Taxiphyllin 6′-O-gallate, actinidioionoside 6′-O-gallate and myricetrin 2′-O-sulfate from the leaves of Syzygium samarangense and their biological activities

Article abstract of DOI:10.1248/cpb.c14-00352

Three new compounds were isolated from a MeOH extract of the leaves of Syzygium samarangense, one new cyanogenic glucoside, taxiphyllin 6′-O-gallate (1), one new megastigmane glucoside, actinidioionoside 6′-O-gallate (2), and one new sulfated flavonoid rhamnoside, myricetrin 2″-O-sulfate (3), together with 14 known compounds, lupeol (4), demethoxymatteucinol (5), cryptostrobin (6), betulinic acid (7), β-sitosterol glucoside (8), 2R-prunasin (9), myrciaphenone A (10), 1-feruloyl-β-D-glucopyranoside (11), (3S,5R,6R,7E,9S)-3,5,6,9-tetrahydroxymegastigman-7-ene (12), guaijaverin (13), myricetin 4′-methyl ether 3-O-α-L-rhamno-pyranoside (14), myricetrin (15), gallic acid (16) and actinidioionoside (17). The structures of the new compounds were determined through a combination of spectroscopic, HPLC and chemical analyses.

Full text of DOI:10.1248/cpb.c14-00352

October 2014
Note
Chem. Pharm. Bull. 62(10) 1013–1018 (2014)
1013
Taxiphyllin 6ꢀ-O-Gallate, Actinidioionoside 6ꢀ-O-Gallate and Myricetrin
2ꢁ-O-Sulfate from the Leaves of Syzygium samarangense and Their
Biological Activities
,b,c
Nabil Samy Mamdouh,a,b Sachiko Sugimoto,b Katsuyoshi Matsunami,b Hideaki Otsuka,* and
Mohamed Salah Kamela
a Department of Pharmacognosy, Faculty of Pharmacy, Minia University; Minia 61519, Egypt: b Department of
Pharmacognosy, Graduate School of Biomedical and Health Sciences, Hiroshima University; 1–2–3 Kasumi, Minami-
c
ku, Hiroshima 734–8553, Japan: and Department of Natural Products Chemistry, Faculty of Pharmacy, Yasuda
Women’s University; 6–13–1 Yasuhigashi, Asaminami-ku, Hiroshima 731–0153, Japan.
Received May 7, 2014; accepted July 3, 2014
Three new compounds were isolated from a MeOH extract of the leaves of Syzygium samarangense, one
new cyanogenic glucoside, taxiphyllin 6ꢀ-O-gallate (1), one new megastigmane glucoside, actinidioionoside
6ꢀ-O-gallate (2), and one new sulfated flavonoid rhamnoside, myricetrin 2ꢁ-O-sulfate (3), together with 14
known compounds, lupeol (4), demethoxymatteucinol (5), cryptostrobin (6), betulinic acid (7), β-sitosterol
glucoside (8), 2R-prunasin (9), myrciaphenone A (10), 1-feruloyl-β-D-glucopyranoside (11), (3S,5R,6R,7E,9S)-
3,5,6,9-tetrahydroxymegastigman-7-ene (12), guaijaverin (13), myricetin 4ꢀ-methyl ether 3-O-α-L-rhamno-
pyranoside (14), myricetrin (15), gallic acid (16) and actinidioionoside (17). The structures of the new com-
pounds were determined through a combination of spectroscopic, HPLC and chemical analyses.
Key words Syzygium samarangense; Myrtaceae; taxiphyllin 6′-O-gallate; actinidioionoside 6′-O-gallate;
myricetrin 2″-O-sulfate
Syzygium samarangense (BLUME) MERR. & L. M. PERRY and actinidioionoside (17)17) by comparing their spectroscopic
(Myrtaceae) (syns. Eugenia javanica L.)1) is commonly data with those reported in the literature.
known as wax apple, wax jambu, rose apple and Java apple.
Compound (1), [α]D22 −51.4, was obtained as an amorphous
Wax apple is widely cultivated throughout Malaysia and also powder and has the molecular formula C21H21O11N as deter-
grown throughout southeast Asian countries such as Thailand, mined by positive-ion mode high-resolution (HR)-electrospray
Indonesia and Taiwan as well as other tropical countries. ionization (ESI)-mass spectrometry (MS). The IR spectrum
The pear-shaped fruits are usually pink, light red or red but of 1 showed bands at 3334 and 1698cm−1 corresponding to
may be greenish-white or cream-colored, and are generally hydroxy and carbonyl group absorptions, respectively. The
crisp, often juicy and refreshing with a subtle sweet taste and 1H-NMR spectrum (Table 1) exhibited a pair of doublets at
aromatic flavor. Wax apple fruit can be eaten raw with salt or δH 6.78 (2H, d, J=8.6Hz) and 7.29 (2H, d, J=8.6Hz) which
cooked as a sauce. The fruit has been used for a wide variety were characteristic of a p-disubstituted benzene ring, and a
of ailments, including cough, diabetes, dysentery, inflamma- singlet for one oxymethine proton at δH 5.57 (1H, s), along
tion and ringworm.1–3)
with one anomeric proton signal at δH 4.18 (1H, d, J=7.4Hz)
The present study deals with the isolation and structural for a β-glucopyranosyl moiety. The 13C-NMR spectrum (Table
elucidation of three new compounds (1–3), together with 14 2) exhibited six signals assignable to a β-glucopyranosyl moi-
known compounds from the leaves of this plant (Fig. 1).
ety,16) six aromatic carbon signals and a methine carbon at δC
68.2, as well as a singlet carbon at δC 119.3, the chemical shift
of which suggested a nitrile functional group.9) The presence
Results and Discussion
A methanol extract of the leaves of S. samarangense was of the nitrile group was supported by the IR absorption band
fractionated by solvent–solvent partitioning. The EtOAc and at 2248cm−1 for a triple bond and the index of hydrogen defi-
1-BuOH-soluble fractions were separated by means of various ciency calculated from the positive-ion mode HR-ESI-MS. In
chromatographic procedures including column chromatogra- accordance with these data, the aglycone was established to be
phy (CC) on silica gel and reversed-phase octadecyl silica gel a p-disubstituted aromatic ring linked to the methine carbon,
(ODS) CC, and HPLC, 17 compounds (1–17) being isolated; a which in turn formed a glycosidic linkage with the anomeric
new cyanogenic glucoside (1), a new megastigmane glucoside carbon, as well as a linkage with the nitrile functional group.
(2) and a new sulfated flavonoid rhamnoside (3), in addition Further NMR analyses involving 1H–1H correlation spec-
to 14 known compounds, which were identified as lupeol troscopy (COSY), heteronuclear single quantum coherence
(4),4) demethoxymatteucinol (5),5) cryptostrobin (6),6) betulinic (HSQC) and heteronuclear multiple-bond correlation (HMBC)
acid (7),7) β-sitosterol glucoside (8),8) 2R-prunasin (9),9) myr- spectroscopies were performed (Fig. 2). In the HMBC spec-
ciaphenone A (10),10) 1-feruloyl-β-D-glucopyranoside (11),11) trum of 1, H-2 (δH 5.57) showed cross peaks with C-1′, C-1
(3S,5R,6R,7E,9S)-3,5,6,9-tetrahydroxymegastigman-7-ene and C-3 (δC 101.0, 119.3 and 124.8, respectively). The signal
(12),12) guaijaverin (13),13) myricetin 4′-methyl ether 3-O-α-L- of the anomeric proton of the glucopyranosyl moiety (δH 4.18)
rhamnopyranoside (14),14) myricetrin (15),15) gallic acid (16)16) was correlated with C-2 (δC 68.2), confirming the sugar link-
age to the hydroxy group at C-2. The presence of glucose was
confirmed by HPLC analysis of a hydrolyzate of 1 and the
The authors declare no conflict of interest.
© 2014 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: hotsuka@hiroshima-u.ac.jp

1014
Vol. 62, No. 10
Fig. 1. Structures of the Isolated Compounds (1–17)
absolute configuration of the sugar was simultaneously deter- were observed. The 13C-NMR spectrum displayed six signals
mined to be in the D-series using a chiral detector. The singlet assignable to a β-glucopyranosyl moiety, and six aromatic
at δH 7.15 for two aromatic protons observed in 1H-NMR signals and one carbonyl carbon signal. The remaining 13
and one carbonyl carbon signal at δC 168.3, and six aromatic resonances comprised those of four methyls, two methylenes,
carbon signals at δC 110.3×2, 140.0 and 146.6×2 seen in two methines, a disubstituted trans double bond and three
13C-NMR (Tables 1, 2) corresponded to a galloyl moiety. The quaternary carbons. From the above evidence, the structure of
location of the galloyl moiety was found to be at C-6′ of the 2 was expected to be a megastigmane O-β-D-glucopyranoside
glucose unit on the basis of the downfield shifts of C-6′ and with a galloyl moiety. The absolute configuration of glucose
H-6′, as well as the upfield shift of C-5′. The linkage was con- was determined by HPLC analysis of a hydrolyzate of 2
firmed by HMBC correlations between H-6′a and 6′b (δH 4.58, using a chiral detector. The NMR spectroscopic data for the
4.44) and C-7″ (δC 168.3). Mild alkaline hydrolysis of 1 with megastigmane skeleton were essentially the same as those
CH3ONa gave a glucoside (1a) and methyl gallate (1b) and the for actinidioionoside (17), a known megastigmane glucoside
NMR spectra of 1a were the same as those of taxiphyllin18) isolated from Eugenia uniflora.19) The downfield shift of C-6′
with the R configuration. Thus compound 1 was characterized was expected to be induced by esterification of the galloyl
as taxiphyllin 6′-O-gallate as shown in Fig. 1.
moiety, and the linkage was confirmed by HMBC correlations
Compound (2), [α]D21 −43.2, was isolated as an amorphous between H-6′a and 6′b (δH 4.48, 4.32) and C-7″ (δC 168.5) (Fig.
powder and its molecular formula was determined to be 3). On mild alkaline hydrolysis of 2 with CH3ONa, a glucoside
C26H38O13 by HR-ESI-MS. The IR spectrum exhibited ab- (2a) and methyl gallate were obtained. The NMR spectra of
sorptions for hydroxy groups (3366cm−1), a conjugated ester 2a were the same as those of 17 and the HPLC retention times
carbonyl group (1699cm−1), and an aromatic ring (1557cm−1). of 17 and 2a were found to be the same at 16min under the
The UV spectrum also indicated the presence of an aromatic same conditions. Therefore, compound 2 was elucidated to
1
ring (285nm). In the H-NMR spectrum, signals for one dou- be (3S,5R,6R,9R)-3,6,7,9-tetrahydroxymegastigman-7-ene 9-O-
blet and three singlet methyls, one singlet aromatic proton, a β-D-(6′-O-galloyl)glucopyranoside, namely actinidioionoside
pair of trans-olefinic protons and an anomeric proton (δH 4.42) 6′-O-gallate as shown in Fig. 1.

October 2014
1015
Table 1. 1H-NMR Spectral Data for Compounds 1–3, 1a and 2a (CD3OD, 400MHz)
No.
2
1
1a
2
2a
3
5.57 (s)
—
5.77 (s)
—
1.41 (ddd, 11.9, 4.5, 1.6)
1.60 (dd, 11.9, 11.9)
4.00 (m)
1.41 (ddd, 11.9, 4.5, 1.6)
1.60 (dd, 11.9, 11.9)
4.00 (m)
—
3
4
—
—
7.29 (d, 8.6)
7.38 (d, 8.5)
1.70 (dd, 11.9, 11.9)
1.74 (ddd, 11.9, 4.5, 1.6)
—
1.70 (dd, 11.9, 11.9)
1.74 (ddd, 11.9, 4.5, 1.6)
—
5
6
7
8
9
6.78 (d, 8.6)
—
6.83 (d, 8.5)
—
—
—
—
6.18 (d, 1.8)
6.78 (d, 8.6)
7.29 (d, 8.6)
6.83 (d, 8.5)
7.38 (d, 8.5)
6.07 (dd, 15.9, 0.8)
5.81 (dd, 15.9, 7.3)
4.37 (quintet, 6.4)
1.31 (d, 6.2)
1.13 (s)
6.07 (dd, 15.9, 0.8)
5.81 (dd, 15.9, 7.3)
4.37 (quintet, 6.4)
1.30 (d, 6.2)
1.151 (s)
—
6.34 (d, 1.8)
—
—
10
11
12
13
0.80 (s)
0.83 (s)
1.16 (s)
1.158 (s)
1′
4.18 (d, 7.4)
3.35–3.45 (m)
3.35–3.45 (m)
3.35–3.45 (m)
3.27 (m)
4.16 (d, 7.8)
3.27 (m)
4.42 (d, 7.6)
3.20 (m)
4.33 (d, 7.6)
3.27 (m)
—
2′
3′
4′
5′
6′a
6′b
1″
2″
3″
4″
5″
6″
6.96 (s)
—
3.24 (m)
3.33 (m)
3.24 (m)
3.23 (m)
3.28 (m)
3.23 (m)
—
3.17 (m)
3.47 (m)
3.17 (m)
—
4.58 (dd, 11.8, 1.7)
4.44 (dd, 11.8, 5.6)
—
3.89 (dd, 11.8, 2.3)
3.70 (dd, 11.8, 6.1)
4.48 (dd, 11.8, 1.6)
4.32 (dd, 11.8, 4.5)
—
3.80 (dd, 11.7, 1.2)
3.57 (dd, 11.7, 6.0)
6.96 (s)
5.76 (brs)
7.15 (s)
7.10 (s)
4.93 (dd, 3.1, 1.2)
3.89 (dd, 9.5, 3.2)
3.33 (m)
—
—
—
—
—
—
3.42 (dq, 9.5, 6.2)
0.97 (d, 6.2)
7.15 (s)
7.10 (s)
H: Chemical shifts, multiplicity and J in Hz. m: Multiplet or overlapped signals.
Compound (3), [α]D20 −80.5, was isolated as a pale yellow potent DPPH radical scavenging activity (IC50: 14.8 1.56,
amorphous powder and its molecular formula was determined 10.9 0.29, 29.3 2.17, 31.4 3.41, 10.5 0.75 and 9.9 0.55 µM,
to be C21H20O15S by HR-ESI-MS. The 13C-NMR spectrum respectively) comparable with that of the standard trolox
showed good similarity to those of myricetrin (15), except for (16.6 2.2µM). Compounds 1, 3, 6, 7, 15 and 16 exhibited
the downfield shift of C-2″ (+6.1ppm) and the upfield shift of weak growth inhibitory activity toward A549 (IC50 73.9 10.2,
C-1″ (−2.7ppm) and C-3″ (−1.0ppm) of the rhamnopyranosyl 76.0 11.6, 75.4 13.5, 56.4 16.8, 59.4 10.3 and 43.2 23.2 µM,
moiety suggested the attachment of some substituent at this respectively) in the MTT cytotoxic assay. The IC50 value for
position, which was substantiated by measurement of a mass standard doxorubicin was found to be 0.53 0.03µM.
spectrum. The HR-ESI-MS spectrum showed a single peak
at m/z 589.0234 in the positive-ion mode, corresponding to
Experimental
General Experimental Procedures Optical rotation data
C21H19O15Na2S [M−H+2Na]+, showing the attachment of a
sulfuryl moiety at the 2″-position. Consequently, the structure were measured on a JASCO P-1030 polarimeter. IR and UV
of compound 3 was established as myricetrin 2″-O-sulfate spectra were obtained on Horiba FT-710 Fourier transform in-
(Fig. 4).
frared and JASCO V-520 UV/Vis spectrophotometers, respec-
1
Compounds 1−17 were examined for their antibacterial tively. H- and 13C-NMR spectra were recorded on a JEOL
activities, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scav- JNM α-400 spectrometer with tetramethylsilane as an internal
enging activities, anti-Leishmania activities and also tumor standard. HR-ESI mass spectra were taken on a LTQ Orbitrap
cell growth inhibitory activities toward A549 by means of the XL mass spectrometer. Silica gel CC was performed on silica
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide gel 60 [(E. Merck, Darmstadt, Germany) 70–230 mesh]. ODS
(MTT) assay. None of the tested compounds (1–17) showed open CC (RPCC) was performed on Cosmosil 75C18-OPN
any significant activity against Mucor racemosus, Staphylo- (Nacalai Tesque, Kyoto, Japan) (20×400mm, 10g fractions
coccus aureus (MRSA) and Leishmania major (IC50>100 µM) being collected). HPLC was performed on an ODS column
except that compound 6 showed weak activity against M. race- [Inertsil ODS-3; GL Science, Tokyo, Japan (6×250mm, flow
mousus (IC50: 65.4 2.86µM) and L. major (IC50: 86.7 4.6µM). rate: 1.0mL/min), using a refractive index and/or a UV detec-
The results were compared with those of amphotericin B as a tor. Precoated silica gel 60F254 plates (E. Merck; 0.25mm in
positive control (IC50, 0.5 0.2 and 0.39 0.5µM, respectively). thickness) were used for TLC analyses, with visualization by
Compound 1 exhibited weak antibacterial activity against S. spraying with a 10% H2SO4 solution in ethanol and heating to
aureus with an IC50 value of 86.9 17.9 µM (IC50 for oxacil- around 150°C on a hotplate.
lin was 10.3 4.52 µM). Compounds 1, 3 and 13–16 showed
Microorganisms The microorganisms used in this study

1016
Vol. 62, No. 10
Table 2. 13C-NMR Spectral Data for Compounds 1–3, 1a and 2a
(CD3OD, 100MHz)
No.
1
1a
2
2a
3
1
2
3
4
5
6
7
8
9
119.3
68.2
119.7
68.0
40.7
46.4
40.7
46.4
65.3
45.6
77.8
78.9
133.0
134.7
78.7
21.8
26.2
27.5
27.6
102.7
75.4
78.0
71.9
78.2
62.9
159.3
135.7
179.4
163.1
99.8
124.8
130.9
116.8
160.3
116.8
130.9
125.2
130.8
116.7
160.3
116.7
130.8
65.3
45.7
77.8
79.2
Fig. 3. Selected COSY and HMBC Correlations of 2
132.8
134.2
78.4
165.7
94.6
158.4
105.9
10
21.7
11
12
13
26.2
27.4
27.5
1′
101.0
74.7
101.1
74.7
77.9
71.5
78.3
62.8
102.7
75.4
121.9
109.7
146.8
137.9
146.8
109.7
101.0
78.0
2′
3′
4′
5′
6′
1″
2″
3″
4″
5″
6″
7″
77.8
78.0
71.6
71.5
76.0
75.6
64.6
64.7
121.5
110.3
146.6
140.0
146.6
110.3
168.3
121.5
110.3
146.5
139.9
146.5
110.3
168.5
Fig. 4. Selected HMBC Correlations of 3
71.2
tively. The remaining aqueous layer was concentrated to fur-
nish a water-soluble fraction (80.0g).
73.7
72.1
The EtOAc fraction (21.5g) was subjected to silica gel CC
(600 g) (60×550mm). The column was eluted initially with
n-hexane (5L), and then with an n-hexane–EtOAc gradient
mixture, 500mL fractions being collected. Similar fractions
were combined, which afforded 20 fractions. The fourth frac-
tion E-4 gave 4 (207mg). The fifth fraction E-5 afforded 5
(161mg). The sixth fraction E-6 produced 6 (81.2mg). The
seventh fraction E-7 yielded compound 7 (51.4mg). Frac-
tion E-18 (961mg) was purified on RPCC, affording five
fractions. The second fraction E-18-2 (116mg) was purified
by HPLC (30% MeOH) to produce compound 1 (10.9mg).
Fraction E-19 (5.88g) was rechromatographed on silica gel
(200g) (30×620mm), using CHCl3–MeOH gradient system,
200mL fractions being collected and similar fractions were
combined to yield eight fractions. The fourth fraction E-19-4
gave 8 (85.5mg). Fraction E-19-6 was subjected to RPCC,
which gave ten fractions. The third fraction E-19-6-3 (16.1mg)
17.7
Fig. 2. Selected COSY and HMBC Correlations of 1
M. racemousus (IFM52680) and S. aureus (JCM20624) were was purified by HPLC (30% MeOH) to furnish 9 (1.88mg)
purchased from RIKEN BRC (Japan) and Medical Mycology and 10 (3.90mg). The fourth fraction E-19-6-4 (51.6mg) was
Research Center, Chiba University, respectively.
purified by HPLC (30% MeOH) to afford 11 (4.66mg) and
Plant Material The leaves of S. samarangense were col- 12 (17.6mg). The sixth fraction E-19-6-6 (160.8mg) was puri-
lected in May 2010 from Aswan Botanical Garden, Egypt. A fied by HPLC (40% MeOH) to produce 13 (2.66mg) and 14
voucher specimen of the plant was deposited in the Herbarium (10.3mg). The seventh fraction E-19-7 gave 15 (18.8mg).
of the Department of Pharmacognosy, Faculty of Pharmacy,
Minia University, Egypt (Mn-Ph-Cog-Ss).
The 1-BuOH fraction (13.0g) was chromatographed on
silica gel (400g) (50×500mm), using a CHCl3-MeOH gradi-
Extraction and Isolation Air-dried powdered leaves ent system and 200mL fractions being collected and similar
(1.30kg) of S. samarangense were extracted with metha- fractions were combined to yield 14 fractions. Fraction B-8
nol (5L×5) till exhaustion followed by concentration under (1.13g) was purified on RPCC, affording six fractions. The
reduced pressure to yield a viscous gummy material. This second fraction B-8-2 gave 16 (84.5mg). Fraction B-12 (1.26g)
residue (223g) was dissolved in 500mL of water and defatted was rechromatographed over silica gel (75g), (20×500mm),
with n-hexane (1L×5). The aqueous layer was evaporated to using CHCl3–MeOH gradient system, 100mL fractions being
remove a trace amount of the organic solvent, and then ex- collected and the similar fractions were combined to yield
tracted with EtOAc and 1-BuOH, successively (1L×5 each). five fractions. The third fraction B-12-3 (75.9mg) was puri-
The EtOAc and 1-BuOH fractions were concentrated under fied by HPLC (30% MeOH) to produce 17 (22.3mg) and 2
reduced pressure to give 21.5g and 13.0g of residues, respec- (6.50mg). Fraction B-14 (915mg) was rechromatographed over

October 2014
1017
silica gel (50g), (20×350mm), using CHCl3–MeOH gradient
Antimicrobial Susceptibility Assay Susceptibility tests
system, 100mL fractions being collected and the similar frac- were performed using a broth microdilution assay according
tions were combined to yield six fractions. The sixth fraction to the National Committee for Clinical Laboratory Standards
B-12-6 (202mg) was purified by HPLC (30% MeOH) to give reference methods. Assays were performed using Mueller–
3 (27.9mg).
Hinton broth (Difco). The bacterial inocula were adjusted to
Taxiphyllin 6′-O-Gallate (1) Amorphous powder; [α]D22 yield a density of 5×10−5 colony forming units/mL. Samples
−51.4, (c=0.72, MeOH); IR νmax (film) cm−1: 3334, 2946, were diluted directly in 96-well microtiter plates by serial
2842, 2248, 1698, 1610, 1513, 1450, 1342, 1318, 1227, 1026, two-fold dilution using a multi channel pipette. The microtiter
766; UV λmax (MeOH) nm (log ε): 219 (4.26), 267 (3.93), plates were incubated during 24h at 35°C and were read using
335 (3.65); H-NMR (400MHz, CD3OD): Table 1; 13C-NMR a Molecular Device Versamex Tunable Microplate Reader at
1
(100MHz, CD3OD): Table 2; HR-ESI-MS (positive-ion mode) 620nm as well as by visual observation. The MIC50 value was
m/z: 486.1004 [M+Na]+ (Calcd for C21H21O11NNa: 486.1012).
determined as a 50% decrease in the optical density. Ampho-
Actinidioionoside 6′-O-Gallate (2) Amorphous powder; tericin B and oxacillin were used as positive controls.20)
Anti-Leishmania Assay The anti-Leishmaniacidal activ-
[α]D21 −43.2, (c=0.34, MeOH); IR νmax (film) cm−1: 3366, 2931,
1699, 1649, 1615, 1557, 1540, 1508, 1455, 1368, 1339, 1221, ity of the isolated compounds was determined using the colo-
1070, 1028, 669; UV λmax (MeOH) nm (log ε): 213 (3.66), 285 rimetric MTT assay. Medium 199 medium supplemented with
1
(3.16), 304 (3.39), 351 (3.65), 364 (3.78); H-NMR (400MHz, 10% heat-inactivated fetal bovine serum (FBS) and 100µg/
CD3OD): Table 1; 13C-NMR (100MHz, CD3OD): Table 2; HR- mL of kanamycin was used as the cell culture medium. The
ESI-MS (positive-ion mode) m/z: 581.2197 [M+Na]+ (Calcd test compounds were dissolved in dimethyl sulfoxide (DMSO)
for C26H38O13Na: 581.2205).
and then added to the wells of 96-well microtiter plates to the
Myricetrin 2″-O-Sulfate (3) Pale yellow amorphous final concentration of 1%. L. major cells (2×105 cells/well)
powder; [α]D20 −80.5 (c=1.85, MeOH); IR νmax (film) cm−1: were cultured in a CO2 incubator at 25°C for 72h and then a
3294, 2982, 2942, 1653, 1606, 1502, 1447, 1202, 1164, 1084, MTT solution was added to each well, followed by incubation
1042, 988, 841; UV λmax (MeOH) nm (log ε): 225 (4.05), overnight at 25°C. The absorbance was measured at 540nm
258 (4.03), 348 (3.99); +NaOMe 238 (4.16), 263 (4.13), 353 using a microplate reader. Amphotericin B was used as a posi-
(3.93), 397 (3.86); +AlCl3 232 (4.06), 269 (4.03), 417 (4.08); tive control.21)
+AlCl3/HCl 225 (4.06), 269 (4.07), 393 (3.87); +NaOAc 231
(4.11), 268 (4.03), 363 (3.93); 1H-NMR (400MHz, CD3OD): tion:
Table 1; 13C-NMR (100MHz, CD3OD): Table 2; HR-ESI-MS
(positive-ion mode) m/z: 589.0234 [M−H+2Na]+ (Calcd for
The inhibition % was calculated using the following equa-


% inhibition= 1−(Asample− Ablank )/(Acontrol − Ablank ) ×100


C21H19O15Na2S: 589.0235).
where Acontrol is the absorbance of the control reaction mixture
Analysis of the Sugar Moiety About 1mg of compounds (containing DMSO and all reagents, except for the test com-
1–3 were hydrolyzed with 1M HCl (1.0mL) at 80°C for 2h. pounds). IC50 was determined as the concentration of sample
The reaction mixtures were neutralized with Amberlite required to inhibit the formation of MTT formazan by 50%.
IRA96SB (OH−), and then partitioned with an equal amount
DPPH Radical Scavenging Activity The absorbance with
of EtOAc. The water layers were analyzed for their sugar various concentrations of the test compounds dissolved in
components. The sugars were determined by HPLC on an MeOH (100µL) in 96-well microtiter plates was measured at
amino column [Shodex Asahipak NH2P-50 4E (4.6×250mm), 515nm as Ablank. Then, a 200µM DPPH solution (100µL) was
CH3CN–H2O (3:1), 1mL/min], using
a chiral detector add to each well, followed by incubation at room temperature
(JASCO OR-2090plus), in comparison with authentic D-glu- for 30min. The absorbance was measured again as Asample
.
cose and L-rhamnose. Compounds 1 and 2 gave a peak for D-
glucose at a retention time of 7.17min with a positive rotation tion:
sign. Compound 3 gave a peak for L-rhamnose at a retention
time of 5.12min with a negative rotation sign.
The % inhibition was calculated using the following equa-


% inhibition= 1−(Asample− Ablank )/(Acontrol − Ablank ) ×100


Mild Alkaline Hydrolysis About 5.9mg of compound where Acontrol is the absorbance of the control reaction mixture
1 in MeOH (450µL) was added to 1M CH3ONa (50µL), fol- containing DMSO and all reagents, except for the test com-
lowed by standing at 20°C for 4h. The reaction mixture was pound. IC50 was determined as the concentration of sample re-
neutralized with Amberlite IR-120B (H+) and then evaporated quired to inhibit the formation of the DPPH radical by 50%.22)
to dryness. The residue was partitioned with CHCl3 (2 mL)–
Human Cancer Cell Growth Inhibition Assay This
H2O (2mL) to afford a taxiphyllin (1a) (2.3mg) from the assay was performed using a human lung cancer cell line
H2O-soluble fraction and methyl gallate (1b) (1.0mg) from the (A549) and the viability was estimated by means of the
CHCl3-soluble fraction.
colorimetric MTT assay. Dulbecco’s modified Eagle’s medium
In a similar manner to as for 1, compound 2 (2.2mg) gave supplemented with 10% heat-inactivated FBS and 100µg/mL
a megastismane glucoside (actinidioionoside) (2a=17) (1.0mg) of kanamycin was used as the cell culture medium. The test
and methyl gallate (2b) (0.4mg).
compounds were dissolved in DMSO and then added to the
HPLC Analysis Megastigmane glucoside (2a), obtained wells of 96-well microtiter plates to the final concentration
by mild alkaline hydrolysis, was analyzed by HPLC [Inert- of 1%. A549 cells (5×103 cells/well) were cultured in a 5%
sil ODS-3; GL Science, Tokyo, Japan (6×250mm, flow rate: CO2 incubator at 37°C for 72h and then a MTT solution was
1.0mL/min), using a refractive index detector] with MeOH– added to each well and the plates were incubated for a further
H2O (3:7, v/v) which gave a peak at 16min, which was the 1.5h. Then the formazan precipitates were dissolved in DMSO
same retention time as that of 17.
and the optical density value for each well was measured at

1018
Vol. 62, No. 10
K., Cardoso C. A. L., Vilegas W., Leite S. R. A., Sato D. N., Quim.
Nova, 32, 1222–1226 (2009).
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8) Kojima H., Sato N., Hatano A., Ogura H., Phytochemistry, 29,
2351–2355 (1990).
540nm with a microplate reader. Doxorubicin was used as a
positive control.
The cell growth inhibition was calculated using the follow-
ing equation:
9) Seigler D. S., Pauli G. F., Nahrstedt A., Leen R., Phytochemistry,
60, 873–882 (2002).
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Agric. Food Chem., 52, 4664–4668 (2004).


% inhibition= 1−(Asample− Ablank )/(Acontrol − Ablank ) ×100


where Acontrol is the absorbance of the control reaction mixture
containing DMSO and all reagents except for the test com-
pound. IC50 was determined as the concentration of sample
required to inhibit the formation of MTT formazan by 50%.20)
Acknowledgments The authors are grateful for access to
the superconducting 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 Re-
search and Development of the Graduate School of Biomedi-
cal and Health Sciences, Hiroshima University.
16) Ascari J., Takahashi J. A., Boaventura M. A. D., Bol. Latinoamer.
Caribe Plant Med. Aromat., 9, 20–28 (2010).
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Glander K. E., Ebinger J. E., Phytochemistry, 66, 1567–1580 (2005).
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J. Nat. Prod. (India), 7, 37–47 (2014).
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Bull., 54, 546–549 (2006).
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