Hydrolyzable tannins of tamaricaceous plants. V. Structures of monomeric-trimeric tannins and cytotoxicity of macrocyclic-type tannins isolated from tamarix nilotica (1)

Article abstract of DOI:10.1021/np4001625

Three new ellagitannin monomers, nilotinins M5-M7 (1-3), a dimer, nilotinin D10 (4), and a trimer, nilotinin T1 (5), together with three known dimers, hirtellin D (7) and tamarixinins B (8) and C (9), and a trimer, hirtellin T2 (6), were isolated from Tamarix nilotica dried leaves. The structures of the tannins were elucidated by intensive spectroscopic methods and chemical conversions into known tannins. The new trimer (5) is a unique macrocyclic type whose monomeric units are linked together by an isodehydrodigalloyl and two dehydrodigalloyl moieties. Additionally, dimeric and trimeric macrocyclic-type tannins isolated from T. nilotica in this study were assessed for possible cytotoxic activity against four human tumor cell lines. Tumor-selective cytotoxicities of the tested compounds were higher than those of synthetic and natural potent cytotoxic compounds, including polyphenols, and comparable with those of 5-fluorouracil and melphalan.

Full text of DOI:10.1021/np4001625

Article
pubs.acs.org/jnp
Hydrolyzable Tannins of Tamaricaceous Plants. V. Structures of
Monomeric−Trimeric Tannins and Cytotoxicity of Macrocyclic-Type
Tannins Isolated from Tamarix nilotica1
Mohamed A. A. Orabi,† Shoko Taniguchi,‡ Hiroshi Sakagami,§ Morio Yoshimura,⊥ Takashi Yoshida,⊥
and Tsutomu Hatano*,‡
†Faculty of Pharmacy, Al-Azhar University, Assiut 71524, Egypt
‡Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan
§Division of Pharmacology, Department of Diagnostic and Therapeutic Sciences, School of Dentistry, Meikai University, Japan
⊥College of Pharmaceutical Sciences, Matsuyama University, Bunkyo-cho, Matsuyama 790-8578, Japan
S
* Supporting Information
ABSTRACT: Three new ellagitannin monomers, nilotinins
M5−M7 (1−3), a dimer, nilotinin D10 (4), and a trimer,
nilotinin T1 (5), together with three known dimers, hirtellin D
(7) and tamarixinins B (8) and C (9), and a trimer, hirtellin
T2 (6), were isolated from Tamarix nilotica dried leaves. The
structures of the tannins were elucidated by intensive
spectroscopic methods and chemical conversions into known
tannins. The new trimer (5) is a unique macrocyclic type
whose monomeric units are linked together by an isodehy-
drodigalloyl and two dehydrodigalloyl moieties. Additionally,
dimeric and trimeric macrocyclic-type tannins isolated from T.
nilotica in this study were assessed for possible cytotoxic
activity against four human tumor cell lines. Tumor-selective cytotoxicities of the tested compounds were higher than those of
synthetic and natural potent cytotoxic compounds, including polyphenols, and comparable with those of 5-fluorouracil and
melphalan.
llagitannins are a unique group of polyphenols that are
widely distributed in plant foods and beverages.2
HSC-3, and HSC-4) and human promyelocytic leukemia (HL-
60) cell lines and human normal oral cells [gingival fibroblast
(HGF), pulp cell (HPC), and periodontal ligament fibroblast
(HPLF)] to determine the tumor specificity.
E
Ellagitannins from different plant sources have been demon-
strated to have marked antiviral, antimicrobial, immunomodu-
latory, antitumor, and hepatoprotective activities.2−8 Plants
from the family Tamaricaceae have been shown to produce
several types of tannins.1,9−17 Ellagitannins from tamaricaceous
plants were reported to exhibit significant host-mediated
antitumor activities against sarcoma-180 in mice and strong
cytotoxic effects with higher tumor specificity against four
tumor cell lines.11−13 Further investigation of the aqueous
acetone extract of the leaves of Tamarix nilotica (Ehrenb.)
Bunge (Tamaricaceae) has led to the isolation of several
tannins. This paper describes the isolation and identification of
three new ellagitannin monomers, nilotinins M5 (1), M6 (2),
and M7 (3), a new dimer, nilotinin D10 (4), a new
macrocyclic-type trimer, nilotinin T1 (5), and the known but
first time isolated tannins hirtellins T2 (6) and D (7) and
tamarixinins B (8) and C (9), as well as the purification of
additional amounts of the known macrocyclic-type tannins
nilotinin D9 (10) and hirtellins C (11) and F (12)11 from T.
nilotica. We also report the results of assessing the cytotoxic
activities of the macrocyclic-type tannins 5, 8, and 10−12
against the human oral squamous cell carcinoma (HSC-2,
RESULTS AND DISCUSSION
■
An aqueous acetone extract of the dried leaves of T. nilotica was
subjected to Diaion HP 20 column chromatography, and
compounds were eluted with H2O, MeOH/H2O (40:60%, v/
v), MeOH, and acetone, successively. The eluate with H2O/
MeOH (6:4, v/v) was submitted to Toyopearl HW-40,
Sephadex LH-20, and MCI-gel CHP-20P gel chromatography,
followed mainly by preparative HPLC purifications to furnish
five new monomeric−trimeric tannins (1−5), four known
oligomeric tannins, hirtellins T2 (6)17 and D (7)13 and
tamarixinins B (8)15 and C (9),15 and additional amounts of
the known macrocyclic-type tannins nilotinin D9 (10)11 and
hirtellins C (11)11 and F (12).11 Although tamarixinin C (9)
was previously assigned to the structure unambiguously,15 our
results revealed the assignments of the protons and carbons of
Received: February 24, 2013
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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1
Table 1. H NMR Data δH (J in Hz) for the Glucose Protons of 1−5 and 9 (600 MHz, acetone-d6/D2O, 9:1)
1
2
3
9
4
5
glucose-1
1
2
3
4
5
6
5.50, d (8.4)
6.02, d (7.8)
6.02, d (7.8)
6.21, d (8.4)
5.24, d (7.8)
5.31, dd (7.8, 9.6)
5.34, t (9.6)
6.16 br s
a
5.55, dd (8.4, 9.6)
5.47, t (9.6)
5.65, dd (7.8, 9.6)
5.72, t (9.6)
5.63, dd (7.8, 9.6)
5.74, t (9.6)
5.63, dd (8.4, 9.6)
5.81, t (9.6)
5.56
b
5.70, t (9.6)
c
5.11, t (9.6)
5.19, t (9.6)
5.19, t (9.6)
5.22, t (9.6)
3.85, t (9.6)
5.12, t (9.6)
d
e
4.23, dd (6.6, 9.6)
5.25, dd (6.6, 13.2)
3.80, br d (13.2)
4.43, dd (6.6, 9.6)
5.28,dd (6.6, 13.2)
3.83, d (13.2)
4.43, ddd (1.2, 6.6, 9.6)
5.28,dd (6.6, 13.2)
3.83, dd (1.2, 13.2)
4.54, dd (6.6, 9.6)
5.33, dd (6.6, 12.6)
3.92, d (12.6)
∼3.60
4.25, dd (6.6, 9.6)
f
3.78, dd (6.6, 13.2)
5.26, dd (6.6, 13.2)
g
3.93, dd (2.4, 13.2)
3.84, d (13.2)
glucose-2
1
2
3
4
5
6
6.03, d (8.4)
5.44, d (7.8)
5.76 br s
5.63, dd (8.4, 9.6)
5.76, t (9.6)
5.51, dd (7.8, 9.6)
5.59, t (9.6)
5.44, dd (7.8, 9.6)
5.80, t (9.6)
5.19, t (9.6)
5.19, t (9.6)
5.10, t (9.6)
eh
,
4.45, dd (6.6, 9.6)
5.30, dd (6.6, 13.2)
3.85, d (13.2)
4.24, dd (6.6, 9.6)
5.26, dd (6.6, 13.2)
3.81, br d (13.2)
4.34
fi
5.23, , dd (6.6, 13.2)
g
3.76, , d (13.2)
glucose-3
i
1
2
3
4
5
6
5.24
a
5.56
ab
,
5.56
c
5.07, t (10.2)
eh
,
4.34
f
5.36, dd (6.6, 13.2)
g
3.72, d (13.2)
a,h,i
b,c,e−g
d
Proton signals with these chemical shifts overlapped, and thus their coupling constants were not measured.
Interchangeable. Overlapped
by solvent signal.
1
the sugar cores in the NMR spectra should be corrected as
shown in Tables 1 and 2.
Structural Elucidation of Monomeric Ellagitannins.
Nilotinin M5 (1) was isolated as an off-white, amorphous
powder. Its molecular formula was C55H38O36 from the
prominent ion peak ([M + Na]+) at m/z 1297.10358 in the
HRESIMS spectrum. The H NMR spectrum of 1 displayed
aromatic proton signals of two mutually coupled doublets [δH
7.21 and 6.46 (each 1H, d, J = 1.8 Hz)] and a one-proton
singlet (δH 7.06), characteristic of the dehydrodigalloyl moiety
(DHDG).9−11,17 The spectrum also showed a two-proton
singlet (δH 6.96) and one-proton singlet (δH 6.89), character-
istic of an isodehydrodigalloyl moiety (isoDHDG),9−11 and a
second two-proton singlet (δH 6.93) and a pair of one-proton
singlets (δH 6.58 and 6.51) ascribable to galloyl and
hexahydroxydiphenoyl (HHDP) units, respectively.18 The
aliphatic region of the spectrum showed seven sets of well-
resolved signals (δH 5.50−3.80) assignable to the protons of a
fully O-acylated glucose core (Table 1).9−11 The large coupling
constants (J1,2 = 8.4 Hz, J2,3 = J3,4 = J4,5 = 9.6 Hz) of these
proton signals indicated the existence of the glucose core in the
Table 2. 13C NMR Data for the Glucose Carbons of 1−5 and
9 (151 MHz, acetone-d6/D2O, 9:1)
1
2
3
9
4
5
glucose-1
1
93.8
71.4
73.6
70.5
72.8
62.9
93.7
71.9
73.5
70.6
72.8
63.0
93.5
72.0
73.4
70.6
72.7
63.0
93.7
71.7
73.4
70.65
72.9
63.0
93.3
71.5
76.4
68.7
78.0
61.4
93.3
71.6
73.4
70.8
72.9
63.4
a
b
c
2
3
4
4
pyranose form with a C1 conformation and a β-oriented acyl
d
e
5
group at the anomeric carbon. The 13C NMR spectrum of 1
showed the aliphatic, aromatic, and the carbonyl carbon peaks
(Tables 2 and 3), which were assigned on the basis of
correlations in the HSQC and HMBC spectra, characteristic of
the structural moieties of 1. A galloyl unit was placed at C-3 of
the glucose core, as indicated from an HMBC correlation
between a galloyl proton signal (δH 6.93, 2H, s) and a glucose
H-3 signal (δH 5.47, 1H, t) through a common carbonyl peak
(δC 166.6). The acylation of the glucose core C-4/C-6 by an
HHDP unit was suggested by the large chemical shift difference
(ΔδH 1.45) between the geminal coupled C-6 methylene
protons.19 Bridging of the HHDP unit at C-4/C-6 of the
glucose core was further confirmed by HMBC correlations
among the HHDP proton signals (δH 6.58 and 6.51) and
signals of H-6 (δH 5.25) and H-4 (δH 5.11) of the glucose core
through the common carbonyl carbon peaks (δC 168.3 and
167.7), respectively. In the 13C NMR spectrum of 1, the carbon
6
glucose-2
1
93.5
71.9
73.3
70.58
72.8
63.0
93.6
71.6
73.5
70.6
72.8
63.0
93.3
72.5
73.4
70.5
72.7
63.5
2
3
4
d
e
5
6
glucose-3
1
2
3
4
5
6
93.8
71.2
74.4
70.7
a
b
c
d
73.0.
e
63.1
a−e
Interchangeable.
B
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Table 3. 13C NMR Data for the Aromatic Skeleton of the Tannins 1−5 (151 MHz, acetone-d6/D2O, 9:1)
a
1
2
3
4
5
DHDG
1
121.8
106.7
147.6
138.8
146.0
112.7
168.1
113.4
136.8
140.4
140.8
143.4
110.6
164.7
119.9, 122.2
106.6, 107.1
147.4, 147.5
119.3, 119.6
107.2, 107.8
147.7 (2C)
2
3
b
c
c
d
d
4
138.5, 139.5
139.43, 139.7
5
145.7, 145.8
112.6, 112.8
164.3, 168.9
113.1, 113.5
145.6, 145.7
112.2, 112.5
163.4, 164.4
112.3, 112.5
135.8, 137.2
6
7
1′
2′
3′
4′
5′
6′
7′
136.78, 136.84
b
b
c
c
d
d
d
140.3, 140.59
139.9, 140.2
c
c
d
140.62, 141.2
140.1, 140.9
143.2, 143.8
142.6, 143.4
109.6, 110.6
164.0, 164.1
110.18, 110.23
164.8 (2C)
isoDHDG
1
124.6
124.9, 126.5
126.5
110.5
149.4
138.3
168.2
113.5
139.2
140.6
140.1
142.1
108.4
166.8
123.3
2/6
3/5
4
110.8 (2C)
150.3 (2C)
139.3
110.5, 110.9 (2C each)
149.5, 150.7 (2C each)
138.4, 139.8
110.4 (2C)
148.1(2C)
d
139.36
7
164.3
164.8, 168.33
113.5 (2C)
164.9
114.5
1′
2′
3′
4′
5′
6′
7′
114.4
b
e
e
d
139.5
139.06, 139.14
139.6, 140.8
139.7
b
d
139.6
139.65
b
d
139.9
139.3, 140.1
139.9
142.2
108.3
170.3
142.0, 142.3
142.4
108.2
166.3
108.2, 108.5
166.6, 170.7
galloyl
1
119.9
120.1
119.4, 120,0
119.1, 119.7, 120.5
120.4, 120.2, 120.0
2/6
3/5
4
110.16 (2C)
145.7 (2C)
139.0
110.2 (2C)
145.7 (2C)
139.3
110.18, 110.23
145.7, 145.9 (2C each)
139.3, 139.8
110.1, 110.18, 110.3 (2C each)
145.8 (4C), 145.7 (2C)
139.2, 139.3, 139.8
110.3 (2C), 110.4 (4C)
145.7, 145.8, 145.9 (2C each)
139.23, 139.26 139.6
7
166.6
166.8
165.2, 166.8
164.6, 166.8, 167.1
167.0 (2C), 167.4
HHDP
1
115.7
115.6
115.6
115.7
115.6, 115.8 (2C)
1′
115.8
115.9
115.9
115.8
115.86, 115.93, 116.2
2, 2′
3
125.4, 126.0
107.9
125.5, 126.1
108.0
125.4, 126.1
107.9
125.4, 126.0
107.84
125.5, 125.6, 125.8, 126.1, 126.2 (2C)
108.1, 108.2 (2C)
3′
107.8
107.8
107.8
107.78
107.9 (3C)
4, 4′
5
145.1, 145.2
136.3
145.16, 145.23
136.3
145.15, 145.2
136.3
145.1, 145.2
136.3
145.2 (6C)
136.3, 136.4 (2C)
5′
6, 6′
7
136.5
136.6
136.6
136.5
136.5, 136.6, 136.8
144.27 (2C), 144.33 (4C)
168.3, 168.5 (2C)
144.3 (2C)
168.3
144.33, 144.36
168.3
144.3 (2C)
168.3
144.3 (2C)
168.3
7′
167.7
167.8
167.8
167.8
167.75, 167.87, 167.92
a
The assignments of the carbons of the DHDG and isoDHDG moieties in 5 were achieved on the basis of HSQC and comparison with
spectroscopic data of the monomeric tannins 1−3 and those of the previously isolated analogous macrocyclic-type tannins 10−12.11
b−
e
Interchangeable.
peak at δC 150.3 was assigned to C-3/C-5 of an isoDHDG
moiety since it has been demonstrated that C-3/C-5 of the
isoDHDG moiety resonate at lower field (δC ∼150) than C-3/
C-5 of the usual galloyl unit (δC ∼145.7) (Table 3).9,10
Assignment of a two-proton singlet (δH 6.96) to H-2/H-6 of an
isoDHDG moiety was confirmed by HMBC connectivity with
the characteristic carbon peak (δC 150.3) of C-3/C-5 of the
same moiety.9,10 The HMBC correlation of this two-proton
singlet with the glucose H-1 signal (δH 5.50) through the ester
carbonyl carbon (δC 164.3) indicated the location of the
isoDHDG moiety at C-1 of the glucose core. A carboxylic
carbon peak (δC 170.3) was assigned to C-7′ of an isoDHDG
unit, as indicated from its HMBC correlation with the H-6′
signal (δH 6.89) of the same isoDHDG moiety. The absence of
a similar correlation of any of the glucose proton signals with
the latter carboxylic carbon peak satisfied orientation of the
isoDHDG moiety as shown by the formula 1. Similarly,
placement of the DHDG moiety at C-2 of the glucose core was
substantiated from connectivity in the HMBC spectrum
between the H-6′ singlet signal (δH 7.06) of the DHDG
C
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moiety and the glucose H-2 signal (δH 5.55) through an ester
carbonyl carbon peak (δC 164.7). The carboxylic carbon peak
(δC 168.1) was assigned to C-7 of the DHDG moiety because
of the HMBC correlation with the meta-coupled signals (δH
7.21 and 6.46) of the same ring. Lack of HMBC correlations of
any of the glucose proton signals with this carboxylic carbon
peak satisfied the attachment mode of the DHDG moiety as
shown by formula 1 (Figure 1). The chiral HHDP unit in 1 was
shown to have an S configuration, as deduced from a positive
Cotton effect at 236 nm in the electronic circular dichroism
(ECD) spectrum.20 On the basis of these findings, nilotinin M5
was formulated as 1. It is the first monomeric hydrolyzable
tannin including both DHDG and isoDHDG moieties.
Nilotinin M6 (2) was isolated as an off-white, amorphous
powder. Its molecular formula, C55H38O36, the same as that of
1
1, was determined by HRESIMS. The H NMR spectrum
displayed two two-proton singlets (δH 7.06 and 6.99) and a pair
of one-proton singlets (δH 6.87 and 6.84), characteristic of two
isoDHDG moieties.9−11,15 Proton signals due to a galloyl (δH
6.97) and an HHDP (δH 6.60 and 6.49) unit were evident in
the aromatic region.18 The aliphatic proton signals comprised
seven spin systems of well-resolved signals (δH 6.02−3.83) with
large coupling constants (Table 1) assignable to protons of a
4
fully O-acylated glucopyranose core with a C1 conformation
including a β-oriented acyl group at the anomeric center. The
13C NMR spectrum also showed the aliphatic (Table 2),
aromatic, and carbonyl carbon peaks (Table 3) consistent with
the structural moieties of 2. Acylation of the C-4/C-6 of the
glucose core by an HHDP unit was suggested because of the
large chemical shift difference (ΔδH 1.45) between the gem-
coupled proton signals (δH 5.28 and 3.83) of the C-6
methylene protons.19 It was also evidenced by HMBC
correlations between HHDP proton signals (δH 6.60 and
6.49) and signals of H-6 (δH 5.28) and H-4 (δH 5.19) of the
glucose core through common carbonyl carbon peaks (δC 168.3
and 167.8), respectively (Figure 2). A galloyl unit was shown to
be at C-3 of the glucose core by HMBC correlation between
the galloyl proton signal (δH 6.97) and the glucose H-3 signal
(δH 5.72) through a common carbonyl carbon peak (δC 166.8).
Consequently, the isoDHDG moieties should be at C-1 and C-
2 of the glucose core. A pair of two-proton singlets (δH 7.06
and 6.99) was assigned to H-2/H-6 of the two isoDHDG
moieties, as indicated from two-bond HMBC connectivity with
the characteristic carbon peaks (δC 150.7 and 149.5) of C-3/C-
5 of the same moieties, respectivly.9−11,16 The attachment
mode of the isoDHDG moieties in 2 (Figure 1) was
substantiated from HMBC correlations of H-2/H-6 [δH 7.06
(2H, s)] of an isoDHDG moiety and H-6′ [δH 6.84 (1H, s)] of
the other isoDHDG moiety with the glucose H-1 signal [δH
6.02 (1H, d)] and the glucose H-2 signal [δH 5.65 (1H, dd)]
via the carbonyl carbon peaks (δC 164.8 and 166.6),
respectively. On the other hand, the remaining H-6′ signal
[δH 6.87 (1H, s)] of the isoDHDG moiety at C-1 of glucose
and the H-2/H-6 signal [δH 6.99 (2H, s) of the other one
showed HMBC correlations with the carboxylic carbon peaks
(δC 170.7 and 168.33), respectively. Lack of HMBC
correlations from the aliphatic proton signals to these
carboxylic carbon peaks satisfied orientations of the isoDHDG
moieties as shown by formula 2 (Figure 1). A positive Cotton
effect at 236 nm in the ECD spectrum indicated an S
configuration of the chiral HHDP unit.20 On the basis of this
spectroscopic evidence, nilotinin M6 was assigned the structure
2 (Figure1), which is isomeric to nilotinin M5 (1), where the
Figure 1. Structures of the new tannins 1−3 and the known one
remurin A. The arrows (H→C) indicate important HMBC
correlations.
DHDG moiety at C-2 of the glucose core in 1 was replaced
with an isoDHDG moiety in 2. It is the first monomeric
hydrolyzable tannin including two isoDHDG moieties.
Nilotinin M7 (3) was isolated as an off-white, amorphous
powder. Its molecular formula, C48H34O31, the same as that of
remurin A,9 was determined from a prominent ion peak at m/z
D
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Figure 2. Structures of the new tannins 4 and 5 and the known tannin 6. The arrows (H→C) indicate important HMBC correlations.
E
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1
1129.09566 ([M + Na]+) in the HRESIMS spectrum. The H
NMR spectrum indicated the presence of two galloyl units [δH
7.07 and 6.965 (each 2H, s)], an HHDP unit [δH 6.60 and 6.48
(each 1H, s)], an isoDHDG moiety [δH 6.968 (2H, s) and 6.81
difference (ΔδH 1.45) between the C-6 gem-proton signals (δH
5.26 and 3.81). Conversely, the C-4 and C-6 hydroxy groups of
glucose-1 were unacylated because of the upfield shifts (δH
3.78−3.93) of the glucose-1 H-4 and H-6. The HMBC
spectrum showed correlations among the galloyl proton signals
[δH 7.03, 6.948, 6.92 (each 2H, s)] and signals of H-3 (δH 5.34)
of glucose-2 and H-1 (δH 5.44) and H-3 (δH 5.59) of glucose-1
through the respective carbonyl carbon peaks (δC 167.1, 164.6
and 166.8). The remaining hydroxy groups of the glucose cores
(at C-1 and C-2 of glucose-2 and C-1 of glucose-1) were also
O-acylated because of the downfield shifts of the corresponding
proton signals (Table 1). Consequently, three galloyl parts of
the two DHDG moieties should be placed at these hydroxy
groups. The HMBC correlations among two DHDG H-6′
signals [δH 7.05 and 7.02 (each 1H, s)] with the glucose-1 H-2
signal (δH 5.31) and the glucose-2 H-2 signal (δH 5.51) via two
equivalent carbonyl carbon peaks (δC 164.8, 2C) substantiated
attachment of the galloyl parts bearing an isolated hydrogen
(G1 and G3) of the DHDG moieties to C-2 of both glucose
cores. The orientation of the galloyl portion (G4) of the
DHDG moiety was confirmed as shown in Figure 2 on the
basis of the HMBC correlations of the meta-coupled proton
signals due to the DHDG H-2 (δH 6.37) and H-6 (δH 7.13)
with the carboxylic carbon peak (δC 168.9, DHDG C-7). In
spite of the overlapping of the glucose-1 H-1 signal (δH 5.24)
with the glucose-2 H-6 signal (δH 5.26) and that of the DHDG
H-6 signal (δH 6.95) with a galloyl (H-2/H-6) signal (δH 6.948)
4
(1H, s)], and a fully O-acylated β-glucose core in the C1
conformation (Table 1). The 13C NMR spectrum showed
carbon peaks (Tables 2 and 3) consistent in numbers and
chemical shifts with the carbons in the structural components
of 3. Locations of the HHDP and galloyl units were the same as
those of remurin A by close similarity of the respective glucose
signals in the H and 13C NMR spectra and also HMBC
1
correlations as shown in Figure 1. As mentioned for 2, the two-
proton singlet (δH 6.968) of H-2/H-6 of the isoDHDG moiety
was differentiated from those of the galloyl (H-2/H-6) signals
[δH 6.965 and 7.07 (each 2H, s)] on the basis of HMBC
connectivity of the former proton signal with the carbon peak
(δC 149.4) diagnostic of C-3/C-5 of the isoDHDG moiety.9,10
One of the galloyl units was confirmed at C-1 of the glucose
core by HMBC correlations between the galloyl-A proton
signal [δH 7.07 (2H, s)] and the glucose H-1 signal [δH 6.02
(1H, d)] through the carbonyl carbon peak (δC 165.2). It was
noteworthy that the H-6′ signal [δH 6.81 (1H, s)] of the
isoDHDG and the H-2/H-6 signal [δH 6.965(2H, s)] of the
galloyl-B moieties exhibited HMBC correlations with the
glucose H-2 [δH 5.63 (1H, dd)] and H-3 [δH 5.74 (1H, t)]
signals through two carbonyl carbons resonating at δC 166.8
(2C). This did not permit exact placement of these structural
moieties on the glucose core on the basis of spectroscopic
evidence. Alternatively, since isomerization of the isoDHDG
moiety into DHDG through Smiles rearrangement in a weak
alkaline solution (phosphate buffer, pH 7.4) was reported,9,10,16
this reaction was applied here, and the rearranged product from
3 was identified as remurin A (Figure 1), to secure structure 3
including the S configuration of the HHDP unit for nilotinin
M7.
1
in the H NMR spectrum, and also the lack of HMBC cross-
peaks for these proton signals, the galloyl part (G2) bearing
two meta-coupled proton signals (δH 6.31 and 6.95) of the
other DHDG moiety should necessarily be attached to C-1 of
glucose-1. A positive Cotton effect at 238 nm in the ECD
spectrum of 4 indicated an S configuration for the HHDP
unit.20 On the basis of the above spectroscopic findings,
nilotinin D10 was assigned the structure 4 (Figure 2).
Nilotinins M5−M7 (1−3) are further examples of the unique
hydrolyzable tannins of T. nilotica. Among these tannins, the
anomeric position, unless esterified with a galloyl unit, is always
esterified by the galloyl part bearing two hydrogens of the
DHDG or the isoDHDG moiety, while C-2 esterification of
glucose was always by the galloyl part bearing an isolated
hydrogen of the DHDG or the isoDHDG moiety. The upfield
shift of H-1 (δH 5.50) of the glucose core in 1 relative to that
(δH 6.02) of the glucose cores in 2 and 3 could be attributable
to the anisotropic effect of the DHDG moiety at C-2 of the
glucose core (Figure 1).9,13
Structural Elucidation of the Trimeric Ellagitannin.
Nilotinin T1 (5) was indicated to be a trimeric ellagitannin
possessing the molecular formula C123H84O78, as revealed by
the ion peak ([M +Na]+) at m/z 2831.24916 in the HRESIMS
1
spectrum and the spectroscopic data shown below. The H
NMR spectrum showed proton signals [δH 6.97 (2H, br s) and
6.76 (1H, br s)] diagnostic for an isoDHDG moiety. Two pairs
of mutually coupled broad signals [δH 6.81 and 6.44 (each 1H,
br s), and δH 7.04 and 6.48 (each 1H, br s)] and a pair of one-
proton singlets (δH 7.15 and 6.99) indicated the presence of
two DHDG moieties.9−11,16 Proton signals attributable to three
HHDP units [δH 6.51, 6.52, 6.53, 6.58, 6.60, and 6.62 (each 1H,
s)] and three galloyl units [δH 7.09, 6.972, and 6.968 (each 2H,
s)] were also evident in the aromatic region of the spectrum.
Structural Elucidation of the Dimeric Ellagitannin.
Nilotinin D10 (4) was isolated as an off-white, amorphous
powder. Its HRESIMS exhibited a prominent ion peak ([M +
Na]+) at m/z 1763.17637, corresponding to the molecular
formula C75H56O49. The 1H NMR spectrum exhibited 11
signals due to three galloyl units [δH 7.03, 6.948, 6.92 (each 2H,
s)], an HHDP unit [δH 6.58 and 6.51 (each 1H, s)], and two
DHDG moieties [δH 7.13, 6.95, 6.37, and 6.31 (each 1H, d, J =
1.8 Hz), DHDG (H-2 and H-6) × 2, δH 7.06, 7.02 (each 1H, s),
DHDG H-6′ × 2]. In the upper-field region of the spectrum,
two seven-spin aliphatic proton systems with large coupling
1
The H NMR spectrum also showed three seven-spin systems
assignable to protons of three glucose cores (Table 1). Among
these signals, three broad signals (δH 6.16, 5.76, and 5.24) were
assigned to anomeric protons of the glucose-1, 2, and 3
moieties on the basis of HSQC correlations with anomeric
carbon peaks [δC 93.3 (2C) and 93.8], respectively. Despite the
broadening of these anomeric proton signals, the 4C1
conformations of the glucose cores were implied from the
large coupling constants of the remaining proton signals (Table
1). Chemical shifts of the glucose proton signals (Table 1)
indicated acylation of all of the hydroxy groups on the
glucopyranose rings. The 13C NMR spectrum of 5 showed
carbon peaks (Tables 2 and 3) consistent with the presence of
these acyl moieties and the glucose cores. The locations of the
1
constants (Table 1) were distinguished by the H−1H COSY
experiment, indicating the presence of two β-glucopyranose
cores in the 4C1 conformation. The 13C NMR spectrum
exhibited carbon peaks (Tables 2 and 3) consistent with the
structural units of 4 (Figure 2). An HHDP unit was located at
C-4/C-6 of glucose-2, as indicated by the large chemical shift
F
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galloyl units were assigned to C-3 of each of the glucose cores,
as determined from the HMBC correlations of the galloyl
proton signals (δH 7.09, 6.972, and 6.968) with the H-3 signals
(δH 5.80, 5.70, and 5.56) of the three glucose cores through
common carbonyl carbon peaks [δC 167.4 and 167.0 (2C)].
Bridging of an HHDP unit at C-4/C-6 of each glucose core was
evidenced from the large chemical shift difference (ΔδH 1.42−
1.64) between the C-6 methylene proton signals (δH 5.26/3.84,
5.23/3.76, and 5.36/3.72) of the glucosyl moieties. The
location of the HHDP units was further confirmed by
HMBC correlations among the HHDP proton signals (δH
6.51, 6.52, 6.53, 6.59, 6.60, and 6.63) and signals of H-4 (δH
5.07, 5.12, and 5.19) and H-6 (δH 5.23, 5.26, and 5.36) of the
glucose cores through common ester carbonyl carbon peaks [δC
167.75, 167.87, 167.92, 168.3, and 168.5 (2C)]. Broadening of
the ester carbonyl carbon peaks of the DHDG (δC 163.4, 164.0,
164.1, and 164.4) and the isoDHDG (δC 164.9 and 166.3)
moieties suggested a macrocyclic structure for nilotinin T1,
since analogous features were also observed in the 13C NMR
spectra of macrocyclic-type tannins with related structures such
as nilotinin D9 (10) and hirtellins C (11) and F (12).11
Consequently, the two DHDG and the isoDHDG moieties in 5
were presumed to be bridged between C-1 of a glucose core
and the C-2 of another to form a macrocyclic structure. Linking
modes of the DHDG and isoDHDG moieties to the glucose
residues could not be established by HMBC due to the
broadening of their proton signals. The structure of nilotinin
T1 was subsequently formulated as 5 by its chemical
conversion into hirtellin T2 (6) (Figure 2), a known tannin
with the same molecular formula, through Smiles rearrange-
ment of the less stable isoDHDG into the more stable DHDG
unit under mild conditions. It is noteworthy that despite the
lack of HMBC cross-peaks among the proton signals of the
DHDG and isoDHDG moieties with their associated carbons,
HSQC correlations of these proton signals with the
corresponding carbons were clearly detected. Thus, assign-
ments of the carbon resonances of 5 (Tables 2 and 3) were
determined on the basis of HSQC data and comparison with
the corresponding signals of the monomeric nilotinins M5−M7
and with signals of the analogous macrocyclic dimers nilotinin
D9 (10) and hirtellins C (11) and F (12) (Figure 3).
Cytotoxic Activity of Tannins. Studies conducted with
tumor cell lines have shown that several monomeric−
oligomeric ellagitannins exhibit potent cytotoxicity against
carcinoma cell lines and lower cytotoxicity to normal
cells.21,22 In our preceding paper,11 we presented the effects
of some monomeric and dimeric tannins on oral squamous cell
carcinoma (HSC-2, HSC-3, and HSC-4) and promyelocytic
leukemia (HL-60) cell lines compared with their effect on
human oral normal cells (HGF, HPC, and HPLF). In our
efforts to find candidates for developing into efficient antitumor
agents, five macrocyclic-type tannins, nilotinin T1 (5, Figure 2),
tamarixinin B (8), nilotinin D9 (10), and hirtellins C (11) and
F (12) (Figure 3), were also investigated against these cell lines
following the procedures shown in the Experimental Section.
Compounds examined in the present study showed
significant cytotoxic effects with medium to high tumor-
specificity (TS) indices against all of the tested tumor cell lines,
as shown in Table 4. Although the effects of these macrocyclic-
type tannins (5, 8, and 10−12) (TS = 2.2−2.9) are relatively
lower than the linear and hellinoyl-type tannins isolated from
the same plant (TS = 2.1−7.1),11 their potencies are still higher
than synthetic ketones (TS = 1.2−1.8), natural potent cytotoxic
Figure 3. Structures of the known tannins 7−12.
polyphenols (flavones, flavonols, and prenylated flavonoids)
(TS = 1.2−2.3), terpenes (triterpene aglycones and triterpene
glycosides) (TS = 1.2−1.6),23 and coumarins (TS = 2.4) and
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Table 4. Cytotoxicity of Tannins 5, 8, and 10−12 against Human Normal and Tumor Cells
a
CC50 (μM)
human oral squamous cell carcinoma cells
promyelocytic leukemia cells
HL-60
human normal oral cells
bc
,
tannin
MW
HSC-2
HSC-3
HSC-4
HGF
HPC
HPLF
TS
nilotinin T1 (5)
tamarixinin B (8)
nilotinin D9 (10)
hirtellin C (11)
hirtellin F (12)
2808
1872
1570
1872
1570
22.3 2.0
23.0 1.8
18.5 0.30
19.9 1.2
20.2 0.75
34.2 1.1
47.8 1.9
21.9 0.98
24.3 2.3
20.8 0.90
42.9 0.31
43.0 0.78
24.5 4.6
28.7 2.6
26.6 1.6
30.5 0.23
55.9 15.7
70.8 17.0
54.2 8.3
96.6 2.5
93.6 3.2
>100
90.7 2.1
88.3 1.5
81.0 1.7
84.3 2.1
80.3 2.1
>100
>100
>100
>100
>100
2.9
>2.2
>2.8
>2.9
>2.9
95.0 1.6
62.0 0.48
>100
a
b
c
Each value represents the mean of at least three independent experiments. TS, tumor specificity. TS = {[CC50(HGF) + CC50(HPC) +
CC50(HPLF)]/[CC50(HSC-2) + CC50(HSC-3)] + CC50(HSC-4) + CC50(HL-60) ]} × (4/3).
Japan. The normal cells were prepared from periodontal tissues,
comparable with those of 5-fluorouracil (5-FU) (TS > 3.35)
and melphalan (TS = 4.09).24
according to the guidelines of the intramural board of Meikai
University Ethics Committee (no. A0808) after obtaining informed
consent from the patients. Because HGF, HPC, and HPLF cells have a
limited lifespan due to in vitro senescence, these cells were used at a
population doubling level of 5−8.
Plant Material. Leaves of T. nilotica were collected at Al-Wadi Al-
Assiuty, 20 km northeast of Assiut, Egypt, in October 2006, and
identified by Prof. Mo’men Mostafa Zareh, Department of Botany,
Faculty of Science, Assiut University. A voucher specimen (No. 1024)
is deposited in the same department.
The ellagitannins, hirtellins A and B and tamarixinin A, from
Tamarix plants exhibited strong antitumor activity upon single
intraperitoneal injection to mice four days before intra-
peritoneal inoculation of the sarcoma-180 cells,25 which agrees
with our results of the cytotoxic activity study of the same and
other tannins11 and with the results of the cytotoxic
investigation shown in this paper. These results support the
possibility of developing antitumor ellagitannins with low
toxicity from tamaricaceous plants.
T. nilotica has been known since pharaonic times and has
been mentioned in medical papyri to relieve headache and as an
antipyretic, an anti-inflammatory, and an aphrodisiac.6 In our
preceding paper on T. nilotica we reported several ellagitannins
with strong cytotoxic effects.11 We report here the isolation and
structural elucidation of five new monomeric−trimeric
ellagitannins, together with four known ones, from T. nilotica.
Cytotoxic effects of macrocyclic-type tannins are also discussed.
The mode of action of these compounds remains to be
established.
Extraction and Isolation. Air-dried, powdered leaves (1 kg) were
homogenized in H2O/acetone (30:70, v/v, 22 L) at room temper-
ature. The filtered homogenate was concentrated in vacuo to 1.5 L at
temperatures not exceeding 40 °C. The concentrated homogenate was
applied to a Diaion HP-20 (5 i.d. × 50 cm) column and eluted with
H2O, MeOH/H2O (40:60, v/v), MeOH, and acetone, successively, to
give the corresponding H2O (116 g), MeOH/H2O (40:60) (52 g),
MeOH (12.4 g), and acetone (0.629 g) fractions, respectively. The
respective fractions were fractionated by monitoring normal- and
reversed-phase HPLC. The MeOH/H2O (40:60, v/v) fraction (52 g)
was divided into two equal portions (26 g each). Each portion was
separately subjected to a Toyopearl HW-40 (coarse) column (2.2 i.d.
× 65 cm) and eluted with EtOH/H2O (50:50 → 70:30, v/v), EtOH/
H2O (70:30, v/v)/acetone/H2O (70:30, v/v) (90:10 → 80:20 →
70:30 → 50:50, v/v), and acetone/H2O (70:30, v/v), successively,
collecting 500 mL fractions. The corresponding fractions from both
Toyopearl columns were combined as seven Toyopearl (T1) fractions
(T1−7).
The T1 fraction 3 (6.12 g), eluted with EtOH/H2O (70:30, v/v)/
acetone/H2O (70:30, v/v) (90:10, v/v), was subjected to the same
Toyopearl (coarse) column and eluted with EtOH/H2O (70:30, v/v),
EtOH/H2O (70:30, v/v)/acetone/H2O (70:30, v/v) (90:10), and
acetone/H2O (70:30, v/v), successively, collecting 900 drop fractions,
to give 700 Toyopearl (T2) fractions. The T2 fractions 158−183 (322
mg), eluted with EtOH/H2O (70:30, v/v), were subjected to an MCI-
gel CHP-20P column (1.1 i.d. × 35 cm) with H2O and H2O/MeOH
(85:15 → 80:20 → 75:25 → 70:30 and 0:100, v/v). The H2O−MeOH
(80:20, v/v) eluate (31 mg) was rechromatographed over an MCI-gel
CHP-20P column (1.1 i.d. × 21 cm) with H2O and H2O/MeOH
(85:15 → 80:20 and 0:100, v/v). A preparative HPLC purification
with solvent I of the late eluate with H2O/MeOH (85:15, v/v) (7 mg)
yielded a pure sample of nilotinin M5 (1) (2.1 mg). Rechromatog-
raphy of the H2O/MeOH (80:20, v/v) eluate (63 mg) over the same
MCI-gel CHP-20P column and by the same elution pattern afforded a
H2O/MeOH (75:25, v/v) eluate (19.8 mg), which upon preparative
HPLC purification with solvent II gave nilotinin D10 (4) (4.5 mg).
The T2 fractions 184−210 (297 mg), eluted with EtOH/H2O (70:30,
v/v), were subjected to an MCI-gel CHP-20P column (1.1 i.d. × 35
cm) with H2O and H2O/MeOH (85:15 → 80:20 → 75:25 → 70:30
and 0:100, v/v). The early eluate with H2O/MeOH (80:20, v/v) (33.7
mg) was rechromatographed over an MCI-gel CHP-20P column (1.1
i.d. × 21 cm) isocratically with H2O/MeOH (85:15, v/v), followed by
preparative HPLC purification of the eluate (21 mg) with solvent II,
affording an additional pure sample (8.2 mg) of nilotinin M5 (1). The
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
recorded on a JASCO DIP-1000 digital polarimeter. UV spectra
were measured on a JASCO V-530 spectrophotometer, and ECD
spectra on a JASCO J-720W spectrophotometer. ESIMS were
performed using a Micromass Auto Spec OA-Tof spectrophotometer
in positive ion mode. The solvent used was 50% MeOH + 0.1%
NH4OAc, and the flow rate was set at 20 μL/min. HRESIMS was
performed using a micrOTOF-Q (Bruker Daltonics, USA) mass
spectrometer. The 1H and 13C NMR spectra were recorded on a
Varian INOVA AS 600 instrument (600 MHz for 1H and 151 MHz for
13C NMR). Chemical shifts are given in δ (ppm) values relative to
those of the solvent signal [acetone-d6 (δH 2.04; δC 29.8)] on the
tetramethylsilane scale. The standard pulse sequences programmed
into the instrument (Varian INOVA AS600) were used for each 2D
measurement. The JCH value was set at 5 Hz in the HMBC spectra.
Normal-phase HPLC was conducted on a YMC-Pack SIL A-003
(YMC, Kyoto, Japan) column (4.6 i.d. × 250 mm) developed with n-
hexane/MeOH/THF/formic acid (47:39:13:1) containing oxalic acid
(450 mg/L) (flow rate, 1.5 mL/min; 280 nm UV detection) at room
temperature. Analytical reversed-phase HPLC was performed on a
YMC-Pack ODS-A A-303 column (4.6 i.d. × 250 mm) eluted with
0.01 M H3PO4/0.01 M KH2PO4/MeOH (2:2:1) (flow rate, 1 mL/
min; 280 nm UV detection) at 40 °C. Preparative reversed-phase
HPLC was performed at 40 °C on a YMC-Pack ODS-A A-324 column
(10 i.d. × 300 mm) using 0.01 M H3PO4/0.01 M KH2PO4/MeOH
[either 43:43:16 (solvent I), 40:40:20 (solvent II), 41:41:18 (solvent
III), 41.5:41.5:17 (solvent IV), or 37.5:37.5:25 (solvent V), v/v], at a
flow rate of 2 mL/min with detection at 280 nm UV. The tumor cell
lines were obtained from Riken Bioresource Center, Tsukuba, Ibaraki,
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late eluate (30 mg) with H2O/MeOH (80:20, v/v) was also
rechromatographed over an MCI-gel CHP-20P column (1.1 i.d. ×
21 cm) with H2O and H2O/MeOH (90:10 → 85:15 → 80:20 →
75:25 and 0:100, v/v), and preparative HPLC purification of the H2O/
MeOH (75:25, v/v) eluate (55 mg) with solvent II yielded nilotinin
M7 (3) (9.0 mg). The T2 fractions 211−264 (555 mg), eluted with
EtOH/H2O (70:30, v/v), were subjected to an MCI-gel CHP-20P
column (1.1 i.d. × 37 cm) with H2O and H2O/MeOH (90:10 →
85:15 → 80:20 → 75:25 →70:30 and 0:100, v/v). The late eluate with
H2O/MeOH (80:20, v/v) (43 mg) with preparative HPLC
purification with solvent II yielded nilotinin M6 (2) (7.2 mg). The
T2 fractions 265−318 (535 mg), early eluted with EtOH/H2O (70:30,
v/v)/acetone/H2O (70:30, v/v) (90:10, v/v), were subjected to an
MCI-gel CHP-20P column (1.1 i.d. × 37 cm) with H2O and H2O/
MeOH (90:10 → 85:15 → 80:20 → 75:25 →70:30 and 0:100, v/v),
and the late eluate with H2O/MeOH (75:25, v/v) (59 mg) was
rechromatographed over the same column with H2O/MeOH (80:20
→ 75:25 → 70:30 and 0:100, v/v). The eluate (42.0 mg) with H2O/
MeOH (75:25 and 70:30, v/v) was subjected to preparative HPLC
purification with solvent III to yield nilotinin D9 (10) (5.4 mg) and
hirtellin F (6.3 mg), respectively.
A part (3.6 g) of the T1 fraction 5 (4.6 g), eluted with EtOH−H2O
(70:30, v/v)/acetone/H2O (70:30, v/v) (70:30, v/v), was rechroma-
tographed on a Toyopearl HW-40 (coarse) column (2.2 i.d. × 45 cm)
with EtOH/H2O (70:30, v/v)/acetone/H2O (70:30, v/v) (90:10 →
80:20 → 70:30, v/v), successively. The eluate (1.4g) with EtOH/H2O
(70:30, v/v)/acetone/H2O (70:30, v/v) (80:20) was subjected to an
MCI-gel CHP-20P column (1.1 i.d. × 36 cm) with H2O and H2O/
MeOH (95:5 → 90:10 → 85:15 → 80:20 → 75:25 → 70:30 → 60:40
and 0:100, v/v), yielding 600 MCI fractions (MCIfrs, 600 drops each).
The MCIfrs 11−60 (159 mg) and the MCIfrs 61−195 (235 mg),
eluted with H2O/MeOH (85:15, v/v), were separately rechromato-
graphed over a Sephadex LH-20 column (2.2 i.d. × 21 cm) with
EtOH/H2O (70:30, v/v), EtOH/H2O (70:30, v/v)/acetone/H2O
(70:30, v/v) (90:10, v/v), and acetone/H2O (70:30, v/v), and the
crude tannins (69 mg) in the eluate with EtOH/H2O (70:30, v/v)/
acetone/H2O (70:30, v/v) (90:10, v/v) from these Sephadex columns
yielded hirtellin T2 (6) (15.2 mg) upon preparative HPLC purification
with solvents I and II, respectively. The MCIfrs 196−300 (268 mg),
eluted with H2O/MeOH (80:20, v/v), were subjected to an MCI-gel
CHP-20P column (1.1 i.d. × 37 cm) with H2O and H2O/MeOH
(95:5 → 90:10 → 85:15 → 80:20 → 75:25 → 70:30 → 60:40 and
0:100, v/v), and the early eluate (57 mg) with H2O/MeOH (70:30, v/
v) was purified by preparative HPLC with solvent IV to afford hirtellin
T2 (6) (7.55 mg) and nilotinin T1 (5) (16.6 mg). The MCIfrs 301−
471 (496 mg), eluted with H2O/MeOH (75:25, v/v), were subjected
to a Sephadex LH-20 column (1.1 i.d. × 37 cm) with EtOH/H2O
(70:30, v/v), EtOH−H2O (70:30, v/v)/acetone/H2O (70:30, v/v)
(90:10 → 80:20, v/v), and acetone/H2O (70:30, v/v), and the crude
tannins (32 mg) in the EtOH/H2O (70:30, v/v) eluate yielded
hirtellin C (11) (7 mg) upon preparative HPLC purification with
solvent II. The early eluate with EtOH/H2O (70:30, v/v)/acetone/
H2O (70:30, v/v) (90:10, v/v) (88.7 mg) from the Sephadex column
was dissolved in MeOH. Tamarixinin B (8) (29 mg) was obtained as
an MeOH-insoluble fraction, whereas the MeOH-soluble fraction was
purified by preparative HPLC with solvent IV and yielded additional
crops of tamarixinin B (8) (10 mg) and nilotinin T1 (5) (9.3 mg). The
MCIfrs 523−572 (72 mg), eluted with H2O/MeOH (60:40, v/v),
were rechromatographed over an MCI-gel CHP-20P column (1.1 i.d.
× 21 cm) with H2O and H2O/MeOH (70:30 → 60:40 and 0:100, v/
v). Preparative HPLC purification of the eluate with H2O/MeOH
(60:40, v/v) (17 mg) with solvent V afforded tamarixinin C (9) (5.7
mg).
6.46 (1H, d, J = 1.8 Hz, DHDG H-2), and glucose protons (Table 1);
13C NMR assignments, see Tables 2 and 3; FABMS m/z 1297 [M +
Na]+; HRESIMS m/z 1297.10358 [M + Na]+ (calcd for C55H38O36
Na, 1297.10350).
Nilotinin M6 (2): off-white, amorphous powder; [α]18D +20.5 (c 1.0,
MeOH); UV (MeOH) λmax (log ε) 218.5 (5.43), 264 (5.04); CD
(MeOH) [θ] (nm) +1.5 × 105 (236), −7.6 × 104 (262), +4.8 × 104
1
(286); H NMR (acetone-d6/D2O, 9:1) δH 7.06, 6.99 [each 2H, s,
isoDHDG (H-2/H-6) × 2], 6.97 [2H, s, galloyl (H-2/H-6)], 6.87,
6.84 [each 1H, s, isoDHDG (H-6′) × 2], 6.60 (1H, s, HHDP H-3),
6.49 (1H, s, HHDP H-3′), and glucose protons (Table 1); 13C NMR
assignments, see Tables 2 and 3; HRESIMS m/z 1297.10145 [M +
Na]+ (calcd for C55H38O36Na, 1297.10351).
Nilotinin M7 (3): off-white, amorphous powder; [α]25D +21.4 (c 1.0,
MeOH); UV (MeOH) λmax (log ε) 217.5 (5.11), 274 (4.74); CD
(MeOH) [θ] (nm) +1.1 × 105 (237), −3.3 × 104 (262), +2.3 × 104
1
(283); H NMR (acetone-d6/D2O, 9:1) δH 7.07 [2H, s, galloyl-A (H-
2/H-6)], 6.968, [2H, s, isoDHDG (H-2/H-6)], 6.965 [2H, s, galloyl-B
(H-2/H-6)], 6.81 (1H, s, isoDHDG H-6′), 6.60 (1H, s, HHDP H-3),
6.48 (1H, s, HHDP H-3′), and glucose protons (Table 1); 13C NMR
assignments, see Tables 2 and 3; FABMS m/z 1229 [M + Na]+;
HRESIMS m/z 1129.09566 [M + Na]+ (calcd for C48H34O31Na,
1129.09763).
Nilotinin D10 (4): off-white, amorphous powder; [α]27 +53.1 (c
D
1.0, MeOH); UV (MeOH) λmax (log ε) 218.5 (5.43), 277 (5.08); CD
(MeOH) [θ] (nm) +0.9 × 105 (238), −1.6 × 104 (266), +2.6 × 104
(291); 1H NMR (acetone-d6/D2O, 9:1) δH 7.13, 6.95 [each 1H, d, J =
1.8 Hz, (DHDG H-6) × 2], 7.06, 7.02 [each 1H, s, (DHDG H-6′) ×
2], 7.03, 6.948, 6.92 [each 2H, s, galloyl (H-2/H-6) × 3], 6.58 (1H, s,
HHDP H-3), 6.51 (1H, s, HHDP H-3′), 6.37, 6.31 [each 1H, d, J =
1.8 Hz, (DHDG H-2) × 2], and glucose protons (Table 1); 13C NMR
assignments, see Tables 2 and 3; FABMS m/z 1763 [M + Na]+;
HRESIMS m/z 1763.17637 [M + Na]+ (calcd for C75H56O49 Na,
1763.17824).
Nilotinin T1 (5): off-white, amorphous powder; [α]31 −5.8 (c 1.0,
D
MeOH); UV (MeOH) λmax (log ε) 218 (5.63), 274.5 (5.28); CD
(MeOH) [θ] (nm) +4.2 × 105 (235), −1.9 × 105 (262), +1.2 × 105
1
(286); H NMR (acetone-d6/D2O, 9:1) δH 7.15, 6.99 [each 1H, s,
(DHDG H-6′) × 2], 7.04, 6.81 [each 1H, br s, (DHDG H-6) × 2],
6.48 and 6.44 [each 1H, br s, (DHDG H-2) × 2], 6.97 (2H, br s,
isoDHDG H-2/H-6), 6.76 (1H, br s, isoDHDG H-6′), 7.09, 6.972,
and 6.968 [each 2H, s, galloyl (H-2/H-6) × 3], 6.62, 6.60, and 6.58
[each 1H, s, HHDP (H-3′) × 3], 6.53, 6.52, 6.51 [each 1H, s, HHDP
(H-3) × 3], and glucose protons (Table 1); 13C NMR assignments,
see Tables 2 and 3; HRESIMS m/z 2831.24916 [M + Na]+ (calcd for
C123H84O78Na, 2831.24989).
Chemical Conversion of Nilotinin M7 (3) into Remurin A. A
solution of 3 (2 mg) in 200 μL of phosphate buffer (pH 7.4)
containing 1 drop of acetone was warmed at 37 °C for 1 h. The
reaction mixture was acidified with 1 N HCl and poured into a Sep-
Pak C18 cartridge. Inorganic solutes were washed out from the
cartridge with H2O, while adsorbed tannin was recovered (1.9 mg)
with MeOH. Conversion of 3 into remurin A was monitored by co-
chromatographic analyses with standard samples isolated from the
plant on normal-phase (tR 6.48 min, remurin A and 3) and reversed-
phase HPLC (tR 7.02 min for remurin A and tR 9.70 min for 3) and
1
was confirmed by the H NMR data comparison.
Chemical Conversion of Nilotinin T1 (5) into Hirtellin T2 (6). A
solution of 5 (2 mg) in 200 μL of phosphate buffer (pH 7.4)
containing 1 drop of acetone was incubated at 37 °C for 2 h. The
experiment was completed as mentioned above. A conversion of 5 into
6 was detected by co-chromatographic analyses with standard samples
isolated from the plant on normal-phase (tR 15.56 min, 5 and 6) and
reversed-phase HPLC (tR 5.76 min for 1 and tR 5.28 min for 6) and
Nilotinin M5 (1): off-white, amorphous powder; [α]26D +23.6 (c 1.0,
MeOH); UV (MeOH) λmax (log ε) 218.5 (5.05), 270 (4.7); CD
(MeOH) [θ] (nm) [θ] +0.75 × 105 (236), −1.8 × 104 (265), +1.3 ×
104 (285); 1H NMR (acetone-d6/D2O, 9:1, 600 MHz) δH 7.21 (1H, d,
J = 1.8 Hz, DHDG H-6), 7.06 (1H, s, DHDG H-6′), 6.96 [2H, s,
isoDHDG (H-2/H-6)], 6.93 [2H, s, galloyl (H-2/H-6)], 6.89 (1H, s,
isoDHDG H-6′), 6.58 (1H, s, HHDP H-3), 6.51 (1H, s, HHDP H-3′),
1
the H NMR data comparison.
Cytotoxic Activity Assay. HL-60 cells (Riken, Tsukuba, Japan)
were cultured at 37 °C in RPMI1640 supplemented with 10% heat-
inactivated fetal bovine serum (FBS) (Sigma Chemical Corp., St.
Louis, MO, USA), under a humidified 5% CO2 atmosphere. Human
oral squamous cell carcinoma cell lines (HSC-2, HSC-3, HSC-4)
I
dx.doi.org/10.1021/np4001625 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(kindly provided by Professor Nagumo, Showa University, Japan)
were cultured in Dulbecco’s modified Eagle medium (Gibco BRL,
Grand Island, NY, USA) supplemented with 10% heat-inactivated
FBS. Normal human oral cells, HGF, HPC, and HPLF were prepared
from periodontal tissues, as previously reported,24 and used at 8−15
population doubling levels. The cells (other than HL-60) were
inoculated at 5 × 103 cells/well in 96-microwell plates (Becton
Dickinson, Franklin Lakes, NJ, USA) unless otherwise stated. After 48
h, the medium was removed by suction with an aspirator and replaced
with 0.1 mL of fresh medium containing different test compound
concentrations. Each test compound was dissolved in DMSO at a
concentration of 20 mM. The first well contained 100 μM of the test
compound and was sequentially diluted 2-fold, with three replicate
wells for each concentration. The cells were incubated for an
additional 48 h, and the relative viable cell number was determined
by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) method. In brief, the cells were washed with phosphate-
buffered saline without calcium and magnesium, which was replaced
with fresh culture medium containing 0.2 mg/mL MTT, and the cells
were incubated for another 4 h. The cells were lysed with 0.1 mL of
DMSO, and the absorbance of the cell lysate at 540 nm (A540) was
determined using a microplate reader (Biochromatic Labsystem,
Helsinki, Finland).26 The A540 of the control cells was usually in the
range from 0.40 to 0.90. The HL-60 cells were inoculated at 3.0 × 104
cells/0.1 mL in 96-microwell plates, and different concentrations of
test compounds were added. After a 48 h incubation, the viable cell
number was determined with a hemocytometer under a light
microscope after trypan blue staining. The CC50 was determined
from the dose−response curve.
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra for 1−5 are available free of charge
(22) (a) Sakagami, H.; Jiang, Y.; Kusama, K.; Atsumi, T.; Ueha, T.;
Toguchi, M.; Iwakura, I.; Satoh, K.; Ito, H.; Hatano, T.; Yoshida, T.
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in Food; Shahidi, F., Ed.; ACS Series 662, American Chemical Society:
Washington, DC, 1997; Chapter 14, pp 245−259. (c) Zunino, F.;
Capranico, G. In Cancer Therapeutics: Experimental and Clinical Agents;
Teicher, B. A., Ed.; Cancer Drug Discovery and Development;
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C. C.; Chen, L. G.; Yang, L. L. Cancer Lett. 1999, 140, 195−200.
(e) Yang, L. L.; Lee, C. Y.; Yen, K. Y. Cancer Lett. 2000, 157, 65−75.
(23) Sakagami, H.; Kobayashi, M.; Chien, C.-H.; Kanegae, H.;
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via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: +81-86-251-7936. Fax: +81-86-251-7926. E-mail:
hatano@pharm.okayama-u.ac.jp.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank the SC-NMR Laboratory of Okayama
University for experiments using the NMR instrument. This
work was supported in part by a Grant-in-Aid for Scientific
Research from the Japan Society for the Promotion of Science.
(24) Koh, T.; Machino, M.; Murakami, Y.; Umemura, N.; Sakagami,
H. In Vivo 2013, 27, 85−96.
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