New isolinariins C, D and E, flavonoid glycosides from Linaria japonica

Article abstract of DOI:10.1248/cpb.c16-00073

Three new flavonoid glycosides named isolinariins C, D and E (1-3), two known flavonoid glycosides (4, 5) and three known flavonoids (6-8) were isolated from the whole plant of Linaria japonica. The structures of these compounds were determined mainly by

Full text of DOI:10.1248/cpb.c16-00073

Vol. 64, No. 5
Chem. Pharm. Bull. 64, 517–521 (2016)
517
Note
New Isolinariins C, D and E, Flavonoid Glycosides from Linaria japonica
a,b
a
a
b
c
Retno Widyowati, Sachiko Sugimoto, Yoshi Yamano, Sukardiman, Hideaki Otsuka, and
,
a
Katsuyoshi Matsunami*
a
b
Graduate School of Biomedical & Health Sciences, Hiroshima University; Hiroshima 734–8553, Japan: Faculty of
c
Pharmacy, Airlangga University; Surabaya 60286, Indonesia: and Graduate School of Pharmacy, Yasuda Women’s
University; Hiroshima 731–0153, Japan.
Received January 23, 2016; accepted February 17, 2016
Three new flavonoid glycosides named isolinariins C, D and E (1–3), two known flavonoid glycosides (4,
5
) and three known flavonoids (6–8) were isolated from the whole plant of Linaria japonica. The structures of
these compounds were determined mainly by spectroscopic analyses. The bioactivities of these isolated com-
pounds were evaluated for their inhibitory activities against human cell line A549, collagenase, and advanced
glycation end product (AGE) formation. Among the isolated compounds, isolinariins C, D and E (1, 2 and 3)
showed inhibition toward AGE formation (IC50 values of 34.8, 35.0 and 19.5µM, respectively). And linariin
(4), pectolinarin (5) and luteolin (8) were found to be active against collagenase with IC50 values of 79.4, 78.6
and 40.5µM, respectively, without significant cytotoxicity at these concentrations.
Key words Linaria japonica; cytotoxicity; collagenase; advanced glycation end product; flavonoid glycoside
Linaria japonica MIQ. (Scrophulariaceae) is a perennial Results and Discussion
herb with elliptic and fleshy leaves. The whole plant extract is
The mixture of hexane and ethyl acetate layers of a MeOH
used as a Japanese folk medicine due to its diuretic, purgative extract of Linaria japonica was fractionated by various types
1)
and laxative properties. On our reinvestigation of the same of chromatography to afford eight compounds (1–8).
plant, collected in sandy seashore areas of Tottori Prefecture, Isolinariin C (1), [α] −4.8, was obtained as a pale yel-
D
flavonoids, phenylethanoids, iridoids and monoterpene glu- low powder with the molecular formula C H O as deter-
3
3
38 17
2
)
+
cosides have been isolated so far. On further investigation mined by HR-ESI-MS at m/z 729.1998 [M+Na] (calcd for
of the non-polar fraction, i.e. a mixture of hexane and ethyl 729.2001). The IR spectrum indicated the presence of hydroxy
−1
−1
acetate layers of the same plant, five new diterpenoids were (3437 cm ), ester carbonyl (1746cm ), aromatic ring (1509
3)
−1
−1
isolated. The present study on the same non-polar fraction and 1460cm ) and ether (1182 and 1054cm ) functions.
1
has demonstrated the presence of three new flavonoid glyco-
The H-NMR spectrum (Table 1) displayed signals due to
sides (1–3) along with linariin (4), pectolinarin (5), pectoli- a methyl of a rhamnose at δH 1.17 (d, J=6.2Hz), two singlet
4)
narigenin (6), apigenin (7) and luteolin (8) (Fig. 1) through methyl signals of acetyl groups at δ 1.75 and 1.93, two oxy-
H
the isolation by various chromatographic techniques such genated methylene protons of glucose at δH 3.76 (m) and 4.05
as silica gel, octadecylsilyl (ODS) and HPLC. The chemical (brd, J=9.9Hz), a singlet signal of two methoxy groups at δH
structures of these compounds were determined mainly by 3.89 (6H, s), two anomeric protons at δ 4.72 (brs) and 5.19 (d,
H
spectrometric analyses such as UV, IR, high resolution elec- J=7.2Hz), two aromatic protons at δ 6.68 (s) and 6.89 (s), and
H
trospray ionization (HR-ESI)-MS, one and two dimensional (1 an AAʹBBʹ type coupling system at δ 7.08 (2H, d, J=8.1Hz)
H
and 2D)-NMR. We also report here the inhibitory activity for and 7.95 (2H, d, J=8.1 Hz).
13
collagenase and advanced glycation end products (AGEs) for-
The C-NMR spectrum (Table 1) of 1 showed 31 carbon
mation of the isolated compounds along with the cytotoxicity resonances that were classified by chemical shift values and
against human cell line.
heteronuclear single quantum coherence (HSQC) spectrum
as; two acetyl groups (δC 20.6, 20.8, 171.5 and 172.1), two
Fig. 1. Structures of Compounds 1–8
*
To whom correspondence should be addressed. e-mail: matunami@hiroshima-u.ac.jp
©
2016 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
Vol. 64, No. 5 (2016)
1
13
Table 1. H- and C-NMR Spectroscopic Data for Compounds 1–3
1
2
3
Position
δC
δH Multi (J in Hz)
δC
δH Multi (J in Hz)
δC
δH Multi (J in Hz)
2
3
4
5
6
7
8
9
0
1
2
3
4
6
4
1
2
3
4
5
6
166.9
104.5
184.6
154.4
134.0
158.0
95.5
—
6.68s
—
166.5
104.5
184.5
154.36
134.6
157.8
95.8
—
6.68s
166.9
104.6
184.6
154.4
134.7
157.8
96.1
—
6.70s
—
—
—
—
—
—
—
—
—
—
—
6.89s
—
6.90s
6.95s
154.6
108.0
124.7
129.8
115.8
164.5
61.7
154.41
107.8
124.6
129.6
115.9
164.6
61.7
—
154.5
107.9
124.6
129.7
115.9
164.7
61.7
—
1
—
—
—
′
—
—
—
′, 6′
′, 5′
′
7.95d (8.1)
7.08d (8.1)
—
7.94d (8.1)
7.07d (8.1)
—
7.97d (8.1)
7.09d (8.1)
—
-OCH3
3.89s
3.89s
5.19d (7.2)
3.58t (8.3)
3.52t (8.9)
3.43m
3.73m
3.76m
3.90s
3.90s
′-OCH3
56.2
56.3
3.89s
56.3
3.88s
″
″
″
″
″
″
101.6
74.8
101.6
74.9
5.20d (7.2)
3.59t (8.3)
3.53m
3.45m
3.73m
3.76m
4.03brd (9.9)
4.74brs
3.98m
5.02dd (9.9, 3.3)
4.98d (9.9)
3.84m
0.96d (6.2)
—
101.8
75.0
5.19d (7.2)
3.58t (8.3)
3.52t (8.9)
3.48m
77.9
78.0
77.9
71.6
71.3
70.9
77.3
77.2
76.9
3.69m
67.5
67.1
66.8
3.75m
4
.05brd (9.9)
3.99m
1
2
3
4
5
6
2
‴
‴
‴
‴
‴
‴
99.3
71.1
73.3
71.3
70.0
18.0
20.6
4.72brs
101.6
70.0
73.4
72.6
67.7
17.7
—
98.8
73.9
68.6
75.5
67.7
17.7
20.8
172.1
—
4.73brs
5.06dd (3.3, 1.6)
3.97dd (9.9, 3.3)
4.78d (9.9)
3.77m
5.15dd (3.3, 1.6)
5.01dd (9.9, 3.3)
3.42d (9.9)
3.77m
1.17d (6.2)
1.93s
0.92d (6.2)
2.03s
‴-OAc
1
71.5
20.8
72.1
—
3
‴-OAc
‴-OAc
1.75s
—
20.8
171.9
20.9
1.82s
1.96s
—
1
4
21.1
2.03s
1
71.9
172.4
Recorded at 600MHz in CD OD. Chemicalshifts (δ) are expressed in ppm. m: multiplet or overlappedsignals.
3
sugars (rhamnose: δ 18.0, 70.0, 71.1, 71.3, 73.3 and 99.3, and 5.15) and H-3‴ (δH 5.01) to the carbonyl carbon signals at
C
glucose: 67.5, 71.6, 74.8, 77.3, 77.9 and 101.6), two methoxy δC 171.5 (2‴-OAc) and 172.1 (3‴-OAc), respectively (Fig. 2).
2
carbons (δ 56.2 and 61.7), four sp methine carbons (δ 95.5, Acid hydrolysis of 1 with 1N HCl liberated D-glucose and L-
1
(
C
C
2
7)
04.5, 115.8 (2×C), 129.8 (2×C)), eight sp quaternary carbons rhamnose by HPLC analysis with optical rotation detector.
δC 108.0, 124.7, 134.0, 154.4, 154.6, 158.0, 164.5, 166.9) and a The coupling constant (J=7.2Hz) of H-1″ indicated β linkage
carbonyl carbon at δ 184.6. The heteronuclear multiple bond for glucose and the chemical shift values of rhamnose moiety
connectivity (HMBC) spectrum exhibited correlations be- were indicative of α-L-rhamnopyranose. Based on the NMR
tween δ 6.68 (H-3) and δ 124.7 (C-1′), 166.9 (C-2) and 184.6 data and acid hydrolysis, the structure of 1 was determined to
C-4), and between δH 6.89 (H-8) and δC 134.0 (C-6), 158.0 be pectolinarigenin-7-O-(2,3-diacetyl-α-L-rhamnopyranosyl)-
C-7), 154.6 (C-9) and 108.0 (C-10) revealed the presence of a (1→6)-β-D-glucopyranoside.
flavone skeleton (Fig. 2). Furthermore, the HMBC correlations Isolinariin D (2), [α] −8.6, was obtained as a pale yel-
of two methoxy groups at δ 3.89 (6H, s) with δ 134.0 (C-6) low powder and its molecular formula was determined to
C
5
)
H
C
(
(
D
H
C
and 164.5 (C-4′), and the correlations of two anomeric protons be C H O from its positive-ion mode HR-ESI-MS data
3
3
38 17
+
1
at δH 4.72 (H-1‴) and 5.19 (H-1″) with 67.5 (C-6″) and 158.0 at m/z 729.1997 [M+Na] (calcd for 729.2001). The H- and
13
(
C-7), respectively, confirmed the linkage of these functional
C-NMR spectra (Table 1) of 2 were very similar to those of
1
13
groups (Fig. 2). The H- and C-NMR spectroscopic data of 1 except for the signals of H-2‴ and H-4‴ of rhamnose, which
5,6)
1
were closely similar to those of linariin
except for the indicated that 2 was a positional isomer of 1. The positions of
chemical shift values of position-2‴ and 3‴ of the rhamnose the two acetyl groups were deduced to be at C-3‴ and C-4‴ by
moiety. The lower field shifted protons at δ 5.15 and 5.01 of 1 analysis of the HMBC data showing correlations of H-3‴ and
H
suggested the presence of the acetyl groups on C-2‴ and C-3‴, H-4‴ to two carbons at δ 171.9 (Fig. 2). The acid hydrolysis
C
which was confirmed by the HMBC correlations of H-2‴ (δH of 2 also yielded the aglycone (pectolinarigenin), D-glucose

Vol. 64, No. 5 (2016)
Chem. Pharm. Bull.
519
Fig. 2. HMBC and COSY Correlations of 1–3
Table 2. Bioactivities of Isolated Compounds (1–8)
rhamnose may contribute to the inhibitory activity against
AGEs formation.
A549 cytotoxic AGE formation Collagenase
Collagenase is an enzyme that is known to be a member
of matrix metalloproteinase (MMP) family. The agents that
inhibit collagenase may have beneficial effects for maintain-
ing healthy skin by preventing dermal matrix degradation.
Therefore, the isolated compounds (1–8) were evaluated the
collagenase inhibitory activity (Table 2). The results showed
that linariin (4) and pectolinarin (5) exhibited weak inhibition
with IC50 values of 79.4 and 78.6µM, respectively. Luteolin
Isolated compounds
(
IC , µM)
(IC , µM)
(IC , µM)
5
0
50
50
Isolinariin C 1
Isolinariin D 2
Isolinariin E 3
Linariin 4
—
—
—
—
—
34.8± 5.6
35.0± 8.8
19.5± 2.0
—
—
—
—
79.4± 3.8
78.6± 2.4
—
Pectolinarin 5
Pectolinarigenin 6
Apigenin 7
—
91.1± 6.7
—
—
—
—
(8, 40.5µM) showed stronger inhibitory activity than that of
Luteolin 8
82.6± 5.4
n.d.
85.8± 6.8
1290± 31.5
n.d.
40.5± 3.2
n.d.
a positive control, caffeic acid (an IC50 value of 120µM) as
Aminoguanidine
Caffeic acid
10)
reported previously.
n.d.
120± 1.8
n.d.
In summary, chemical investigation of the non-polar frac-
tion of L. japonica led to the isolation of eight compounds
Doxorubicin
0.7± 0.06
n.d.
—
: >100µM, n.d.: not determined.
(1–8), including three new flavonoid glycosides (1–3). These
isolated compounds were examined for their cytotoxicity, col-
and L-rhamnose, thus the structure of 2 was determined lagenase and AGEs formation. New compounds 1, 2, and 3
as pectolinarigenin-7-O-(3,4-diacetyl-α-L-rhamnopyranosyl)- showed moderate inhibition of AGEs formation, and 4, 5 and
(
1→6)-β-D-glucopyranoside.
8 for collagenase, without any cytotoxicity at the concentra-
Isolinariin E (3), [α] −6.6 was also a pale yellow powder. tions of each IC50 value (Table 2), which indicated that these
D
The NMR data together with molecular ion at m/z 729.1997 compounds and crude extract of L. japonica may become an
[
+
M+Na] in HR-ESI-MS indicated that 3 was also another useful remedy for the AGEs associated diseases and skin de-
positional isomer of 1. The lower field shift of H-4‴ [δH 4.78 terioration.
(
1H, d, 9.9Hz)] and the upper field shift of H-3‴ [δ 3.97 (1H,
H
dd, 9.9, 3.3Hz)] suggested that the acetylated position was Experimental
1
13
changed from C-3‴ to C-4‴ in 3, which was further supported
General H- and C-NMR spectra were taken on a Bru-
by a correlation between the proton at δH 4.78 (H-4‴) and ker Ultrashield 600 spectrometer at 600MHz and 150MHz,
carbon signal at δ 172.4 (Fig. 2) in the HMBC spectrum. The respectively, with tetramethylsilane (TMS) as an internal stan-
C
acid hydrolysis of 3 also showed the aglycone (pectolinari- dard. IR and UV spectra were measured on Horiba FT-720
genin), D-glucose and L-rhamnose. Therefore, the structure of and Jasco V-520 UV/Vis spectrophotometers, respectively.
3
was elucidated as pectolinarigenin-7-O-(2,4-diacetyl-α-L- Optical rotations were measured on a Jasco P-1030 digital
polarimeter and a Jasco J-720 spectropolarimeter, respectively.
rhamnopyranosyl-(1→6)-β-D-glucopyranoside.
AGEs are well known to cause aging, hyperglycemia and Positive ion HR-ESI-MS was performed with an Applied
diabetic complications. Therefore, inhibitors of AGEs for- Biosystems QSTAR XL NanoSpray™ system. Silica gel open
mation are demanded as potential therapeutic remedy. The column chromatography (CC) and reversed-phase [octadecyl
inhibitory effects of the isolated compounds (1–8) on AGEs silylated silica gel (ODS)] CC were performed on silica gel
8
,9)
formation were evaluated using a fluorescent method (Table 60 (E. Merck, Darmstadt, Germany) and Cosmosil 75C18-
). The results showed that new compounds 1, 2 and 3 showed OPN (Nacalai Tesque, Kyoto, Japan; Φ=35mm, L=350mm)
stronger inhibitory activity (IC =34.8, 35.0 and 19.5µM, columns, respectively. HPLC was performed on an ODS col-
2
5
0
respectively) than that of a reference compound, aminogua- umn (Inertsil ODS-3, GL Science, Tokyo, Japan; Φ=6mm,
nidine (1.29mM). Thus, the presence of acetyl groups on the L=250mm) and the eluate was monitored with a Jasco RI-930

5
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Chem. Pharm. Bull.
Vol. 64, No. 5 (2016)
intelligent detector and a Jasco PU-1580 intelligent pump. All ture was neutralized by addition of amberlite IRA96SB (OH−
reagents were purchased from TCI (Tokyo, Japan), Wako Pure form) and filtered. The filtrate was dried and partitioned with
Chemical Industries, Ltd. (Osaka, Japan) and Nacalai Tesque EtOAc–H O mixture (1:1) two times. The combined EtOAc
2
unless otherwise specified.
layer was evaporated to afford an aglycone, pectolinarigenin
Plant Material Whole plants of Linaria japonica were (6), which was identified with NMR, MS and/or HPLC analy-
collected in late July 1990 in seashore areas of Tottori Pre- sis with authentic sample isolated in this study. The water
fecture, Japan, and a voucher specimen (90-LJ-Tottori) was layer was dried in vacuo and dissolved in 0.2mL of pyridine
deposited at the Department of Pharmacognosy, Faculty of containing L-cysteine methyl ester (15mg/mL) and reacted at
Pharmaceutical Sciences, Hiroshima University.
60°C for 1h. To the mixture, a solution (0.1mL) of o-toryl
Extraction and Isolation The air-dried plants (2.30kg) isothiocyanate in pyridine (5mg/mL) was added, and it was
were extracted with MeOH (15L) two times. The MeOH heated at 60°C for 1h. The final mixture was directly ana-
extract was concentrated to 5L and adjusted to 95% aque- lyzed by HPLC [Cosmosil 5C18 AR II (250×4.6mm i.d., Na-
ous MeOH by the addition of H O. This solution was then calai Tesque); 25% CH CN in 50mM H PO ; flow rate 0.8mL/
2
3
3
4
partitioned with n-hexane (1.5L) two times. The remaining min; column temperature 35°C; detection 250nm]. The tR of
aqueous MeOH layer was evaporated, resuspended in 1.5L of the peak at 18min coincided with that of D-glucose. The t of
R
7
)
water, and then partitioned with ethyl acetate (1.5L) two times the L-rhamnose was 30min.
and 1-butanol (1.5L) three times successively. Cytotoxicity Assay Human lung cancer cells (A549)
The non-polar fraction (60.5g, as a mixture of the ethyl were obtained from Riken cell bank (RCB3677) and cultured
acetate and n-hexane layers) was separated on a silica gel in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-
(
300g) CC with increasing polarity [hexane:CHCl3 (1:1), Aldrich Japan) supplemented with 10% heat-inactivated fetal
4
L, CHCl :MeOH (50:1, 40:1, 30:1, 20:1, 15:1, 10:1, 7:1, bovine serum, kanamycin (100µg/mL) and amphotericin B
3
5
:1, 3:1, 2:1, MeOH, each 2L)] yielding 12 fractions (Frs. (0.5 µg/mL). Into a 96-well plate, aliquots of the dimethyl sulf-
Lj1–Lj12). Fractions Lj3 (9.61g), Lj6 (1.07g), Lj8 (3.43g) and oxide (DMSO) solution of the test compounds (1% final con-
3
Lj10 (2.86g) were subjected to open reversed-phase (ODS) centration) were incubated with A549 cells (5×10 cells/well)
CC in a 10% aq. MeOH (400mL) to 100% MeOH (400mL) in a CO incubator at 37°C for 72h. 3-[4,5-Dimethylthiazol-
2
linear gradient, which led to 19 fractions (Frs. Lj3-1–Lj3-19, 2-yl]-2,5-diphenyltetrazolium bromide (MTT) was added into
Frs. Lj6-1–Lj6-19, Frs. Lj8-1–Lj8-19 and Frs. Lj10-1–Lj10-19, each well and the plate was further incubated for 1.5h. Ab-
respectively). The residue of fraction Lj3-11 (62.9mg) was sorbance was measured at 540nm using a 2300 EnSpire Mul-
recrystallized with MeOH to give pectolinarigenin (6, 4.0mg). timode plate reader (PerkinElmer, Inc.). DMSO was used as
Fraction Lj6-10 (33.4mg) was also purified by HPLC (67.5% a negative control and doxorubicin as a positive control. The
aq. MeOH) to give luteolin (8, 12.1mg). The other residue of viability was compared to that of control cells incubated in
fraction Lj6-11 (37.5mg) was purified by preparative HPLC the same medium without the test compounds. Measurements
(
45% aq. acetone) to give 7 (apigenin, 5.1mg). Fraction Lj8-11 were performed in triplicate and the IC5 of the intensity of
0
1
1)
(
238mg) was purified by preparative HPLC (55% aq. MeOH). absorbance were determined graphically.
Three peaks appeared at 18, 25 and 35min and were collected
AGEs Assay The reaction mixture, 10mg/mL of bovine
to give isolinariin C (1, 11.6mg), isolinariin D (2, 18.0mg) and serum albumin (Sigma-Aldrich Japan) in 50mM phosphate
isolinariin E (3, 5.3mg), respectively. Then, fractions Lj10-10 buffer (pH 7.4) containing 0.02% sodium azide, was added to
(
186mg) and Lj10-11 (379mg) were recrystallized with MeOH a 0.5M ribose solution. The reaction mixture was then mixed
to give 4 (linariin, 15.5mg) and 5 (pectolinarin, 71.1mg), re- with the test compounds. After incubation at 37°C for 24h,
spectively.
the fluorescent reaction products were assayed with a spec-
2
5
Isolinariin C (1), Pale yellow powder; [α]D −4.8 (c=0.77, trofluorometric detector (EnSpire, PerkinElmer, Inc., Japan;
MeOH); UV (EtOH) λmax (logε) nm: 324 (3.83), 274 (3.86), Ex: 370nm, Em: 440nm). Measurements were performed in
−
1
2
32 (3.83); IR (film) vmax cm : 3437, 2933, 1746, 1653, 1606, triplicate and the IC50 of the intensity of fluorescent were de-
1
8)
1
566, 1509, 1460, 1361, 1251, 1182, 1054, 837, 667; H- and termined graphically.
C-NMR, see Table 1; positive HR-ESI-MS m/z 729.1998
13
Collagenase Inhibition Assay Collagenase inhibitory
+
[
M+Na] (calcd for C H O Na: 729.2001).
activity was examined using the modified method described
33
38 17
2
6
10)
Isolinariin D (2), Pale yellow powder; [α]D −8.6 (c=1.20, by Teramachi et al. Briefly, the test compounds, 10µg/mL
MeOH); UV (EtOH) λmax (logε) nm: 322 (3.85), 272 (3.85), of enzyme (collagenase from Clostridium histolyticum (Sigma-
−1
2
1
33 (3.86); IR (film) vmax cm : 3443, 2932, 1735, 1653, 1607, Aldrich Japan)) and 50mM Tricine buffer (pH 7.5) were added
1
562, 1510, 1458, 1360, 1250, 1182, 1044, 836, 669; H-NMR to a 96-well microtiter plate and preincubated for 10min at
and C-NMR, see Table 1; positive HR-ESI-MS m/z 729.1997 37°C. Afterwards, the substrate solution ((7-methoxycou-
13
+
β
[
M+Na] (calcd for C H O Na: 729.2001).
marin-4-yl)
acetyl-L-prolyl-L-leucylglycyl-L-leucyl-[N -(2,4-
33
38 17
2
6
Isolinariin E (3), Pale yellow powder; [α]D −6.6 (c=0.35, dinitrophenyl)-L-2,3-diaminopropionyl]-L-alanyl-L-arginine
MeOH); UV (EtOH) λmax (logε) nm: 340 (4.30), 276 (3.76), amide) (PEPTIDE INSTITUTE, Osaka, Japan) at a final
−1
2
1
29 (3.78); IR (film) vmax cm : 3361, 2931, 1735, 1652, 1603, concentration of 10µM was added to initiate the reaction. The
1
560, 1508, 1457, 1360, 1250, 1182, 1051, 837, 670; H-NMR fluorescence values were measured at an excitation of 320nm
and C-NMR, see Table 1; positive HR-ESI-MS m/z 729.1997 and an emission of 405nm after 0 and 30min incubation at
13
+
[
M+Na] (calcd for C H O Na: 729.2001).
37°C using a fluorescence plate reader (EnSpire; PerkinElmer,
33
38 17
Acid Hydrolysis of 1, 2 and 3 A solution of isolinariin Inc., Japan). These assays were performed in triplicate using
C (1), D (2) and E (3) (5mg each) in 1N HCl (0.2mL) was caffeic acid as a positive control, and the IC50 of the intensity
heated at 90–100°C in a screw-capped vial for 2h. The mix- of fluorescent were determined graphically.

Vol. 64, No. 5 (2016)
Chem. Pharm. Bull.
521
2
)
Otsuka H., Phytochemistry, 39, 1111–1114 (1995), and references
cited therein.
Widyowati R., Sugimoto S., Yamano Y., Sukardiman, Otsuka H.,
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Acknowledgments This work was supported by JSPS
KAKENHI Grant numbers 15H04651, 26460122 and
5860078. The authors are grateful for access to the supercon-
3)
2
ducting NMR instrument, UV and ESI-MS at the Analytical
Center of Molecular Medicine, the Analysis Center of Life
Science and the Natural Science Center for Basic Research
and Development of the Graduate School of Biomedical and
Health Sciences, Hiroshima University. The authors would
like to express their sincere thanks to the Indonesian Director-
ate General of Higher Education (DIKTI) for doctoral scholar-
ships.
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Conflict of Interest The authors declare no conflict of
interest.
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