Tetrahedron 75 (2019) 4042e4052

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Tetrahedron
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Characterization and cytotoxicity of ellagitannins from Stachyurus

praecox fruit
Joanna Orejola a, b, Mark Anthony Luz c, d, Yosuke Matsuo a, Yoshinori Saito a,
Kouichi Morita d, Takashi Tanaka a, *
a
Department of Natural Product Chemistry, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
b
College of Pharmacy, University of the Philippines, Taft Ave., Manila 1000, Philippines
c
Department of Virology, Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan
d
Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan

a r t i c l e i n f o a b s t r a c t

Article history: This study aimed to characterize and evaluate the cytotoxicities of ellagitannins from S. praecox fruit.
Received 6 May 2019 Fractionation of an acetone extract of the fruit led to isolation of five new ellagitanninsdstachyuranin D
Received in revised form (15), stachyuranin E (16), stachyuranin F (17), stachyugluconin (18), and stachyuglyconin (19)dalong
13 June 2019
with seven ellagitannins previously isolated from S. praecox leaves and seven known ellagitannins iso-
Accepted 22 June 2019
lated from Stachyuraceae for the first time. Two-dimensional NMR and other spectroscopic methods
Available online 24 June 2019
enabled complete elucidation of the structures of the new compounds. Structure of flavanoellagitannin
(16) was confirmed by 1H NMR comparison with synthetic analogs. The configuration of stachyuglyconin
Keywords:
Stachyurus praecox
(19) with a C8-aldonic acid core was established with the aid of density functional theory calculations. A
Ellagitannin MTT assay revealed 5-desgalloylpterocarinin A (11) and hippophaenin B (9) had the highest cytotoxicities
Stachyuranin among the compounds against HeLa (IC50 ¼ 35.35 mM) and HepG2 (IC50 ¼ 60.00 mM) cells, respectively.
Stachyugluconin © 2019 Elsevier Ltd. All rights reserved.
Stachyuglyconin
Cytotoxicity

1. Introduction [20e22], antitumor, and cytotoxic activities [23e25]. Previous

Stachyurus praecox Sieb. & Zucc. (Stachyuraceae) is a deciduous
shrub endemic to Japan [1]. The tannin-rich fruits have traditionally
been used as a surrogate of sumac gallnut; however, the plant is
now considered more valuable horticulturally because of its light
yellow blossoms in early spring. An aqueous extract of the fruit was
previously demonstrated to have strong inhibitory action against
certain strains of Gram-negative bacteria [2] and cytotoxicity
against certain cancer cells [3]. This study suggested the ellagi-
tannins present in S. praecox were responsible for the reported
activity. Okuda and colleagues [4e7] previously isolated casuarinin,
casuarictin, pedunculagin, casuariin, tellimagrandin I, stachyurin,
and strictinin, and later on, praecoxins AeE, 2,3-(S)-hexahydrox-
ydiphenoyl (HHDP)-D-glucose, rugosins C and F, stachyuranins
AeC, and guavin C from the leaves. Ellagitannins or ellagitannin-
enriched fractions have been shown to exhibit antiparasitic [8],
antibacterial, antimalarial [2,9], antiviral [10e14], a-glucosidase
inhibitory [15e17], anti-inflammatory [18,19], anti-mutagenic
studies have demonstrated that the antitumor and cytotoxic ac-
tivities of ellagitannins are structure-specific [25,26]. A more
detailed investigation of the structureeactivity relationship (SAR)
to DNA topoisomerase II inhibitory action demonstrated the influ-
ence of the nature (alkyl or phenolic), steric demand, and orien-
tation of substituents at C-1 of 4,6-HHDP-bearing C-ellagitannins
on selectivity of action towards the a-isoform of the enzyme [27].
Evaluation of the SAR towards any target-specific activity may lead
to further exploration of ellagitannins as sources or molecular
templates of new drugs for treatment of cancer and other diseases.
This study focuses on the isolation and characterization of ellagi-
tannins present in unripe S. praecox fruit and evaluation of the
cytotoxic activities of these compounds against cervical adenocar-
cinoma (HeLa) and hepatocellular carcinoma (HepG2) cell lines.
2. Results and discussion
2.1. Isolation and structure determination

A comparison between HPLC profiles of ethanolic extracts of

* Corresponding author. fresh leaves and unripe fruit showed that the overall quantity of
E-mail address: t-tanaka@nagasaki-u.ac.jp (T. Tanaka). ellagitannins was higher in the fruit than in the leaves (Fig. S1,

https://doi.org/10.1016/j.tet.2019.06.037
0040-4020/© 2019 Elsevier Ltd. All rights reserved.
 J. Orejola et al. / Tetrahedron 75 (2019) 4042e4052 4043

Supplementary Data). Marked differences in the ellagitannin con-
tents were also observed, with pedunculagin (1) being the major
component in the unripe fruit. This preliminary investigation
suggests that the fruit are a better source of ellagitannins than the
leaves. A series of chromatographic separations of an aqueous
acetone extract of fresh unripe S. praecox fruit using Diaion HP20SS,
Sephadex LH-20, and Chromatorex ODS columns led to isolation of
19 compounds. All the ellagitannins isolated were monomeric and
most contained a C-glycosidic linkage. Comparison of the 1H NMR
spectra to existing literature enabled identification of 5-
desgalloylstachyurin (6) [28,29], alnusjaponin B hydrolysate (8)
[30], hippophaenin B (9) [31], 5-desgalloylpterocarinin A (11) [32],
hippophaenin C (10) [33], sanguiin H-5 (14) [34], and pterocarinin A
(12) [32] as known ellagitannins isolated from Stachyuraceae for the
first time (Fig. 1). Pedunculagin (1), casuarictin (2), praecoxin A (3),
strictinin (4), casuariin (5), casuarinin (7), and 2,3-(S)-HHDP-D-
glucose (13) were also isolated and identified, and have been pre-
viously reported for the leaves of S. praecox. [3e6] Stachyuranin D
(15), stachyuranin E (16), stachyuranin F (17), stachyugluconin (18),
and stachyuglyconin (19) were identified as new compounds
(Fig. 2) by 1H, 13C NMR, HSQC, HMBC, 1He1HeCOSY, NOE, and CD
spectroscopy.
The 1H NMR data (Table 1) of 15 revealed a doublet at dH 5.58
(J ¼ 4.9 Hz) and a doublet of doublets at dH 4.05 (J ¼ 3.2, 8.7 Hz),
which were ascribed to glucose H-1 of a C-glycosidic ellagitannin
and H-5 with a free OH group. These signals both suggest a
casuariin-type glucose core because of the resemblance of the
chemical shifts and coupling patterns to those in a previous report
[4] and those of casuariin isolated from the same material. Four
aromatic singlet signals at dH 6.14, 6.39, 6.73, and 7.10 (1H each)
suggested the presence of a triphenoyl group in addition to a C-
glycosidic HHDP group [5,7,31]. This was confirmed by appearance

Fig. 1. Structures of known ellagitannins 1e14.

Fig. 2. Structures and key HMBC correlations of new ellagitannins 15e20.

4044 J. Orejola et al. / Tetrahedron 75 (2019) 4042e4052

Table 1
1 13
H (500 MHz) and C NMR (126 MHz) data of 15, 18, and 19 in acetone-d6 þD2O.

Position 15 18 19

dC dH (J in Hz) dC dH (J in Hz) dC dH (J in Hz)

Glucose or Aldonic Acid 1 67.4 5.58 d (4.9) 173.4 174.9

2 76.9 4.63 dd (2.6, 4.9) 70.4 4.40 d (2.9) 73.1 4.50 d (6.6)
3 70.8 5.43 t (2.6) 73.5 5.70 dd (2.9, 6.3) 45.1 3.53 dd (6.6, 1.5)
4 76.9 5.01 dd (2.6, 8.7) 70.4 5.77 dd (6.3, 8.3) 5.11 brs
5 68.1 4.05 dd (3.2, 8.7) 72.0 5.60 dd (8.3, 2.6) 75.9 5.04 t (2.1)
6 68.1 4.57 dd (3.2, 12.6) 64.8 4.89 dd (2.6, 13.1) 75.4 5.61 dd (2.1, 8.8)
3.73 d (12.6) 4.05 d (13.1) 74.2
7 70.7 5.24 dd (8.8, 3.4)
8 64.6 4.78 dd (3.4, 13.3)
3.95 d (13.3)
A 1 116.3 116.3 114.0
2 120.1a 127.1 124.3a
3 116.7 108.8 6.51 s 117.5
4 143.6 145.25 143.0
5 138.6 136.1 137.2
6 143.7 144.5a 149.3
COO 165.2 167.7 169.5
B 10 116.1 115.1 116.3
20 124.9a 125.5 124.5a
30 105.1 6.39 s 107.2 6.67 s 105.4 6.46 s
40 145.7 145.34 145.8
50 134.9 135.9 135.0
60 145.2 144.6a 149.8
COO 170.2 169.3 169.4
C 1 115.8 120.5 115.2
2 126.9a 110.42 7.08 s 126.5b
3 108.3 6.73 s 145.93 107.1 6.85 s
4 145.0 139.3 145.2
5 136.6 145.93 136.9
6 144.7 110.42 7.08 s 149.3
COO 168.9 166.45 168.8
D 1 116.7 120.7 116.4
2 127.5a 110.24 7.13 s 127.3b
3 104.4 6.14 s 146.15 104.6 6.14 s
4 146.9 139.5 147.0
5 136.4 146.15 136.7
6 146.1 110.24 7.13 s 149.8
COO 169.2 166.53 168.6
E 10 115.3 117.2
20 139.95 140.0
30 140.02 139.9
40 137.4 137.1
50 143.1 143.3
60 109.7 7.10 s 109.7 7.06 s
COO 167.2 167.3
F 1 120.5
2,6 110.0 7.02 s
3,5 145.7
4 139.2
COO 166.1
15
same/overlapping chemical shift assignment.
aec
interchangeable assignment.

of a [M þ H]þ peak at m/z 953.0895 in HRFABMS (C41H29O27,
953.0891). The observed HMBC cross peak (Fig. 2) between the
more upfield aromatic proton (dH 6.14) and the aromatic carbon
corresponding to galloyl-substituted biphenyl C-4 (dC 146.9)
confirmed the presence of a valoneoyl moiety [7,30,31]. The
observed HMBC cross signal between glucose H-4 at dH 5.01 (dd,
J ¼ 2.6, 8.7 Hz) and a carbonyl carbon at dC 168.9 belonging to the
valoneoyl moiety suggested this moiety was linked to glucose C-4
and C-6. The same carbonyl carbon shared a HMBC cross signal
with the valoneoyl H-3 (dH 6.73), which was correlated to the
valoneoyl C-4 with an unsubstituted hydroxyl group (dC 145.0)
[3,7]. This establishes the orientation of the valoneoyl moiety on
glucose chain. The positive Cotton effect observed at 220e240 nm
in the CD spectrum indicated the HHDP configuration at the
glucose 2,3-position was S (Fig. 3) [7].
According to the observed m/z 1377.1691 [M þ H]þ in HRFABMS
(C63H45O36, 1377.1685), stachyuranin E (16) was identified as a
flavanoellagitannin with catechin as the flavan-3-ol component
and galloylated C-glycosidic ellagitannin having both HHDP and
valoneoyl moieties. A broad singlet at dH 4.64, doublet of doublets
at dH 5.64 (J ¼ 8.6, 1.9 Hz), and peak at dH 5.32 (J ¼ 8.6, 3.5 Hz) in the
1
H NMR spectrum (Table 2) corresponded to glucose H-1, H-4, and
H-5, respectively. These peaks suggested the presence of a sta-
chyurin moiety [4]. Despite the similarity in coupling constants and
shielding pattern to those previously reported for stachyurin,
similar chemical shift values were only observed for glucose H-4
and H-5, whereas those corresponding to glucose H-1, H-2, and H-3
resembled those reported for another C-glycosidic flavanoellagi-
tannin, stenophyllanin B [4,36]. This implies that catechin is b-
linked through glucose C-1 and HHDP through C-2 and C-3. The
 J. Orejola et al. / Tetrahedron 75 (2019) 4042e4052
Fig. 3. CD spectra of 15e19.
large upfield shift observed for glucose C-1 of 16 (dC 38.2) relative to
that of 15 (dC 67.4) indicates further CeC linkage at C-1 instead of
hydroxylation. The corresponding H-1 observed as a broad singlet
at dH 4.64 is also indicative of b-orientation of the attached flavan-
3-ol [36]. The linkage of the HHDP moiety to glucose C-2 and C-3
was confirmed by HMBC cross peaks observed between glucose H-
2 at dH 4.74 (brs) and H-3 at dH 5.18 (t, J ¼ 1.9 Hz) and HHDP carbonyl
carbons at dC 168.0 and 169.8, respectively. The occurrence of the
valoneoyl group was substantiated by distinct and intense one-
proton singlets located upfield (dH 6.17) and downfield (dH 7.07),
which were not observed in stenophyllanin B with its two HHDP
moieties [5,7,31,36]. Furthermore, the observed upfield shift for the
glucose H-4, H-5, and H-6 signals relative to those of stenophyllanin
B hinted at valoneoyl substitution at the 4,6-position. The orien-
tation of the valoneoyl moiety was established through HMBC
(Fig. 2). The more shielded valoneoyl H-3 (dH 6.17) correlated to the
less shielded valoneoyl C-4 (dC 147.0) and shared a cross peak with
the carbonyl carbon, which showed a three-bond correlation with
glucose H-4. This led to the conclusion that the gallic acid moiety
was linked to the biphenyl part connected to glucose C-4. The ab-
solute configurations of both the HHDP and valoneoyl moieties
were assigned as S based on the observed positive Cotton effect at
220e240 nm [7]. The 1H NMR signals at dH 6.70 (dd, J ¼ 8.2, 1.9 Hz),
dH 6.75 (d, J ¼ 8.2 Hz), and dH 6.87 (d, J ¼ 1.9 Hz) were characteristic
of catechol B-ring H-60 , H-50 , and H-20 , respectively [36], and the
doublet at dH 4.47 (J ¼ 8.1 Hz) connected to the carbon at dC 82.2
was indicative of a 2,3-trans configuration of the flavan ring [37].
From these results, catechin was designated as the flavan-3-ol
substituent at glucose C-1. This was further supported by the
marked similarity in chemical shift and coupling pattern to those
reported for the catechin moiety of stenophyllanin B [36]. The re-
sults inferred that the A-ring C-6 of catechin was connected to
glucose C-1 because distinct rather than broad 1H NMR signals
were observed for C-ring H-2, and B-ring H-20 , H-50 , and H-6'.
Conversely, peak broadening was observed for the C-ring H-4 and
H-3 because of dynamic rotational isomerism as a consequence of
the proximity of these protons to the ellagitannin moiety [36]. To
validate the A-ring C-6 linkage, 5-desgalloylstenophyllanin B and
stenophyllanin C were both prepared from pedunculagin by heat-
ing with (þ)-catechin at 80  C in pH 5 acetate buffer for 4 h. In
contrast to stenophyllanin C, comparison of the 1H NMR spectra
revealed the chemical shift and coupling pattern of catechin pro-
tons of 16 were similar to those observed for 5-
desgalloylstenophyllanin B (Fig. 4). The absolute configuration of
the catechin moiety cannot be fully designated using the CD
4045
spectrum (Fig. 3) because of the strong signal generated by the
HHDP and valoneoyl moieties. However, the chemical shifts of the
flavan-3-ol unit of 16 were very similar to those of 5-
desgalloylstenophyllanin B synthesized from (þ)-catechin, which
strongly suggested that the catechin unit of 16 was in the (þ)-form.
On the basis of these results, we concluded that 16 was stachyur-
anin E.
The HRFABMS of stachyuranin F (17) showed a [M þ H]þ peak at
m/z 1073.1471, indicating 17 was a flavanoellagitannin with a
pyrogallol-type flavan-3-ol and C-glycosidic ellagitannin bearing
two HHDP groups. Three prominent singlets at dH 6.45, 6.51, and
6.80 (1H each) suggested the presence of two HHDP groups, one of
which was connected to the glucose C-1 through a C-glycosidic
linkage [4]. In addition, the chemical shifts of the signals at dH 4.65
(brs), dH 4.76 (brs), and dH 5.21 (t, J ¼ 2.2 Hz), which corresponded
to glucose H-1, H-2, and H-3, respectively, were very similar to
those of 16, indicating that 17 was a stachyurin-type ellagitannin
with a b-linked flavan-3-ol at C-1. On the other hand, the signals of
glucose H-4, H-5, and H-6 and lack of galloyl signals were similar to
the results reported for 5-desgalloylstachyurin (6) [28,29]. The
flavan-3-ol group was confirmed to be linked through C-1 as evi-
denced by the HMBC cross peak between glucose H-1 and a non-
hydroxylated A-ring carbon at dC 107.1 (Fig. 2). The sharp singlet
at dH 6.40 attributable to H-20 and H-60 of the pyrogallol B-ring and
the 2,3-trans C-ring methine protons at dH 3.91 (dd, J ¼ 7.8, 13.2 Hz)
and dH 4.39 (d, J ¼ 7.8 Hz) suggested the presence of gallocatechin.
The 2,3-trans configuration was supported by the C-2 carbon signal
at dC 82.1 [38]. The chemical shifts of the gallocatechin protons of 17
were analogous to those of strobilanin [39]; however, rotational
isomerism observed for the C-8-linked gallocatechin unit of stro-
bilanin was not observed, as evidenced by distinct signals for C-ring
H-2, H-3, and pyrogallol protons. This led to the conclusion that the
5-desgalloylstachyurin moiety was linked to the A-ring C-6 of
gallocatechin. The positive Cotton effect observed at 220e240 nm
in the CD spectrum (Fig. 3) revealed the absolute configuration of
(S)-HHDP [7], but the absolute configuration of gallocatechin could
not be designated for the same reasons as stated for compound 16.
Stachyugluconin (18) is an aldonic acid-based ellagitannin with
one HHDP and two galloyl groups, as evidenced by appearance of
the [M þ H]þ peak at m/z 803.0942 in the HRFABMS (C34H27O23,
803.0938). The 13C signals of aldonic acid were observed at dC 64.8,
70.4 (2C), 72.0, and 73.5, for five hydroxylated aliphatic carbons,
and dC 173.4 for the carboxylic acid (Table 1) [40]. One-proton
singlets at dH 6.51 and dH 6.67 and two-proton singlets at dH 7.08
and dH 7.13 confirmed the presence of one HHDP and two galloyl
moieties. Locations of the HHDP and two galloyl groups were
assigned by comparing the 1H NMR data of 18 with those of
punigluconin [40], which has the same shielding pattern and
coupling values as the aldonic acid core, differing only in the
chemical shifts of the H-2 and H-3 signals. The observed upfield
shift of H-2 (dH 4.40) and downfield shift of H-3 (dH 5.70) relative to
those of punigluconin hinted at location of the galloyl group at the
3-position instead of the C-2. The HMBC cross peaks observed be-
tween the H-3 and H-5 of the aldonic acid moiety and the two
galloyl carbonyl carbons finally confirmed the 3,5-di-O-galloyl
structure, and the cross peak observed between H-2 and the
carboxylate carbon confirmed linkage of the carboxyl group to C-2.
The positive Cotton effect observed at 220e240 nm in the CD
spectrum of 18 led to establishment of the (S)-HHDP configuration
(Fig. 3). The HHDP configuration, coupling constants (J2,3 ¼ 2.9 Hz,
J3,4 ¼ 6.3 Hz), and observed strong NOE correlations between H-5
and H-2, H-2 and H-3, and H-3 and H-5 (Fig. 5) inferred that the
aldonic acid moiety adopted a 2R,3S configuration. These results all
led to the conclusion that 18 was 3,5-di-O-galloyl-4,6-(S)-HHDP-D-
gluconic acid.
4046 J. Orejola et al. / Tetrahedron 75 (2019) 4042e4052

Table 2
1 13
H (500 MHz) and C NMR (126 MHz) data of 16 and 17 in acetone-d6 þ D2O.

Position 16 17

dC dH (J in Hz) dC dH (J in Hz)

Glucose 1 38.2 4.64 brs 38.0 4.65 brs

2 81.4 4.74 brs 81.4 4.76 brs
3 74.9 5.18 t (1.9) 75.8 5.21 t (2.2)
4 73.2 5.64 dd (8.6,1.9) 76.3 5.26 dd (7.9, 2.2)
5 71.0 5.32 dd (8.6, 3.5) 68.5 4.1 dd (7.9, 2.6)
6 64.6 4.85 dd (3.5, 13.3) 67.81 4.73 dd (2.6, 12.3)
3.98 d (13.3) 3.82 d (12.3)
A 5 154.7 154.72
6 107.2 107.13
7 155.0 154.72
8 96.2 5.91 brs 95.9 5.90 brs
9 152.8 154.72
10 101.0 100.7
B 10 131.6 130.9
20 115.3 6.87 d (1.9) 107.13 6.40a s
30 145.6 146.04
40 145.71 133.1
50 116.0 6.75 d (8.2) 146.04
60 120.1 6.70 dd (8.2, 1.9) 107.13 6.40a s
C 2 82.2 4.47 d (8.1) 82.1 4.39 d (7.8)
3 68.1 3.94e3.99 m 67.81 3.91 dd (7.8, 13.2)
4 29.3 2.52 brs 28.8 2.45e2.47 m
2.87 brs 2.78e2.80 m
D 1 116.9 115.9
2 123.7 123.7
3 122.6 122.5
4 142.96 143.0
5 137.6 135.8
6 143.0 143.15
COO 168.02 168.0
E 10 116.8 116.9
20 124.6 125.4a
30 105.8 6.53 s 105.6 6.45 s
40 145.71 145.9
50 135.1 135.0
60 143.13 143.15
COO 169.8 170.6
F 1 115.6 115.7
2 126.6a 127.9a
3 108.7 6.89 s 108.7 6.80 s
4 145.4 145.0
5 136.84 137.5
6 144.8b 144.36
COO 168.7 168.4
G 10 117.3 115.3
20 127.8a 127.2a
30 104.8 6.17 s 107.0 6.51 s
40 147.0 145.1
50 136.84 136.4
60 145.1b 144.36
COO 168.9 169.5
H 100 116.4
200 140.2
300 140.0
400 137.4
500 143.13
600 109.8 7.07 s
COO 168.02
I 1 120.7
2,6 110.1 7.06 s
3,5 146.0
4 139.4
COO 166.2
16
same/overlapping chemical shift assignment.
a,b
interchangeable assignment.

According to HRFABMS, which showed a [M þ Na]þ peak at m/ (Table 1) showed eight aliphatic proton signals, six of which were
z1185.0887 (C50H34O33Na, 1185.0880), stachyuglyconin (19) is a C- attributed to one methylene and five methines analogous to those
glycosidic ellagitannin with a novel eight-carbon aldonic acid core reported for the glucose moiety of stachyurin [4]. The remaining
and HHDP, valoneoyl, and galloyl esters. The 1H NMR spectrum two methine signals at dH 4.50 (d, J ¼ 6.6 Hz) and dH 3.53 (dd, J ¼ 6.6,
 J. Orejola et al. / Tetrahedron 75 (2019) 4042e4052
Fig. 4. 1H NMR spectra of 16 (a), 5-desgalloylstenophyllanin B (b), and stenophyllanin C (c).
1.5 Hz) were assigned as H-3 and H-2, and the 1He1H COSY data
revealed they were part of the aldonic acid chain (Fig. 2). Moreover,
Fig. 5. Key NOESY correlations of 18.
4047
the HMBC cross peak of H-3 to the C-1 carboxyl carbon (dC 174.9)
confirmed the location of the carboxyl group. Aromatic one-proton
signals at dH 6.14, 6.46, 6.85, and 7.06 attributed to valoneoyl and C-
glycosidically linked HHDP moieties and a two-proton singlet at dH
7.02 of the galloyl group were similar to those of 9 and 10.5,31,35
Analogous chemical shifts and coupling patterns to those of glucose
H-2 and H-3 of stachyurin suggested the C-glycosidically linked
HHDP esters were located at the 4,5-position of the aldonic acid
moiety [4]. This was confirmed through HMBC, where aldonic acid
H-4 and H-5 exhibited cross peaks with HHDP carbonyl carbons at
dC 169.5 (A-ring) and dC 169.4 (B-ring), respectively. Similarities
between the chemical shifts and coupling constants and those of
glucose H-4 and H-6 of 16 suggested the valoneoyl moiety was
linked through aldonic acid C-6 and C-8. This was further sub-
stantiated by the HMBC cross peaks between aldonic acid H-8 and a
carbonyl carbon belonging to the valoneoyl (C-ring) moiety (dC
168.8) (Fig. 2). The same carbonyl carbon shared a HMBC cross
signal with the valoneoyl H-3, which showed a three-bond corre-
lation with a valoneoyl C-4 (dC 145.2) and was characteristic of an
unsubstituted valoneoyl-4-OH functionality [5,35]. This led to the
conclusion that the orientation of the valoneoyl moiety was the
same as that of 16. The positive Cotton effect observed at
220e240 nm indicated S configurations for both the HHDP and
4048 J. Orejola et al. / Tetrahedron 75 (2019) 4042e4052

Fig. 6. Four possible conformers of model compound of (2S)-stachyuglyconin, (2S)-20A, B, C, D, with the calculated Boltzmann weighted values.

valoneoyl moieties (Fig. 3). The absolute configuration of aldonic
acid C-2 could not be established using NMR and CD spectroscopy;
hence density functional theory (DFT) calculations were employed.
To reduce the computational cost, a conformational study of a
model compound (20) (Fig. 2), in which the valoneoyl group of 19
was replaced by a HHDP group, was performed. After a conforma-
tional search of (2S)-20 and (2R)-20, the obtained conformers were
optimized at the B3LYP/6-31G(d,p) level. The DFT-optimized con-
formers were classified into four groups for (2S)-20 and three
groups for (2R)-20. The lowest-energy conformers in each group,
along with the DFT-calculated 1H NMR coupling constants between
H-2 and H-3, are shown in Figs. 6 and 7. The conformers with high
populations for (2S)-20 were (2S)-20A (DG ¼ 0.0 kcal/mol, 83.5%)
and (2S)e20B (DG ¼ þ1.0 kcal/mol, 15.5%). The calculated 1H NMR
coupling constants between H-2 and H-3 were 2.7 Hz (dihedral
angle: 75.3 ) for (2S)-20A and 2.6 Hz (64.6 ) for (2S)e20B. These
values are very small compared with the experimental value
(6.6 Hz). The conformers with high populations for (2R)-20 were
(2R)-20A (DG ¼ 0.0 kcal/mol, 71.0%) and (2R)-20B (DG ¼ 0.6 kcal/
mol, 26.7%). In this case, the experimental coupling constant be-
tween H-2 and H-3 (6.6 Hz) was intermediate to those calculated
for (2R)-20A (10.0 Hz, 176.8 ) and (2R)-20B (5.3 Hz, 66.9 ), and
the Boltzmann-weighted value of (2R)-20 (8.6 Hz) was more similar
to the experimental value than that of (2S)-20. From these results,

Fig. 7. Three possible conformers of model compound of (2R)-stachyuglyconin (2S)-20A, B, C, with the calculated Boltzmann weighted values.
 J. Orejola et al. / Tetrahedron 75 (2019) 4042e4052 4049

the absolute configuration of 19 was concluded to be 2R. 4. Experimental

Stachyuglyconin is the first ellagitannin with a C-8 aldonic acid
instead of C-6 aldonic acid core. We proposed that 19 was formed
by condensation of a C-glycosidic ellagitannin precursor with gly-
colic acid (Scheme S1). However, our initial attempts to prepare the
C-8 aldonic acid moiety by condensation of glycolic acid with 5-
desgalloylstachyurin in an aqueous (pH 5) or nonaqueous (1% TFA
in dioxane) medium failed. Hence, the feasibility of the proposed
mechanism warrants further investigation.
2.2. Evaluation of cytotoxicity
All ellagitannins isolated from S. praecox fruits were screened for
cytotoxicity against HeLa and HepG2 cell lines using the MTT assay
[41]. After incubation with the cells for 72 h, all the ellagitannins
showed concentration-dependent cytotoxicity against HeLa cells,
with 11 showing the highest cytotoxicity (IC50 ¼ 35.4 mM). The IC50
values for all ellagitannins, except for 2 (IC50 ¼ 66.4 mM), were
similar to each other (Table 3). This suggests there is no discernible
SAR for the cytotoxicities of these ellagitannins against the HeLa
cell line. Against HepG2, only compounds 1, 5, 6, 8, 9, 11, 15, and 16
demonstrated cytotoxicity, with 9 being the most cytotoxic at
IC50 ¼ 60.0 mM, followed by the structurally-related 8, 15, and 16,
and then the less structurally-related 11. Although the results
suggest the valoneoyl moiety is common to active compounds 8, 9,
15, and 16, compound 10 with the same acyl group did not show
cytotoxic activity.
3. Conclusions
In this study, we identified five new ellagitannins from
S. praecox fruit. Among them, stachyuglyconin (19) is a novel ella-
gitannin with an unusual C-8 aldonic acid core, which is presumed
to be biosynthesized by substitution of a glycolic acid unit at C-1 of
the C-glycosidic ellagitannin. The configuration of the additional C-
2 unit was determined using a DFT calculation. The major ellagi-
tannin in the fruits was identified as pedunculagin, and biosyn-
thetically related C-glycosidic ellagitannins were isolated as minor
tannins. The presence of ellagitannins with a valoneoyl group is
also characteristic of this plant. Stachyuranin E (16) was charac-
terized as a new flavanoellagitannin, and its structure was deter-
mined by comparison of NMR data with synthesized analogs.
Furthermore, the cytotoxic activities of the ellagitannins were
compared, but a clear SAR was not observed.
4.1. General experimental procedures
NMR spectra were recorded in acetone-d6 (Wako Pure Chemical
Industries Ltd., Osaka, Japan), with a Varian Unity Plus 500 spec-
trometer (Palo Alto, CA, USA) operating at 500 MHz for 1H and
125 MHz for 13C, and with a JEOL JNM-AL 400 spectrometer (JEOL
Ltd, Tokyo, Japan) at 400 MHz for 1H and 100 MHz for 13C.
HRFABMS spectra were recorded on a JMS 700N spectrometer (JEOL
Ltd., Tokyo, Japan) in positive ion mode with glycerol or m-nitro-
benzyl alcohol, with or without NaCl, as the matrix. UV spectra
were recorded in MeOH with a Jasco V-560 UV/Vis spectrometer
(Jasco Co. Ltd., Tokyo, Japan). The same solvent was used for the CD
spectroscopic analysis using a Jasco-725N spectrometer (Jasco Co.
Ltd., Tokyo, Japan), and optical rotation measurements using a Jasco
P-1020 polarimeter (Jasco Co. Ltd., Tokyo, Japan). IR spectra were
recorded using a Jasco FT/IR-410K IR spectrometer (Jasco Co. Ltd.,
Tokyo, Japan). Column chromatography was performed using
Sephadex LH-20 (25e100 mm, GE Healthcare UK Ltd., Little Chal-
font, England), Diaion HP20SS (Mitsubishi Chemical Co., Tokyo,
Japan), and Chromatorex ODS (Fuji Silysia Chemical Ltd., Kasugai,
Japan) columns. TLC was performed both on 0.25-mm thick, pre-
coated silica gel 60 F254 (Merck, Darmstadt, Germany) with
tolueneeethyl formateeformic acid (1:7:1, v/v/v) and on 0.1-mm
thick, pre-coated cellulose F (Merck, Darmstadt, Germany) with
2% aqueous acetic acid. Spots were detected by illumination under
short wavelength UV (254 nm), followed by spraying with 2%
ethanolic FeCl3. Analytical HPLC of the crude ethanolic extract of
the leaves and fruit was performed with a gradient elution of 4%e
30% (39 min), 30%e75% (15 min), and 75%e95% (6 min) of aceto-
nitrile (Kanto Chemical Co., Inc., Tokyo, Japan) in 50 mM phosphoric
acid (Kishida Chemical Co., Osaka, Japan). A Cosmosil 5C18-ARII
column (4.6  250 mm; Nacalai Tesque, Inc., Kyoto, Japan) was used
with a mobile phase flow rate of 0.8 mL/min. The HPLC system
consisted of a Jasco DG-2080-53 Plus degasser, Jasco PU-2080 Plus
pump, Jasco AS-2055 Plus autosampler, Jasco CO-2065 Plus column
oven (maintained at 35  C), and Jasco MD-2018 Plus PDA detector
(Jasco Co. Ltd., Tokyo, Japan). HPLC during fraction monitoring was
performed with a gradient elution of 4%e27.3% (35 min) and
27.3%e90% (10 min) of acetonitrile (Kanto Chemical Co., Inc., Tokyo,
Japan) in 50 mM phosphoric acid (Kishida Chemical Co., Osaka,
Japan). The column was a Cosmosil 5C18-ARII (3  150 mm; Nacalai
Tesque, Inc.) and the mobile phase flow rate was 0.4 mL/min. The
HPLC system consisted of a Jasco PU-4180 RHPLC pump, Jasco AS-
4050 autosampler, Jasco CO-4061 column oven (maintained at
40  C), and Jasco MD-4017 PDA detector (Jasco Co. Ltd., Tokyo,
Japan).
4.2. Plant material

Fresh unripe S. praecox fruit were collected from Nagayo District,

Table 3
IC50 of ellagitannins from S. praecox fruits against HeLa and HepG2.
Nagasaki Prefecture, Japan on July 15, 2017, and extracted on the
same day. A voucher specimen was deposited at the Nagasaki
Compound IC50 (mM) Compound IC50 (mM)
University Graduate School of Biomedical Sciences.
HeLa HepG2 HeLa HepG2

Doxorubicin HCl 1.3 5.3 10 37.5 >100.0 4.3. Extraction and isolation
1 38.1 92.5 11 35.4 77.5
2 66.4 >100.0 12 38.1 >100.0 About 1.97 kg of the fresh unripe fruit, including the rachis, was
3 38.7 >100.0 13 35.9 >100.0
comminuted using a blender and then macerated with 5 L of 80%
4 38.9 >100.0 14 41.0 >100.0
5 35.8 82.6 15 39.0 63.3 acetone for 24 h. After collection of the first crop of acetone liquid
6 39.5 85.8 16 40.8 73.3 extract, the resulting debris was extracted again with 70% acetone
7 44.5 100.0 17 41.1 >100.0 for 24 h (Fig. S3). This step was repeated three times. The resulting
8 38.9 63.5 18 39.6 >100.0 liquid extracts were combined, followed by removal of the solvent
9 41.0 60.0 19 38.3 >100.0
in vacuo at 40  C. The aqueous concentrate was then fractionated
4050 J. Orejola et al. / Tetrahedron 75 (2019) 4042e4052
using a Diaion HP20SS column (8  50 cm) and a gradient elution
with 0%e100% acetonitrile. This yielded four fractions (Fr 1eFr 4),
which were all subjected to a series of chromatographic separation
steps. Fraction 1 (151.18 g) was fractionated using a Sephadex LH-20
column (8  29 cm) and eluted with methanol (0%e100% gradient),
followed by washing with 60% acetone. This gave six fractions and
all except for Fr 1e6 were subjected to further fractionation. Frac-
tion 1e2 (9.81 g) was separated on a Sephadex LH-20 column
(8  29 cm) with a gradient elution of 0%e60% acetone to yield six
fractions, including 13 (6.44 g). Elution of Fr 1e3 (10.12 g) on a
Sephadex LH-20 column (4  30 cm) with 0%e60% acetone yielded
16 fractions, including 6 (5.38 g), Fr 1-3-8, Fr 1-3-14, and Fr 1-3-16.
From Fr 1-3-16, 11 (80.4 mg), 18 (83.1 mg), 15 (89.9 mg), and 19
(21.7 mg) were isolated using a Chromatorex ODS column
(1.5  16 cm) and a gradient elution with methanol in water (0%e
100%). Fr 1-3-14-1 (348.1 mg) was further purified on a Sephadex
LH-20 column (2  12 cm) using 0%e100% methanol, and washed
with 60% acetone to give 8 (119.0 mg). Fr 1e4 (7.25 g) was frac-
tionated with a Sephadex LH-20 column (4  30 cm) using 0%e60%
acetone as the eluent to obtain 6 (450.3 mg), 5 (3.7 g), and 1 (1.58 g).
Fr 1e5 (39.89 g) was fractionated on a Sephadex LH-20 column
(5.5  29 cm) with 0%e100% methanol and washed with 60%
acetone to yield seven fractions, including 11 (142.2 mg) and 1
(34.1 g). Fr 1-5-3 (2.69 g) yielded 6 (450.3 mg), 5 (476.5 mg), 1
(59.7 mg), and Fr 1-5-3-4 (1.34 g) after elution with 0%e100%
methanol and pure acetone from a Diaion HP20SS column
(3  28 cm). Fr 1-5-3-4 gave 1 (262.2 mg) and Fr 1-5-3-4-2
(996.1 mg) after fractionation on a Cosmosil C75 OPN column
(3  20 cm) with 0%e100% methanol. Fr 1-5-3-4-2 was further
separated on a Sephadex LH-20 column (2  19 cm) with 0%e100%
methanol and washed with 60% acetone to yield 14 (7.7 mg) and Fr
1-5-3-4-2-4 (13.7 mg), from which 12 (10.7 mg) was isolated after
purification on a Chromatorex ODS column (1.5  16 cm) with 0%e
100% methanol. Fractionation of Fr 1-5-6 (1.75 g) on a Diaion
HP20SS column (3  28 cm) with 0%e100% methanol and washing
with pure acetone produced seven fractions, including 10
(133.2 mg) and 7 (305.4 mg). A series of separation steps of Fr 1-5-
6-1 (33.8 mg) on a Sephadex LH-20 column (2  12 cm) with 0%e
100% methanol and 60% acetone, and on a Chromatorex ODS col-
umn (1.5  16 cm) with 0%e100% methanol gave 17 (19.0 mg). Pu-
rification of Fr 1-5-6-4 (268.7 mg) with 0%e100% methanol and
washing with 60% acetone on a Sephadex LH-20 column
(2  12 cm) gave 3 (21.4 mg). Fractionation of Fr 2 (21.31 g) on a
Diaion HP20SS column (5  32 cm) with 0%e100% acetonitrile
yielded five fractions, including 1 (11.78 g). Fr 2-1 (623.0 mg) yiel-
ded six fractions including 13 (176.4 mg) and 6 (116.8 mg) after
elution from a Sephadex LH-20 column (1.5  24 cm) with 0%e
100% methanol followed by washing with 60% acetone. Fraction
2e3 (5.68 g) produced 4 (245.0 mg), 7 (1.06 g), and Fr 2-3-10
(657.5 mg), along with seven other fractions after separation on a
Sephadex LH-20 column (3  23 cm) using 0%e100% methanol for
elution and 60% acetone for washing. Chromatographic separation
of Fr 2-3-10 on a Chromatorex ODS column (1.5  16 cm) with 0%e
100% methanol gave four fractions, among which Fr 2-3-10-2
(119.9 mg) yielded 16 (62.8 mg) after purification on a Sephadex
LH-20 column (2  12 cm) with 0%e100% methanol and 60%
acetone. Sequential separation of Fr 3 (21.31 g) on a Diaion HP20SS
column (5  32 cm) with 0%e100% acetonitrile and on a Diaion
HP20SS column (3  15 cm) with 10%e100% acetone led to isolation
of 2 (35.3 mg). Separation of Fr 4 (8.4 g) on a Sephadex LH-20 col-
umn (4  25 cm) with 0%e100% methanol for elution and 60%
acetone for washing gave six fractions, with only Fr 4e6 (3.71 g)
being subjected to further purification. Chromatographic separa-
tion of Fr 4e6 on a Sephadex LH-20 column (4  47 cm) with 3:2
acetoneeurea (pH 2) yielded three fractions, and Fr 4-6-1 and Fr 4-
6-3 were purified further. Purification of acetone-free Fr 4-6-1 on a
Diaion HP20SS column (3  19 cm) with 0%e100% methanol and
then pure acetone gave 678.6 mg of a polymeric fraction. Parti-
tioning of acetone-free Fr 4-6-3 on a Diaion HP20SS column
(3  24 cm) with 0%e100% methanol and pure acetone yielded four
fractions, and Fr 4-6-3-1 (52.2 mg) and Fr 4-6-3-2 (352.7 mg) were
subjected to further purification. Fr 4-6-3-1 yielded 7 (9.5 mg) after
elution with 0%e100% methanol from a Chromatorex ODS column
(2.5  20 cm), whereas Fr 4-6-3-2 gave seven fractions, including 9
(5.7 mg) and 2 (69.3 mg), after elution with 0%e100% methanol
from a Chromatorex ODS column (3  30 cm).
4.3.1. Stachyuranin D (15)
Yellowish brown, amorphous powder; [a]16 D 143.6
 (c 0.9,
MeOH); UV (MeOH) lmax (log ε) 261 (4.54) sh, 232 (4.82) sh, 208
(4.89) nm; IR (film) nmax 3380, 1728, 1613, 1505, 1454, 1317,
1180 cm1; HRFABMS m/z 953.0895 [MþH]þ (Calcd for C41H29O27,
953.0891).
4.3.2. Stachyuranin E (16)
Brown, amorphous powder; [a]16 
D 106.8 (c 0.8, MeOH); UV
(MeOH) lmax (log ε) 279 (4.57) sh, 205 (5.16) nm; IR (film) nmax 3411,
1714, 1613, 1506, 1454; 1313, 1187 cm1; HRFABMS m/z 1377.1691
[MþH]þ (Calcd for C63H45O36, 1377.1685).
4.3.3. Stachyuranin F (17)
Off-white, amorphous powder; [a]16 
D 132.5 (c 0.8, MeOH); UV
(MeOH) lmax (log ε) 266 (4.59) sh, 233 (4.95) sh, 204 (5.16) nm; IR
(film) nmax 3432, 1723, 1605, 1506, 1446, 1312,1182 cm1; HRFABMS
m/z 1073.1471[MþH]þ (Calcd for C49H37O28, 1073.1466).
4.3.4. Stachyugluconin (18)
Yellowish brown, amorphous powder; [a]26 
D 48.2 (c 0.8, MeOH);
UV (MeOH) lmax (log ε): 264 (4.84) sh, 208 (5.33) nm; IR (film) nmax
3419, 1715, 1614, 1514, 1455, 1316, 1185 cm1; HRFABMS m/z
803.0942 [MþH]þ (Calcd for C34H27O23, 803.0938).
4.3.5. Stachyuglyconin (19)
Brown, amorphous powder; [a]26 
D 69.5 (c 0.1, MeOH); UV
(MeOH) lmax(log ε) 273 (4.52) sh, 207 (4.96) nm; IR (film) nmax
3408, 1721, 1612, 1311, 1189 cm1; HRFABMS m/z 1185.0887
[MþNa]þ (Calcd for C50H34O33Na, 1185.0875).
4.4. Preparation of 5-desgalloylstenophyllanin B and
stenophyllanin C
Pedunculagin (1) (1.0 g) isolated from S. praecox in this study
was reacted with 0.37 g of (þ)-catechin in 200 mL of pH 5 acetate
buffer at 80  C for 4 h. The resulting reaction medium was subjected
to chromatographic separation on a Sephadex LH-20 column
(3  25 cm), eluted with 0%e100% MeOH, and washed with 60%
acetone to give 5-desgalloylstenophyllanin B (134.3 mg) and sten-
ophyllanin C (260.3 mg). The identities of the products were
confirmed by comparison of the obtained 1H NMR data with re-
ported data [36].
4.5. DFT calculation
A conformational search was performed using the Monte Carlo
method and the MMFF94 force field with Spartan ’14 (Wave-
function, Irvine, CA, USA). The obtained low-energy conformers
within a 6 kcal/mol window were optimized at the B3LYP/6-
31G(d,p) level in acetone (PCM). The vibrational frequencies were
calculated at the same level to confirm their stability, and no
imaginary frequencies were found. The DFT-optimized conformers
 J. Orejola et al. / Tetrahedron 75 (2019) 4042e4052
were classified and 1H NMR coupling constants of the lowest-
energy conformers in each classified group were calculated at the
B3LYP/6-31G(d,p)uþ1s (using only the Fermi contact term) level
(PCM) and scaled using a slope parameter of 0.94 [42]. The calcu-
lated values were averaged using Boltzmann distribution theory at
298 K from their relative Gibbs free energies. All DFT calculations
were performed using Gaussian 16 [43]. Three-dimensional struc-
tures of the molecules were generated using GaussView [44].
4.6. Cell culture
HepG2 (ATCC HB 8065) and HeLa (ATCC CCL2) cells were
maintained as monocultures in minimum essential medium (Gibco
by Life Technologies, Carlsbad, CA, USA), supplemented with 10%
fetal bovine serum (HyClone Laboratories, Inc., Logan, UT, USA) and
incubated at 37  C in humidified air with 5% CO2.
4.7. Preparation of test compounds
To prepare 500 mM stock solutions, the ellagitannins were
weighed, dissolved in 40 mL of water, and then diluted with PBS to
2 mL. These solutions were then filtered using 0.22-mm PVDF filter
membranes (Merck Millipore, Ltd., Cork, Ireland). The same
waterePBS solvent system (1 in 50) was used as a diluent to pre-
pare solutions of the test compounds with concentrations in the
range of 0.5e500 mM, and was also used as the solvent control in
the MTT assay.
4.8. Cytotoxicity assay
The cytotoxicities of compounds 1e19 were assessed using the
MTT assay [41] with some modifications. About 5,000 cells of both
HepG2 and HeLa lines were grown in 96-well flat bottom plates
(Corning Inc., NY, USA). After 24 h of pre-incubation, the medium in
each well was replaced with 100 mL of fresh complete medium, to
which 25 mL of the test compound solution was added. For the
negative control and blank, the wells were replenished with 125 mL
of fresh complete medium. Doxorubicin HCl (Wako Pure Chemical
Industries Ltd.) was used as the positive control. The cells were then
incubated for another 72 h. The medium in each well was replaced
with 100 mL of a 0.5 mg/mL solution of MTT (Tokyo Chemical In-
dustry, Co., Ltd., Tokyo, Japan) in minimum essential medium
without fetal bovine serum, and then incubated for 3 h at 37  C in
humidified air with 5% CO2 and protected from light. The formazan
crystals that formed were dissolved in 100 mL of 10% SDS in 0.01 N
HCl overnight at room temperature. The absorbance was measured
at 570 nm using a microplate reader (Bio-rad iMark, Bio-Rad Lab-
oratories, K.K., Tokyo, Japan). The percentage cytotoxicity was
calculated as [(AS  AT)/(AT  A0)]  100%, where AS is the absor-
bance of the solvent control, AT is the absorbance of the test com-
pound, and A0 is the absorbance of the blank. Tests were performed
in triplicate and the results are expressed as the mean ± standard
error of the mean. The IC50 was calculated by extrapolation of the
concentration corresponding to 50% cytotoxicity from the linear
part of the percentage cytotoxicity curve derived from the triplicate
set of data points.
Acknowledgments
This work was supported by the Japan Society for the Promotion
of Science KAKENHI [grant numbers 17K08338 and 16K07741]. The
authors are grateful to N. Tsuda, at the Center for Industry, Uni-
versity and Government Cooperation, Nagasaki University, for
recording the MS data. In addition, J.O. would like to express her
sincere thanks to the Japanese Government (MEXT) for a doctoral
4051
scholarship. The computations were partly carried out using com-
puter resources at the General Projects by Research Institute for
Information Technology, Kyushu University. We thank Edanz Group
(www.edanzediting.com/ac) for editing a draft of this manuscript.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tet.2019.06.037.
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