144.33 (α-HHDP-6,6′), 144.37, 144.45 (β-HHDP-6,6′), 145.07 (α-
HHDP-4′), 145.09 (β-HHDP-4′), 145.12 (α-HHDP-4), 145.2 (β-
HHDP-4), 145.8 (α-HBCHT2,2′), 145.9 (β-HBCHT-2,2′), 146.0 (β-
1
(−21.7); H NMR (500 MHz, acetone-d6) δ 3.96 (dd, J = 0.7, 13.7
Hz, α-HBCHT-1′), 3.96 (dd, J = 0.7, 13.7 Hz, α-HBCHT-1′), 4.06
(dd, J = 0.7, 13.7 Hz, α-HBCHT-1), 4.21 (dd, J = 3.2, 10.7 Hz, α-glc-
6), 4.46 (s, α-DHHDP-1), 4.54 (ddd, J = 3.2, 9.9, 10.7 Hz, α-glc-5),
4.88 (dd, J = 3.3, 10.3 Hz, α-glc-2), 4.91 (t, J = 10.7 Hz, α-glc-6), 5.54
(t, J = 9.9 Hz, α-glc-4), 5.62 (brd, J = 2.7 Hz, α-glc-1), 5.97 (d, J =
10.3 Hz, α-glc-3), 6.54 (s, α-DHHDP-3′), 6.66 (d, J = 0.7 Hz, α-
HBCHT-3), 6.73 (d, J = 0.7 Hz, α-HBCHT-3′), 6.83 (s, α-DHHDP-
3), 6.855 (brs, α-glc-2-OH); 13C NMR (126 MHz, acetone-d6) δ 44.2
(DHHDP-1), 48.5 (HBCHT-1′), 48.6 (HBCHT-1), 65.5 (α-glc-5),
66.5 (α-glc-6), 72.8 (α-glc-3), 74.4 (α-glc-4), 76.8 (α-glc-2), 91.2 (α-
glc-1), 91.5 (DHHDP-6), 91.6 (HBCHT-5), 91.7 (HBCHT-5′), 97.0
(DHHDP-5), 103.6 (HBCHT-6′), 103.7 (HBCHT-6), 106.5
(DHHDP-3′), 112.0 (DHHDP-1′), 124.6 (DHHDP-2′), 129.8
(HBCHT-3), 130.4 (HBCHT-3′), 131.4 (DHHDP-3), 135.4
(DHHDP-5′), 142.9 (DHHDP-6′), 145.90 (HBCHT-2), 145.94
(HBCHT-2′), 146.3 (DHHDP-4′), 150.1 (DHHDP-2), 165.6
H
https://dx.doi.org/10.1021/acs.jnatprod.0c00893
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
HBCHT-2,2′), 166.09 (α-HBCHT-7′), 166.11 (β-HBCHT-7′),
166.47 (β-HBCHT-7), 166.54 (α-HBCHT-7), 168.6 (β-HHDP-7),
168.7 (α-HHDP-7), 168.9 (α-HHDP-7′), 169.0 (β-HHDP-7′), 192.1
(β-HBCHT-4,4′), 192.2 (α-HBCHT-4,4′).
(d, J = 3.4 Hz, α-glc-1), 6.45 (s, α-HHDP-6), 6.61 (s, β-HHDP-6),
6.67 (s, β-DHHDP-3), 6.70 (s, α-DHHDP-3), 6.71 (s, α-HHDP-6′),
6.78 (s, DHHDP-3′), 6.79 (s, DHHDP-3′), 7.18 (s, β-HHDP-6′);
13C NMR (100 MHz, acetone-d6 + D2O) δ 43.2 (α-DHHDP-1′),
43.4 (β-DHHDP-1′), 64.0 (α-glc-6), 66.0 (α-glc-5), 66.8 (β-glc-6),
68.9 (β-glc-5), 70.9 (α-glc-2), 73.0 (β-glc-4), 73.3 (α-glc-4), 74.5 (α-
glc-3), 78.3 (β-glc-3), 79.1 (β-glc-2), 91.6 (α-glc-1), 91.9 (α,β-
DHHDP-6′), 95.2 (β-glc-1), 96.9 (α, β-DHHDP-5′), 107.4 (α-
HHDP-3), 107.9 (β-HHDP-3), 108.2 (α-DHHDP-3), 108.4 (β-
DHHDP-3), 108.5 (α-HHDP-3′), 111.7 (β-HHDP-3′), 113.2 (α,β-
DHHDP-1, α-HHDP-1), 114.9 (β-HHDP-1), 117.2 (α-HHDP-1′),
117.6 (β-HHDP-1′), 120.5 (α,β-HHDP-2), 122.8 (α,β-HHDP-2′),
124.2 (α,β-DHHDP-2), 131.2 (α-DHHDP-3′), 131.3 (β-DHHDP-
3′), 136.0 (α-HHDP-5, α,β-DHHDP-5), 136.5 (β-HHDP-5), 137.2
(α-HHDP-5′), 138.2 (β-HHDP-5′), 142.6 (β-DHHDP-6), 142.7 (α-
DHHDP-6), 144.0 (β-HHDP-6′), 144.9 (α-HHDP-6′), 145.2 (α-
HHDP-4′), 145.3 (α-HHDP-6), 145.6 (β-HHDP-6), 145.7 (β-
HHDP-4′), 146.4 (α-HHDP-4, β-DHHDP-4), 146.5 (α-DHHDP-4,
β-HHDP-4), 151.9 (β-DHHDP-2′), 152.0 (α-DHHDP-2′), 164.78
(β-DHHDP-7′), 164.8 (α-DHHDP-7′), 168.2 (α,β-DHHDP-7),
168.7 (α,β-HHDP-7′), 169.1 (α-HHDP-7), 169.2 (β-HHDP-7),
192.3 (β-DHHDP-4′), 192.4 (α-DHHDP-4′).
Methanolysis of Phenazine Derivatives. Carpinin F (2) (100
mg) and 1,2-phenylenediamine (40 mg) were dissolved in ethanolic
20% HOAc (3 mL) and stirred at room temperature for 12 h. The
mixture was poured into ice-cooled water (10 mL), and the resulting
yellow precipitate was collected by filtration. One portion (95 mg) of
the precipitate (122 mg) was dissolved in MeOH−acetone (3:1, v/v,
3 mL) and treated with CH2N2 in Et2O at room temperature for 15 h.
After evaporation, the residue was heated with a mixture of 5% NaOH
and MeOH (1:2, v/v) at 80°C for 40 min. The solution was acidified
with concentrated HCl and extracted three times with Et2O. The
Et2O layer was concentrated, dissolved in MeOH (1 mL), and treated
with CH2N2 in Et2O. The products were separated by silica gel
column chromatography with n-hexane−acetone (3:2, v/v) and
purified by silica gel column chromatography with acetone in CHCl3
(0−4%, 2% stepwise) to yield 2a [19.2 mg, ECD (MeOH) λmax (Δε)
301 (−2.8), 286 (0), 265 (+9.5), 253 (0), 237 (−6.1)] and 2b (20.7
mg) together with a mixture of 2a and 2b (21.7 mg). From carpinin E
(62.0 mg), 2a (3.5 mg), 2b (1.5 mg), and a mixture of 2a and 2b
(10.0 mg) were obtained by a similar procedure.
Heating of 2 in 1% H2SO4. Compound 2 (500 mg) was
dissolved in 1% H2SO4 (10 mL) and refluxed for 3.5 h. The
precipitate formed was collected by filtration (48.3 mg) and identified
as ellagic acid (11) on the basis of its HPLC retention time and UV
spectrum. The filtrate was separated on a Toyopearl Ether 650M
column (3.0 cm i.d. × 25 cm) with 0−80% MeOH (10% stepwise,
each 100 mL) to give 7 (110.8 mg), 9 (74.5 mg), 10 (32.4 mg), 4
(59.9 mg), and 11 (19.4 mg).
(S)-Methyl 4-Methoxyphenazine-2-carboxylate 3,3′-Dimer
(2b): yellow amorphous powder, [α]D +252 (c 0.1, CHCl3); FABMS
m/z 535 [M + H]+; HRFABMS m/z 535.1616 [M + H]+ (calcd for
C30H23N4O6, 535.1616); IR νmax cm−1 2923, 2850, 1729, 1505, 1455,
1361, 1324, 1243; UV (MeOH) λmax nm (log ε) 371 (4.18), 276
1
(4.75); H NMR (400 MHz, CDCl3) δH 3.71 (6H, s), 4.06 (6H, s),
7.24 (2H, s), 7.88 (4H, m), 8.30 (4H, m), 8.87 (2H, s); ECD
(MeOH) λmax (Δε) 375 (+1.1), 348 (0), 331 (−0.56), 323 (0), 282
(+17.2), 270 (0), 259 (−30.4), 241 (0), 231 (+4.9).
4,6-(S)-DHHDP-D-glucoses (9): pale yellow amorphous powder;
[α]D +115 (c 1.0, acetone); FABMS m/z 497 [M − H]−; anal. calcd
1
Heating of 2 in H2O. An aqueous solution of 2 (500 mg/50 mL)
was heated at 60 °C for 46 h. After concentration using a rotary
evaporator, the residue was separated on a Sephadex LH-20 column
(2.0 cm i.d. × 45 cm) with 7 mol/L urea−acetone (4:6, v/v,
containing 0.5% concentrated HCl)35 into two fractions: Fr. 1,
containing larger molecular weight substances, and Fr. 2, containing
polyphenols with lower molecular weight. Fr. 1 was concentrated and
applied to a Diaion HP20SS column (2.0 cm i.d. × 13 cm) with 0−
80% MeOH (10% stepwise, each 50 mL) to yield polyphenolic
material (39.5 mg), which was detected at the origin on silica gel TLC
plates. Fr. 2 was separated by Diaion HP20SS column chromatog-
raphy (2.5 cm i.d. × 20 cm) with 0−80% MeOH (5% stepwise, each
50 mL) into five fractions: 2-1−2-5. HPLC analysis indicated that
major components of fractions 2-1 (32.4 mg), 2-2 (80.3 mg), and 2-5
(60.5 mg) were 7, 9, and 11, respectively. Fr. 2-3 (195.0 mg) was
separated by chromatography on an Avicel cellulose column (2.0 cm
i.d. × 17 cm) with 2% HOAc into two fractions. Further separation of
Fr. 2-3-1 on a Chromatorex ODS column (2.0 cm i.d. × 15 cm) with
0−40% MeOH (5% stepwise, each 50 mL) yielded 3 (115.8 mg).
Chromatography of Fr. 2-3-2 by MCI-gel CHP20P column
chromatography (2.0 cm i.d. × 10 cm) with 0−60% MeOH (10%
stepwise, each 50 mL) yielded 10 (2.7 mg). Fr. 2-4 (79.5 mg) was
separated by Chromatorex ODS column chromatography (2.5 cm i.d.
× 22 cm) with 0−40% MeOH (5% stepwise, each 50 mL) to give 8
(29.4 mg).
2,3-(R)-HHDP-4,6-(S)-DHHDP-D-glucoses (8): tan amorphous
powder; [α]D +34 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 223
(5.00), 247 (4.81), 357 (3.95); IR νmax cm−1 3394, 1719, 1618, 1511,
1449, 1341; FABMS m/z 823 [M + Na]+; HRFABMS m/z 823.0606
[M + Na]+ (calcd for C34H24O23Na, 823.0601); ECD (MeOH) λmax
(Δε) 200 (+7.93), 217 (−16.12), 261 (+6.50), 294 (−3.96); 1H
NMR (500 MHz, acetone-d6) δ 3.95 (m, β-glc-5), 3.98 (t, J = 9.0 Hz,
β-glc-6), 4.01 (t, J = 10.7 Hz, α-glc-6), 4.17 (ddd, J = 4.5, 9.5, 10.7 Hz,
α-glc-5), 4.64 (s, β-DHHDP-1), 4.65 (dd, J = 7.4, 9.0 Hz, β-glc-2),
4.69 (s, α-DHHDP-1), 4.81 (dd, J = 3.4, 9.5 Hz, α-glc-2), 4.90 (dd, J
= 4.5, 10.7 Hz, α-glc-6), 4.96 (dd, J = 3.5, 9.0 Hz, β-glc-6), 5.10 (d, J
= 7.4 Hz, β-glc-1), 5.14 (t, J = 9.0 Hz, β-glc-3), 5.28 (t, J = 9.0 Hz, β-
glc-4), 5.36 (t, J = 9.5 Hz, α-glc-3), 5.43 (t, J = 9.5 Hz, α-glc-4), 5.49
for C20H18O15 C, 48.20; H, 3.64; found C, 48.40, H, 4.06; H NMR
(400 MHz, acetone-d6 + D2O) δ 3.53 (dd, J = 9.3, 7.8 Hz, β-glc-2),
3.53 (dd, J = 9.4, 3.4 Hz, α-glc-2), 3.67 (ddd, J = 10.5, 10.5, 4.4 Hz, β-
glc-5), 3.68 (t, J = 9.3 Hz, β-glc-3), 3.87 (t, J = 10.7 Hz, α-glc-6), 3.88
(t, J = 10.5 Hz, β-glc-6), 3.95 (t, J = 9.4 Hz, α-glc-3), 4.06 (ddd, J =
10.7, 10.7, 4.9 Hz, α-glc-5), 4.63 (d, J = 7.8 Hz, β-glc-1), 4.69, 4.68
(each s, DHHDP-1), 4.85 (dd, J = 10.5, 4.4 Hz, β-glc-6), 4.91 (dd, J =
10.7, 4.6 Hz, α-glc-6), 5.09 (t, J = 9.2 Hz, α-glc-4), 5.10 (t, J = 9.2 Hz,
β-glc-4) 5.17 (d, J = 3.4 Hz, α-glc-1), 6.71, 6.70 (each s, DHHDP-3),
6.73 (1H, s, DHHDP-3′); 13C NMR (100 MHz, acetone-d6 + D2O) δ
43.1 (DHHDP-1), 76.9, 76.4, 75.3, 74.6, 72.8, 71.2, 68.8, 67.3, 66.7,
64.7 (glc-2,3,4,5,6), 91.7 (DHHDP-6), 93.5 (α-glc-1), 97.0
(DHHDP-5), 97.7 (β-glc-1), 108.8 (DHHDP-3′), 112.9 (DHHDP-
1′), 124.30, 124.21 (DHHDP-2′), 131.46, 131.42 (DHHDP-3),
136.21, 136.18 (DHHDP-5′), 142.63 (DHHDP-6′), 146.3 (DHHDP-
4′), 151.83, 151.80 (DHHDP-2), 165.14, 165.13 (DHHDP-7′),
170.2, 170.1 (DHHDP-7), 193.0 (DHHDP-4).
Reduction of Carpinins D (7) with Ascorbic Acid. An aqueous
solution of 7 (50 mg) and ascorbic acid (150 mg) was heated at 100
°C for 20 min. The reaction mixture was separated on a Sephadex
LH-20 column (2 × 15 cm) with 0−60% MeOH (10% stepwise, each
20 mL) to afford 9 (7.6 mg) as well as unreacted 7 (9.3 mg).
Heating of Carpinins D in H2O. An aqueous solution of 7 (200
mg/10 mL) was heated in a 50 mL screw-capped vial at 100 °C for 5
h. The mixture was separated by chromatography using a
Chromatorex ODS column (2 × 20 cm) with H2O containing
increasing proportions of MeOH (0−50%, 5% stepwise, each 50 mL)
to give 9 (80.9 mg) as well as unreacted 7 (30.0 mg).
Hydrolysis of Trapain. An aqueous solution of 1,2,3-tri-O-galloyl-
4,6-(S)-DHHD-β-D-glucose (10 mg/2 mL) was treated with tannase
(5 mg, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) at
room temperature for 14 h. The mixture was separated by Sephadex
LH-20 column chromatography (0−50% MeOH in H2O, 10%
stepwise, each 20 mL) to give gallic acid (5 mg) and 9 (3.0 mg).
Conversion of 2 to 12. Compound 2 (50 mg) and ascorbic acid
(500 mg) were dissolved in H2O (5.0 mL) and heated at 100 °C for
60 min. The reaction mixture was separated on a Diaion HP20SS
column (2 × 20 cm) with H2O containing increasing proportions of
I
https://dx.doi.org/10.1021/acs.jnatprod.0c00893
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
MeOH (0−50%, 5% stepwise, each 50 mL) to give 12 (25.7 mg,
55%) and 4 (16.5 mg, 34%). The product 12 was identified by
mechanism of reduction−oxidation disproportionation
of the DHHDP group of 1 and 2, reduction of 1 and 2
to 4 with ascorbic acid, possible reduction mechanism of
the DHHDP group with ascorbic acid, HPLC profiles of
4 and 5 on heating with pyridine, 1H, 13C, and 2D NMR
spectra of 1, 2, 3, 4, 8, and 9, and ECD spectra of 1−4
and 7 (PDF)
25
1
comparison of its H and 13C NMR spectra.
Conversion of 2 to 13. Compound 2 (100 mg) was dissolved in
H2O (10 mL) and heated at 100 °C for 20 min. Then, ascorbic acid
(1.2 g) was added, and the mixture was heated at 100 °C for 60 min.
The solution was directly applied to a Diaion HP20SS column (2 ×
20 cm), and products were eluted with H2O containing increasing
proportions of MeOH (0−50%, 5% stepwise, each 50 mL). This
separation yielded 14 (25.7 mg), 13 (19.6 mg), 12 (15.2 mg), and 11
(3.0 mg). Product 13 was identified as the tannase hydrolysate of
cuspinin by comparison of its 1H and 13C NMR spectra with reported
data.19 [α]D −14 (c 0.1, MeOH); ESIMS (negative mode) m/z 783
(M − H)−; ESIMS (positive mode) m/z 807 (M + Na)+; HRESIMS
(negative mode) m/z 783.0707 [M − H]− (calcd for C34H23O22,
783.0687); UV (MeOH) λmax nm (log ε) 228 (4.69), 276 sh (4.34);
AUTHOR INFORMATION
Corresponding Author
■
Takashi Tanaka − Department of Natural Product Chemistry,
Graduate School of Biomedical Sciences, Nagasaki University,
Nagasaki 852-8521, Japan; orcid.org/0000-0001-7762-
7432; Phone: +81-95-819-2432; Email: t-tanaka@
nagasaki-u.ac.jp
1
IR (dry film) νmax cm−1 3421, 1717, 1613, 1513, 1444, 1311; H
NMR (400 MHz, acetone-d6) δ 3.68 (dd, J = 12.7, 1.8 Hz, α-glc-6),
3.73 (d, J = 12.9 Hz, β-glc-6), 4.11 (dd, J = 9.8, 5.6 Hz, β-glc-5), 4.47
(m, α-glc-5), 4.49 (dd, J = 9.8, 7.8 Hz, β-glc-1), 4.69 (dd, J = 9.8, 3.5
Hz, α-glc-2), 4.72 (t, J = 9.8 Hz, β-glc-4), 4.80 (brd, J = 8 Hz, OH),
4.97 (t, J = 9.8 Hz, α-glc-4), 4.97 (brt, J = 8 Hz, β-glc-1), 5.16-5.25
(m, α, β-glc-3, α, β-glc-6), 5.48 (t, J = 3.5 Hz, α-glc-1), 6.28 (brs,
OH), 7.20, 7.05, 6.79, 6.69, 6.66, 6.57 (total 4H, HHDP-3, 3′); 13C
NMR (100 MHz, acetone-d6) δ 63.5 (α, β-glc-6), 79.9, 78.4, 77.20,
77.16, 76.5, 72.0, 70.2, 70.0, 67.3 (glu-2,3,4,5), 91.5 (α-glc-1), 95.5
(β-glc-1), 108.3, 108.2, 108.0, 107.9, 107.48, 107.45 (HHDP-3,3′),
119.7, 117.7, 117.63, 117.61, 117.2, 115.83, 115.78, 115.71, 115.6,
111.9 (HHDP-1,1′), 126.4, 126.3, 126.23, 126.16, 123.2 (HHDP-
2,2′), 138.9, 136.4 (×3), 136.3 (HHDP-5,5′), 145.5, 145.4, 145.3,
145.24, 145.19, 145.14, 145.07, 145.06, 145.0, 144.81, 144.76, 144.74,
144.44, 144.42, 144.28, 144.26 (HHDP-4,6,4′,6′), 169.2, 168.14,
168.08, 168.0, 167.3, 167.2 (COO).
Heating of 4 and 5 in Pyridine. A solution (2 mg/mL) of 4 in
4% pyridine−CH3CN was heated at 80 °C for 90 min. After cooling,
2.5% TFA in H2O (0.1 mL) was added, and the mixture analyzed by
HPLC. The chromatogram showed two peaks corresponding to the
α- and β-anomers of 12. Treatment of 5 under the same conditions
and HPLC analysis of the reaction mixture showed the formation of
2,3-di-O-galloyl-4,6-(S)-HHDP-D-glucose (tR = 18.5 and 22.1 min).
This product was identified by comparison with an authentic sample
(1-desgalloyleugeniin) isolated from fruits of Paeonia lactiflora.28
Reaction of 2 with 15. Reaction A: An aqueous solution of 2 (10
mmol/L, 0.3 mL) was mixed with H2O (0.3 mL) and pH 6 citrate−
phosphate buffer (0.3 mL) in a screw-capped vial and left standing at
room temperature for 3 h. Reaction B: An aqueous solution of 2 (10
mmol/L, 0.3 mL) was mixed with an aqueous solution of 15 (10
mmol/L, 0.3 mL) and pH 6 citrate−phosphate buffer (0.3 mL) in a
screw-capped vial and left standing at room temperature for 3 h.
Then, 0.5% TFA (0.3 mL) and CH3CN (0.3 mL) were added to the
mixtures. The mixtures were analyzed by HPLC on a Cosmosil PAQ
column (250 × 4.6 mm, i.d.; Nacalai Tesque Inc., Kyoto, Japan) with
a gradient elution of 4−25% (40 min) and 25−85% (5 min) CH3CN
in 50 mM H3PO4 at 35 °C (flow rate, 0.8 mL/min). Eluted products
were detected at 220 nm using a JASCO MD-2018 Plus photodiode
array detector. An aqueous solution of 15 alone did not show
significant change in the HPLC profile under these conditions.
Authors
Daisetsu Kojima − Department of Natural Product
Chemistry, Graduate School of Biomedical Sciences, Nagasaki
University, Nagasaki 852-8521, Japan
Kengo Shimizu − School of Pharmaceutical Sciences,
Nagasaki University, Nagasaki 852-8521, Japan
Kosuke Aritake − Laboratory of Chemical Pharmacology,
Daiichi University of Pharmacy, Fukuoka 815-8511, Japan
Manami Era − Department of Natural Product Chemistry,
Graduate School of Biomedical Sciences, Nagasaki University,
Nagasaki 852-8521, Japan
Yosuke Matsuo − Department of Natural Product Chemistry,
Graduate School of Biomedical Sciences, Nagasaki University,
Nagasaki 852-8521, Japan; orcid.org/0000-0002-6462-
9727
Yoshinori Saito − Department of Natural Product Chemistry,
Graduate School of Biomedical Sciences, Nagasaki University,
Nagasaki 852-8521, Japan; orcid.org/0000-0002-3587-
9743
Gen-ichiro Nonaka − Usaien Pharmaceutical Company, Ltd.,
Saga 840-0055, Japan
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c00893
Author Contributions
#D. Kojima, K. Shimizu, and K. Aritake contributed equally to
this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by JSPS KAKENHI Grant Numbers
20K07102, 17K08338, 16K07741, and 26460125. The authors
are grateful to Mr. N. Tsuda, Mr. K. Chifuku, and H. Iwata,
Center for Industry, University and Government Cooperation,
Nagasaki University, for recording NMR and MS data. Some
computations were carried out using the computer facilities at
the Research Institute for Information Technology, Kyushu
University. We thank the Edanz Group for editing a draft of
this manuscript.
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c00893.
Experimental and calculated ECD spectra of 2b, HPLC
profiles of reaction mixtures of 2 after heating in H2O
and 1% H2SO4, 13C NMR spectrum (100 MHz, D2O) of
a mixture of oligomeric products obtained by heating of
2 in H2O, HPLC profiles of 1 and 2 in pH 3−7 citrate−
phosphate buffer solutions after 3 h at rt, possible
REFERENCES
■
(1) Okuda, T.; Yoshida, T.; Hatano, T. Hydrolyzable Tannins and
Related Polyphenols. In Progress in the Chemistry of Organic Natural
Products; Herz, W., Kirby, G. W., Moore, R. E., Steglich, W., Tamm,
C., Eds.; Springer-Verlag: New York, 1995; pp 1−117.
J
https://dx.doi.org/10.1021/acs.jnatprod.0c00893
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
(2) Haddock, E. A.; Gupta, R. K.; Al-Shafi, S. M. K.; Layden, K.;
Haslam, E.; Magnolato, D. Phytochemistry 1982, 21, 1049−1062.
(3) Okuda, T.; Ito, H. Molecules 2011, 16, 2191−2217.
(35) Yanagida, A.; Shoji, T.; Shibusawa, Y. J. Biochem. Biophys.
Methods 2003, 56, 311−322.
(4) Quideau, S.; Jourdes, M.; Lefeuvre, D.; Pardon, P.; Saucier, C.;
Teissedre, P.-L.; Glories, Y. Ellagitannins - An Underestimated Class
of Plant Polyphenols: Chemical Reactivity of C-Glucosidic
Ellagitannins in Relation to Wine Chemistry and Biological Activity.
In Recent Advances in Polyphenol Research; Santos-Buelga, C.,
Escribano-Bailon, M. T., Lattanzio, V., Eds; Wiley-Blackwell Publish-
ing: Oxford, 2010; Vol. 2, pp 81−137.
́
(5) Quideau, S.; Deffieux, D.; Douat-Casassus, C.; Pouysegu, L.
Angew. Chem., Int. Ed. 2011, 50, 586−621.
(6) Niemets, R.; Gross, G. G. Phytochemistry 2005, 66, 2001−2011.
(7) Okuda, T.; Hatano, T.; Yazaki, K. Chem. Pharm. Bull. 1982, 30,
1113−1116.
(8) Okuda, T.; Yoshida, T.; Hatano, T. Phytochemistry 2000, 55,
513−529.
(9) Foo, L.Y. Phytochemistry 1993, 33, 487−491.
(10) Haddock, E. A.; Gupta, R. K.; Haslam, E. J. Chem. Soc., Perkin
Trans. 1 1982, 2535−2545.
(11) Era, M.; Matsuo, Y.; Saito, Y.; Tanaka, T. Molecules 2020, 25,
1051.
(12) Wakamatsu, H.; Tanaka, S.; Matsuo, Y.; Saito, Y.; Nishida, K.;
Tanaka, T. Molecules 2019, 24, 24.
(13) Nonaka, G.; Mihashi, K.; Nishioka, I. J. Chem. Soc., Chem.
Commun. 1990, 790−791.
(14) Okuda, T.; Yoshida, T.; Hatano, T. J. Chem. Soc., Perkin Trans.
1 1982, 9−14.
(15) Okuda, T.; Yoshida, T.; Hatano, T.; Koga, T.; Tho, N.;
Kuriyama, K. Tetrahedron Lett. 1982, 23, 3941−3944.
(16) Esumi, A.; Aoyama, H.; Shimozu, Y.; Tanoguchi, S.; Hatano, T.
Heterocycles 2019, 98, 895−903.
(17) Okuda, T.; Yoshida, T.; Nayeshiro, H. Chem. Pharm. Bull.
1977, 25, 1862−1869.
(18) Okuda, T.; Yoshida, T.; Mori, K.; Hatano, T. Heterocycles 1981,
12, 1323−1348.
(19) Nonaka, G.; Ishimatsu, M.; Ageta, M.; Nishioka, I. Chem.
Pharm. Bull. 1989, 37, 50−53.
(20) Yamaguchi, S.; Hirokane, T.; Yoshida, T.; Tanaka, T.; Hatano,
T.; Ito, H.; Nonaka, G.; Yamada, H. J. Org. Chem. 2013, 78, 5410−
5417.
(21) Tanaka, T.; Nonaka, G.; Nishioka, I. Chem. Pharm. Bull. 1990,
38, 2424−2428.
(22) Nonaka, G.; Matsumoto, Y.; Nishioka, I. Chem. Pharm. Bull.
1981, 29, 1184−1187.
(23) Tanaka, T.; Mine, C.; Inoue, K.; Matsuda, M.; Kouno, I. J.
Agric. Food Chem. 2002, 50, 2142−2148.
(24) Li, Y.; Shibahara, A.; Matsuo, Y.; Tanaka, T.; Kouno, I. J. Nat.
Prod. 2010, 73, 33−39.
(25) Tsujita, T.; Matsuo, Y.; Saito, Y.; Tanaka, T. Tetrahedron 2017,
73, 500−507.
(26) Tanaka, T.; Nonaka, G.; Nishioka, I. Chem. Pharm. Bull. 1990,
38, 2424−2428.
(27) Wilkins, C. K.; Bohm, B. A. Planta Med. 1976, 30, 72−74.
(28) Tanaka, T.; Fukumori, M.; Ochi, T.; Kouno, I. J. Nat. Prod.
2003, 66, 759−763.
(29) Tanaka, T.; Watarumi, S.; Matsuo, Y.; Kamei, M.; Kouno, I.
Tetrahedron 2003, 59, 7939−7947.
(30) Li, Y.; Tanaka, T.; Kouno, I. Phytochemistry 2007, 68, 1081−
1088.
(31) Matsuo, Y.; Li, Y.; Watarumi, S.; Tanaka, T.; Kouno, I.
Tetrahedron 2011, 67, 2051−2059.
(32) Matsuo, Y.; Okuda, K.; Morikawa, H.; Oowatashi, R.; Saito, Y.;
Tanaka, T. J. Nat. Prod. 2016, 79, 189−195.
(33) Okuda, T.; Yoshida, T.; Hatano, T.; Ikeda, Y. Heterocycles 1986,
24, 1841−1843.
(34) Tanaka, T.; Nonaka, G.; Nishioka, I.; Miyahara, K.; Kawasaki,
T. J. Chem. Soc., Perkin Trans. 1 1986, 369−376.
K
https://dx.doi.org/10.1021/acs.jnatprod.0c00893
J. Nat. Prod. XXXX, XXX, XXX−XXX