Phenolic constituents of Acorus gramineus

Article abstract of DOI:10.1007/s12272-011-0808-6

The purification of a MeOH extract from the rhizome of Acorus gramineus (Araceae) using column chromatography furnished two new stereoisomers of phenylpropanoid, acoraminol A (1) and acoraimol B (2). It also furnished 17 known phenolic compounds, β-asarone (3), asaraldehyde (4), isoacoramone (5), propioveratrone (6), (1'R,2'S)-1',2'-dihydroxyasarone (7), (1'S,2'S)- 1',2'-dihydroxyasarone (8), 3',4'-dimethoxycinnamyl alcohol (9), 3',4',5'-trimethoxycinnamyl alcohol (10), kaempferol 3-methyl ether (11), 2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-1,3- propanediol (12), hydroxytyrosol (13), tyrosol (14), (2S,5S)-diveratryl-(3R,4S)-dimethyltetrahydrofuran (15), (7S,8R)-dihydrodehydrodiconiferyl alcohol (16), 7S,8S-threo-4,7,9,9'- tetrahydroxy-3,3'-dimethoxy-8-O-4'-neolignan (17), 7S,8R-erythro-4,7,9,9'- tetrahydroxy-3,3'-dimethoxy-8-O-4'-neolignan (18), and dihydroyashsbushiketol (19). The structures of the new compounds were elucidated by analysis of spectroscopic data including 1D and 2D NMR data. The absolute configurations of 1 and 2 were determined using the convenient Mosher ester procedure. Compounds 5-19 were isolated for the first time from this plant source. The isolated compounds were tested for cytotoxicity against four human tumor cell lines in vitro using a Sulforhodamine B (SRB) bioassay.

Full text of DOI:10.1007/s12272-011-0808-6

Arch Pharm Res Vol 34, No 8, 1289-1296, 2011
DOI 10.1007/s12272-011-0808-6
Phenolic Constituents of Acorus gramineus
Cheol Hyeong Park1, Ki Hyun Kim1, Il Kyun Lee1, Seung Young Lee1, Sang Un Choi2, Jei Hyun Lee3, and
Kang Ro Lee1
2
1Natural Products Laboratory, School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Korea, Korea Research
3
Institute of Chemical Technology, Teajeon 305-600, Korea, and College of Oriental Medicine, Dongguk University, Gyeong
Ju 780-714, Korea
(Received January 3, 2011/Revised April 15, 2011/Accepted April 26, 2011)
The purification of a MeOH extract from the rhizome of Acorus gramineus (Araceae) using col-
umn chromatography furnished two new stereoisomers of phenylpropanoid, acoraminol A (1)
and acoraimol B (
hyde ( ), isoacoramone (
1',2'-dihydroxyasarone (
alcohol (10), kaempferol 3-methyl ether (11), 2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-1,3-
propanediol (12), hydroxytyrosol (13), tyrosol (14), (2 ,5 )-diveratryl-(3 ,4 )-dimethyltet-
rahydrofuran (15), (7 ,8 )-dihydrodehydrodiconiferyl alcohol (16), 7 ,8 threo-4,7,9,9'-tetra-
hydroxy-3,3'-dimethoxy-8- -4'-neolignan (17), 7 ,8 erythro-4,7,9,9'-tetrahydroxy-3,3'-dimeth-
oxy-8- -4'-neolignan (18), and dihydroyashsbushiketol (19). The structures of the new com-
pounds were elucidated by analysis of spectroscopic data including 1D and 2D NMR data. The
absolute configurations of and were determined using the convenient Mosher ester proce-
2
). It also furnished 17 known phenolic compounds,
), propioveratrone ( ), (1' ,2' )-1',2'-dihydroxyasarone (
), 3',4'-dimethoxycinnamyl alcohol ( ), 3',4',5'-trimethoxycinnamyl
β
-asarone (
3
7
), asaralde-
4
5
8
6
R
S
), (1' ,2' )-
S
S
9
S
S
R S
S
R
O
S S-
S R-
O
1
2
dure. Compounds 5-19 were isolated for the first time from this plant source. The isolated
compounds were tested for cytotoxicity against four human tumor cell lines in vitro using a
Sulforhodamine B (SRB) bioassay.
Key words: Acorus gramineus, Araceae, Acoraminol A, Acoraminol B, Cytotoxicity
activities (Lee et al., 2004).
In our continuing study on bioactive natural products
INTRODUCTION
Acorus gramineus (Araceae), which is distributed from Korean traditional medicinal plants, we investi-
throughout Korea, Japan, and China, has been used gated the rhizomes of A. gramineus. In this study, we
as a Korean traditional medicine for learning and isolated two new stereoisomers of phenylpropanoid,
memory improvement, sedation and analgesia (Liao et acoraminol A (
al., 1998). Moreover, this herb has long been used for seventeen known phenolic compounds (
the treatment of stomach ache (Tang and Eisenbrand, four lignan derivatives (15
1
) and acoraminol B (
2
-
), along with
19) including
3
-18) from its MeOH extract.
1992) and swelling as well as for the extermination of Their structures were elucidated by spectroscopic data
insects (Wang et al., 1998). Several pharmacologically including 1D and 2D NMR and comparisons with
active phenolics, such as
β-asarone, α-asarone and reported data. The isolated compounds were tested for
phenylpropenes have been reported from this rhizome cytotoxicity against four human tumor cells in vitro
(Greca et al., 1989). Previous pharmacological studies using a Sulforhodamine B (SRB) bioassay. This paper
on A. gramineus found that its extracts showed describes the isolation and structural elucidation of
neuroprotective (Chun et al., 2008) and antibacterial and , and the cytotoxic activity of all the compounds
19).
1
2
(1-
Correspondence to: Kang Ro Lee, Natural Products Laboratory,
School of Pharmacy, Sungkyunkwan University, Suwon 440-
746, Korea
Tel: 82-31-290-7710, Fax: 82-31-290-7730
E-mail: krlee@skku.ac.kr
MATERIALS AND METHODS
General experimental procedure
Melting points were determined on a Gallenkamp
1289

1290
C. H. Park et al.
melting point apparatus and are uncorrected. Optical mg). The CHCl3 fraction (14 g) was separated over an
rotations were measured on a JASCO P-1020 Polari- RP-C18 silica gel column with 50% MeOH to give seven
meter. IR spectra were recorded on a Bruker IFS-66/S fractions (C1-C7). Fraction C1 (800 mg) was subjected
FT-IR spectrometer. UV spectra were recorded with a to a silica gel column chromatography (CHCl3-MeOH
Schimadzu UV-1601 UV-Visible spectrophotometer. = 40:1) and was purified by an RP-C18 prep. HPLC
NMR spectra were recorded on a Varian UNITY (55% MeOH) to obtain compounds
7 (8 mg) and 8 (63
INOVA 500 NMR spectrometer. EIMS and FABMS mg). Fraction C2 (497 mg) was subjected to a silica
data were obtained on a JEOL JMS700 mass spectro- Lobar A-column (CHCl3-MeOH = 40:1) to give four
meter, and HR-ESIMS data were obtained on an Agilent subfractions (C21-C24). Subfraction C21 (157 mg) was
1100LC/MSD trap SL LC/MS. Preparative HPLC was subjected to Sephadex LH-20 column chromatography
performed using a Gilson 306 pump with a Shodex (CH2Cl2-MeOH = 1:1) and was purified by an RP-C18
refractive index detector and Apollo Silica 5
(250 × 10 mm) or Econosil RP-18 10 column (250 × mg),
µ
column prep. HPLC (65% MeOH) to yield compounds
1 (6
µ
2
(3 mg), (9 mg) and 10 (3 mg). Subfraction C24
9
10 mm). Silica gel 60 (Merck, 70~230 mesh and 230~ (84 mg) was purified by an RP-C18 prep. HPLC (55%
400 mesh) was used for column chromatography. TLC MeOH) to yield compound 17 (40 mg). Fraction C3
was performed using Merck precoated Silica gel F254 (544 mg) was subjected to a silica gel column (CHCl3-
plates and RP-18 F254s plates. The packing material EtOAc-MeOH = 10:1:1) and was purified by an RP-C18
for molecular sieve column chromatography was prep. HPLC (55% MeOH) to give compound 16 (5 mg).
Sephadex LH-20 (Pharmacia Co.). Low-pressure liquid The EtOAc fraction (5 g) was separated over an RP-
chromatography was performed over Merck LiChroprep C18 silica gel column using a solvent system of 50%
Lobar-A Si 60 (240 × 10 mm) or LiChroprep Lobar-A MeOH to give eight fractions (E1-E8). Fraction E2
RP-18 (240 × 10 mm) columns with a FMI QSY-0 (800 mg) was subjected to a silica gel column (CHCl3-
pump (ISCO).
MeOH = 20:1) and was purified by an RP-C18 prep.
HPLC (30% MeOH) to afford compound 13 (9 mg).
Fraction E3 (807 mg) was subjected to a silica gel
Plant materials
The rhizome parts of A. gramineus were collected on column (CHCl3-MeOH = 20:1) to give five subfractions
Jeju Island, Korea in March 2009, and identified by (E31-E35). Subfraction E32 (53 mg) was subjected to
one of us (K.R.Lee). A voucher specimen (SKKU-NPL- Sephadex LH-20 column chromatography (MeOH 80%)
0910) of the plant was deposited in the herbarium of and was purified by a silica gel prep. HPLC using
the School of Pharmacy, Sungkyunkwan University, CHCl3-MeOH (35:1) to yield compound 14 (5 mg).
Suwon, Korea.
Subfraction E33 (53 mg) was subjected to Sephadex
LH-20 column chromatography (MeOH 80%) and was
purified by a silica gel prep. HPLC using CHCl3-MeOH
Extraction and isolation
The rhizome parts of A. gramineus (15 kg) were (25:1) to give compounds 18 (5 mg) and 19 (5 mg).
extracted at room temperature with 80% MeOH to Subfraction E33 (53 mg) was subjected to Sephadex
give a MeOH extract (825 g). The extract was dissolved LH-20 column chromatography (MeOH 80%) and was
in water (2 L) and then successively partitioned with purified by an RP-C18 prep. HPLC (45% MeOH) to
n-hexane, CHCl3, EtOAc, and n-BuOH, yielding 166, afford compound 12 (5 mg). Fraction E7 (90 mg) was
14, 5, and 47 g of residues, respectively. The hexane- subjected to a silica Lobar A-column (CHCl3-MeOH =
soluble fraction (62 g) was separated over a silica gel 25:1) and was purified by a silica gel prep. HPLC
column with
n-hexane-EtOAc (11:1) to yield eight using a solvent system of CHCl3-MeOH (40:1) to yield
fractions (H1-H8). Fraction H3 (24 g) was separated compound 11 (9 mg).
over an RP-C18 silica gel column with 80% MeOH and
was purified by a silica gel prep. HPLC using
hexane-EtOAc (10:1) to yield compounds (2 g) and
(19 mg). Fraction H5 (1 g) was subjected to Sephadex 3410, 2937, 1608, 1512, 1460, 1206; UV (MeOH) λmax
LH-20 column chromatography (CH2Cl2-MeOH = 1:1) nm: 209, 231, 291; HR-ESI-MS : 279.1202 [M +
and was purified by an RP-C18 prep. HPLC (60% Na]+ (calcd for C13H20NaO5, 279.1208); 1H- (CDCl3, 500
MeOH) to give compound
(5 mg). Fraction H6 (606 MHz) and 13C-NMR (CDCl3, 125 MHz): see Table I.
n
4
-
Acoraminol A (1)
White oil; [α]D − c 0.30, CHCl3); IR ν
12.3o ( max cm−1:
25
3
m/z
6
mg) was subjected to Sephadex LH-20 column chro-
matography (CH2Cl2-MeOH = 1:1) and was purified Acoraminol B (2)
14.3o (
c
0.15, CHCl3); IR
max nm: 207, 230,
ν
max cm−1:
25
by using a silica gel prep. HPLC using
EtOAc (2:1) to yield compounds
n
-hexane- Yellow oil; [
α
]
−
D
5
(4 mg) and 15 (6 3419, 2971, 1618, 1512; UV (MeOH)
λ

Phenolic Constituents of Acorus gramineus
1291
290; HR-ESI-MS
C13H20NaO5, 279.1208); 1H- (CDCl3, 500 MHz) and 13C- 6.53 (1H, s, H-3), 6.98 (1H, s, H-6); 13C-NMR (CDCl3,
NMR (CDCl3, 125 MHz): see Table I. 125 MHz): 17.8 (C-3'), 56.4 (-OCH3×2), 56.8 (-OCH3),
m/z
: 279.1203 [M + Na]+ (calcd for (3H, s, -OCH3), 4.08 (1H, m, H-2'), 4.88 (1H, br s, H-1'),
δ
70.6 (C-2'), 73.8 (C-1'), 97.6 (C-3), 112.2 (C-6), 120.2
(C-1), 143.5 (C-5), 149.3 (C-4), 151.1 (C-2).
β
-Asarone (3)
− c 1.85, CHCl3); IR ν
14.0o (
max cm−1:
25
Yellow oil; [α D
2937, 2834, 1608, 1512, 1463, 1209; FAB-MS
[M]+;1H-NMR (CDCl3, 500 MHz):
1.83 (3H, dd, J = 7.0, Yellow oil; [
1.8 Hz, H-3'), 3.74 (3H, s, -OCH3), 3.79 (3H, s, -OCH3), 3389, 2939, 1513, 1207; FAB-MS
3.83 (3H, s, -OCH3), 5.76 (1H, dq, = 10.6, 6.8 Hz, H- NMR (CDCl3, 500 MHz): 1.07 (3H, d,
2'), 6.47 (1H, dq, = 10.6, 1.8 Hz, H-1'), 6.53 (1H, s, H- 3'), 3.83 (3H, s, -OCH3), 3.86 (3H, s, -OCH3), 3.90 (3H,
3), 6.84 (1H, s, H-6); 13C-NMR (CDCl3, 125 MHz):
s, -OCH3), 3.98 (1H, m, H-2'), 4.56 (1H, br s, H-1'), 6.52
14.9 (C-3'), 56.3 (-OCH3), 56.5 (-OCH3), 56.7 (-OCH3), (1H, s, H-3), 6.86 (1H, s, H-6); 13C-NMR (CDCl3, 125
97.9 (C-3), 114.5 (C-6), 118.4 (C-5), 124.5 (C-2'), 125.3 MHz): 18.9 (C-3'), 56.4 (-OCH3×2), 56.8 (-OCH3), 71.5
]
m/z
: 208 (1'S,2'S)-1',2'-Dihydroxyasarone (8)
25
δ
α]
D
+78.7o (
c
1.10, CHCl3); IR
ν
max cm−1:
1
m
/
z
: 242 [M]+; H-
J
δ
J = 6.5 Hz, H-
J
δ
δ
(C-1'), 142.7 (C-1), 148.9 (C-2), 151.8 (C-4).
(C-2'), 75.6 (C-1'), 97.7 (C-3), 112.4 (C-6), 120.7 (C-1),
143.4 (C-5), 149.4 (C-4), 151.3 (C-2).
Asaraldehyde (4)
Colorless oil; [
α
]
25 +39.3o (
c
0.95, CHCl3); IR
ν
max cm−1: 3',4'-Dimethoxycinnamyl alcohol (9)
D
2942, 2833, 1664, 1607, 1514, 1213; FAB-MS
m/z
: 197 Colorless oil; [
3.88 (3H, s, 3361, 2944, 2833; FAB-MS
-OCH3), 3.90 (3H, s, -OCH3), 3.95 (3H, s, -OCH3), 6.48 NMR (CDCl3, 500 MHz):
(1H, s, H-6), 7.30 (1H, s, H-3), 10.29 (1H, s, H-1'); (3H, s, -OCH3), 4.32 (2H, br s, H-3'), 6.26 (1H, dt,
α]
D
25 +18.6o (
c
0.45, CHCl3); IR
ν
max cm−1:
1
1
[M + H]+; H-NMR (CDCl3, 500 MHz):
δ
m
/
z
: 195 [M + H]+; H-
δ
3.89 (3H, s, -OCH3), 3.90
J
=
13C-NMR (CDCl3, 125 MHz):
(-OCH3), 96.3 (C-6), 109.3 (C-3), 117.6 (C-2), 143.8 (C- 6.83 (1H, d,
δ
56.4 (-OCH3×2), 56.5 15.7, 5.8 Hz, H-2'), 6.56 (1H, br d,
J
= 15.7 Hz, H-1'),
J
= 8.0 Hz, H-6), 6.92 (1H, d, J = 8.0 Hz,
4), 156.0 (C-1), 158.9 (C-5), 188.2 (C-1').
H-5), 6.95 (1H, s, H-2); 13C-NMR (CDCl3, 125 MHz):
δ
56.1 (-OCH3), 56.2 (-OCH3), 64.1 (C-3'), 109.2 (C-5),
111.4 (C-2), 119.9 (C-6), 126.8 (C-2'), 130.0 (C-1), 131.4
Isoacoramone (5)
25
Colorless oil; [
α
]
−
17.0o (
c
m/z
0.20, CHCl3); IR
: 225 [M + H]+; 1H-NMR
1.16 (3H, t, = 7 Hz, H-3'), 2.99 3',4',5'-Trimethoxycinnamyl alcohol (10)
(2H, q, J = 7 Hz, H-2'), 3.88 (3H, s, -OCH3), 3.91 (3H, Colorless oil; [ 0.15, CHCl3); IR
25 +10.3o (
s, -OCH3), 3.95 (3H, s, -OCH3), 6.50 (1H, s, H-3), 7.43 3385, 1604; ESI-MS
: 225 [M + H]+; 1H-NMR (CDCl3,
(1H, s, H-6); 13C-NMR (CDCl3, 125 MHz):
8.8 (C-3'), 500 MHz): 3.85 (3H, s, -OCH3), 3.86 (3H, s, -OCH3),
37.3 (C-2'), 56.3 (-OCH3), 56.4 (-OCH3), 56.5 (-OCH3), 3.87 (3H, s, -OCH3), 4.33 (2H, dd, = 5.6, 1.4 Hz, H-
96.8 (C-3), 113.0 (C-6), 119.5 (C-1), 143.3 (C-5), 153.8 3'), 6.29 (1H, dt, = 15.8, 5.6 Hz, H-2'), 6.52 (1H, br d,
= 15.8 Hz, H-1'), 6.63 (2H, s, H-2, H-6); 13C-NMR
(CDCl3, 125 MHz): 56.3 (-OCH3×2), 61.1 (-OCH3),
ν
max cm−1: (C-1'), 149.2 (C-3), 149.3 (C-4).
D
2945, 2832, 1453; ESI-MS
(CDCl3, 500 MHz):
δ
J
α]
D
c
ν
max cm−1:
m/z
δ
δ
J
J
(C-4), 155.3 (C-2), 200.9 (C-1').
J
δ
Propioveratrone (6)
63.9 (C-3'), 103.8 (C-2, C-6), 128.3 (C-2'), 131.4 (C-1'),
Colorless gum; [
α
]
−
10.7o (
c
0.25, CHCl3); IR νmax 132.6 (C-1), 138.2 (C-4), 153.6 (C-3, C-5).
25
D
cm− : 3386, 2940, 1595, 1418; FAB-MS
H]+; 1H-NMR (CDCl3, 500 MHz):
Hz, H-3'), 2.96 (2H, q, J = 7.2 Hz, H-2'), 3.94 (6H, s, Yellow powder; m.p. 270-275oC; [
-OCH3×2), 6.88 (1H, d, J = 8.5 Hz, H-5), 7.54 (1H, s, CH3OH); IR
max cm−1: 3378, 1606; UV (MeOH) λmax
H-2), 7.58 (1H, d, J = 8.0 Hz, H-6); 13C-NMR (CDCl3, nm: 203, 267, 280, 349; ESI-MS : 301 [M + H]+; 1H-
125 MHz): 8.7 (C-3'), 31.5 (C-2'), 56.2 (-OCH3), 56.3 NMR (CD3OD, 500 MHz): 3.78 (3H, s, -OCH3), 6.19
(-OCH3), 110.2 (C-5), 110.5 (C-2), 122.7 (C-6), 130.4 (1H, d, = 2.0 Hz, H-6), 6.39 (1H, d, = 2.0 Hz, H-8 ),
(C-1), 149.3 (C-3), 153.3 (C-4), 199.7 (C-1'). 6.91 (2H, d, = 9.0 Hz, H-3', H-5'), 7.96 (2H, d,
Hz, H-2', H-6'); 13C-NMR (CD3OD, 125 MHz):
m/z: 195 [M +
1
δ
1.22 (3H, t, J = 7.0 Kaempferol 3-methyl ether (11)
+14.0o (
c 0.45,
D
25
α
]
ν
m/z
δ
δ
J
J
J
J
δ
= 9.0
59.4
(1'R,2'
Colorless gum; [α D
S
)-1'
,
2'-Dihydroxyasarone (7)
(-OCH3), 93.6 (C-8), 98.6 (C-6), 104.7 (C-10), 115.4 (C-
25
]
+31.0o (
c
0.40, CHCl3); IR νmax 3', C-5'), 121.4 (C-1'), 130.2 (C-2', C-6'), 138.3 (C-3),
cm−1: 3379 , 2943, 2834, 1207; FAB-MS
1H-NMR (CDCl3, 500 MHz):
1.32 (3H, d,
H-3'), 3.83 (3H, s, -OCH3), 3.86 (3H, s, -OCH3), 3.90
m/z
: 242 [M]+; 156.9 (C-2), 157.3 (C-9), 160.5 (C-4'), 161.9 (C-5), 164.8
δ
J = 6.5 Hz, (C-7), 178.8 (C-4).

1292
C. H. Park et al.
2-[4-(3-Hydroxypropyl)-2-methoxyphenoxy]-1,3-
Colorless oil; [α D
3371, 2939, 1607, 1517, 1460; UV (MeOH) λmax nm:
] c 0.50, MeOH); IR ν
25 +14.2o ( max cm−1:
propanediol (12)
25
1
Colorless gum; [
α]
+18.4o (
c
0.25, MeOH); IR νmax 212, 282; FAB-MS
m/z
: 360 [M]+; H-NMR (CD3OD,
D
cm−1: 3382, 1602; ESI-MS
(CD3OD, 500 MHz):
m
/
z
: 257 [M + H]+; 1H-NMR 500 MHz):
δ
1.80 (2H, m, H-8'), 2.62 (2H, t, = 7.2 Hz,
1.79-1.84 (2H, m, H-2'), 2.63 (2H, H-9'), 3.45 (1H, m, H-8), 3.46 (1H, dd, = 11.2, 6.4 Hz,
t, J = 7.7 Hz, H-1'), 3.55 (2H, t, J = 6.6 Hz, H-3'), 3.74 H-9), 3.56 (2H, t, = 6.4 Hz, H-9'), 3.74 (1H, dd,
(4H, d, J = 5.1 Hz, H-1'', H-3''), 3.84 (3H, s, -OCH3), 4.15 11.2, 5.6 Hz, H-9), 3.79 (3H, s, -OCH3), 3.82 (3H, s,
J
δ
J
J
J =
(1H, q, J = 5.1 Hz, H-2''), 6.73 (1H, dd, J = 8.1, 2.2 Hz, -OCH3), 5.47 (1H, d,
H-6), 6.85 (1H, d, J = 2.2 Hz, H-2), 6.99 (1H, d, J = 8.1 8.0 Hz, H-5), 6.78 (2H, s, H-2', H-6'), 6.83 (1H, dd,
Hz, H-5); 13C-NMR (CD3OD, 125 MHz):
31.5 (C-1'), 8.0, 1.6 Hz, H-6), 6.95 (1H, d,
34.4 (C-2'), 55.3 (-OCH3), 60.9 (C-1'', C-3''), 61.0 (C-3'), NMR (CD3OD, 125 MHz): 31.7 (C-7'), 34.6 (C-8'),
82.2 (C-2''), 112.9 (C-2), 118.3 (C-5), 120.7 (C-6), 137.1 54.2 (C-8), 55.2 (-OCH3), 55.6 (-OCH3), 61.1 (C-9'), 63.8
J
= 6.2 Hz, H-7), 6.72 (1H, d, J
=
J
=
δ
J
= 1.6 Hz, H-2); 13C-
δ
(C-1), 145.7 (C-4), 152.0 (C-3).
(C-9), 87.8 (C-7), 109.4 (C-2), 113.0 (C-2'), 115.0 (C-5),
116.8 (C-6'), 118.6 (C-6), 128.6 (C-5'), 133.6 (C-1), 135.7
(C-1'), 144.0 (C-3'), 146.3 (C-4), 146.4 (C-4'), 147.9 (C-
Hydroxytyrosol (13)
25
Brown oil; [α D
]
+30.6o (
c
0.02, MeOH); IR
: 154 [M]+; H-
2.66 (2H, t, = 7.3 Hz, H-
= 7.3 Hz, H-2'), 6.52 (1H, dd, = 8.0, 8-
2.1 Hz, H-6), 6.59 (1H, d, = 2.1 Hz, H-2), 6.67 (1H, d, Colorless gum; [
= 8.0 Hz, H-5); 13C-NMR (CD3OD, 125 MHz): 38.5 cm−1: 3386, 1604; UV (MeOH)
(C-1'), 63.4 (C-2'), 115.1 (C-2), 115.9 (C-5), 120.0 (C-6), ESI-MS
ν
max cm−1: 3).
1
3375, 2946, 1606, 1447; FAB-MS
NMR (CD3OD, 500 MHz):
1'), 3.67 (2H, t,
m/z
δ
J
7S,8S-threo-4,7,9,9'-Tetrahydroxy-3,3'-dimethoxy-
J
J
O-4'-neolignan (17)
25
J
α
]
+10.2o (
c
0.25, MeOH); IR νmax
λmax nm: 207, 228, 280;
1
D
J
δ
m/z
: 401 [M + Na]+; H-NMR (CD3OD, 500
1.80 (1H, m, H-8'), 2.62 (2H, t, = 7.0 Hz, H-
7'), 3.55 (2H, t, = 6.5 Hz, H-9'), 3.79 (3H, s, -OCH3),
130.6 (C-1), 143.4 (C-4), 145.0 (C-3).
MHz):
δ
J
J
Tyrosol (14)
Brown oil; [α D
3.80 (3H, s, -OCH3), 4.19 (1H, m, H-8), 4.87 (1H, d, J
25
]
+32.6o (
c
0.37, MeOH); IR
ν
max cm−1: = 6.0 Hz, H-7), 6.68-7.02 (6H, m, H-2, H-5, H-6, H-2',
1
3359, 2940, 1514, 1444; FAB-MS
NMR (CD3OD, 500 MHz): 2.71 (2H, t,
1'), 3.67 (2H, t, = 7.3 Hz, H-2'), 6.70 (2H, dd,
2.1 Hz, H-3, H-5), 7.02 (2H, dd, = 8.0, 2.1 Hz, H-2, 114.7 (C-5), 118.5 (C-5'), 119.6 (C-6), 120.9 (C-6'), 132.6
H-6); 13C-NMR (CD3OD, 125 MHz):
38.2 (C-1'), 63.4 (C-1), 137.1 (C-1'), 145.8 (C-4), 146.0 (C-4'), 147.7 (C-
m/z
: 138 [M]+; H- H-5', H-6'); 13C-NMR (CD3OD, 125 MHz):
δ
31.5 (C-7'),
δ
J
= 7.0 Hz, H- 34.4 (C-8'), 55.2 (-OCH3), 55.4 (-OCH3), 60.7 (C-9), 61.0
= 8.0, (C-9'), 73.0 (C-7), 86.6 (C-8), 110.6 (C-2), 112.8 (C-2'),
J
J
J
δ
(C-2'), 114.9 (C-3, C-5), 129.7 (C-2, C-6), 130.7 (C-1), 3), 150.5 (C-3').
155.6 (C-4).
7
S
,8
oxy-8-
Colorless gum; [ 0.25, MeOH); IR νmax
max cm−1: cm−1: 3375, 1513; UV (MeOH)
λmax nm: 209, 227, 280;
1
R
-
erythro-4,7,9,9'-Tetrahydroxy-3,3'-dimeth-
(2
S
,5
S
)-Diveratryl-(3
R
,4
S
)-dimethyltetrahydro-
O-4'-neolignan (18)
25
furan (15)
Colorless oil; [
3381, 2968, 1600; UV (MeOH)
ESI-MS
δ
α]
+16.6o (
c
D
25
α]
−
16.6o (
c
0.30, CHCl3); IR
max nm: 206, 276, 393; ESI-MS
: 373 [M + H]+; 1H-NMR (CDCl3, 500 MHz): MHz):
0.60 (1H, d, = 7.0 Hz, H-9'), 1.01 (1H, d, = 6.3 Hz, 7'), 3.54 (2H, t,
H-9), 2.45 (2H, m, H-8, H-8'), 3.87 (3H, s, -OCH3), 3.88 3.80 (3H, s, -OCH3), 4.28 (1H, m, H-8), 4.83 (1H, d,
(3H, s, -OCH3), 3.89 (3H, s, -OCH3), 3.90 (3H, s, -OCH3), = 4.0 Hz, H-7), 6.65-7.02 (6H, m, H-2, H-5, H-6, H-2',
4.66 (1H, d, = 9.1 Hz, H-7), 5.46 (1H, d, 31.5 (C-7'),
= 5.4 Hz, H-5', H-6'); 13C-NMR (CD3OD, 125 MHz):
H-7'), 6.82-6.87 (4H, m, H-5, H-5', H-6, H-6'), 6.92 (1H, 34.3 (C-8'), 55.2 (-OCH3), 55.3 (-OCH3), 61.0 (C-9), 62.3
d, = 1.3 Hz, H-2'), 6.96 (1H, d,
= 1.8 Hz, H-2); 13C- (C-9'), 73.0 (C-7), 85.5 (C-8), 110.7 (C-2), 112.9 (C-2'),
NMR (CDCl3, 125 MHz): 9.7 (C-9'), 12.1 (C-9), 43.7 114.5 (C-5), 118.5 (C-5'), 119.8 (C-6), 120.7 (C-6'), 133.0
ν
D
λ
m/z
: 401 [M + Na]+; H-NMR (CD3OD, 500
1.80 (1H, m, H-8'), 2.60 (2H, t, = 7.0 Hz, H-
= 6.5 Hz, H-9'), 3.79, (3H, s, -OCH3),
m/z
δ
J
J
J
J
J
J
J
δ
J
J
δ
(C-8), 47.8 (C-8'), 56.1 (-OCH3×2), 56.2 (-OCH3×2), (C-1), 136.9 (C-1'), 145.8 (C-4), 146.1 (C-4'), 147.5 (C-
85.0 (C-7'), 85.9 (C-7), 109.4 (C-2), 109.7 (C-2'), 111.1 3), 150.7 (C-3').
(C-5'), 111.2 (C-5), 118.3 (C-6'), 118.7 (C-6), 133.5 (C-
1'), 135.9 (C-1), 148.0 (C-4), 148.7 (C-3), 149.0 (C-4'), Dihydroyashsbushiketol (19)
25
149.4 (C-3').
Colorless needle; [α D
]
−
215.3o (
c
0.10, MeOH); IR νmax
cm−1: 3394, 2923, 1604, 1424; FAB-MS
m/z: 283 [M +
1
(7 ,8 )-Dihydrodehydrodiconiferyl alcohol (16)
S
R
H]+; H-NMR (CDCl3, 500 MHz):
δ
1.65-1.81 (2H, m,

Phenolic Constituents of Acorus gramineus
1293
H-8'), 2.55 (4H, t,
H-8), 2.90 (2H, m, H-10), 4.03 (1H, q,
7.19 (10H, br s, H-1, H-2, H-3, H-4, H-5, H-1', H-2', H- OV-3 (ovarian cancer cells), SK-MEL-2 (skin melanoma),
3', H-4', H-5'); 13C-NMR (CDCl3, 125 MHz):
29.8 (C- and HCT15 (colon cancer cells). Doxrubicin was used
J
= 7.0 Hz, H-7, H-7'), 2.74 (2H, m, Technology against four cultured human tumor cell
J
= 7.0 Hz, H-9'), lines: A549 (non-small cell lung adenocarcinoma), SK-
δ
7'), 32.0 (C-7), 38.3 (C-8'), 45.3 (C-8), 49.5 (C-10), 67.1 as a positive control. The cytotoxicity of doxorubicin
(C-9'), 126.1 (C-4), 126.5 (C-4'), 128.5 (C-2, C-6), 128.7 against A549, SK-OV-3, SK-MEL-2, and HCT15 cell
(C-2', C-6', C-3', C-5'), 128.8 (C-3, C-5), 140.9 (C-1'), lines were IC50 0.02, 0.01, 0.01, and 0.04 µM, respec-
142.0 (C-1), 211.3 (C-9).
tively.
Preparation of the (R)- and (S)-MTPA ester
RESULTS AND DISCUSSION
derivatives of 1 and 2
Compound (1.5 mg) in deuterated pyridine (0.75
mL) was transferred to a clean NMR tube. ( )-(+)-
methoxy-
ride (10
1
Compound 1 was obtained as white oil. The HR-
S
α-
ESI-MS ([M + Na]+ at
m/z
279.1202, calcd for 279.1208)
gave the
1
α
µ
-(trifluoromethyl)phenylacetyl (MTPA) chlo- and H- and 13C-NMR spectral data of
1
L) was added to the NMR tube immediately molecular formula of C13H20O5. The IR spectrum of 1
under a N2 gas stream. The NMR tube was then indicated the presence of an OH (3410 cm−1) and an
shaken carefully to thoroughly mix the sample and aromatic system (1608 cm−1). The H-NMR spectrum
1
the MTPA chloride. The NMR reaction tube was left of
at room temperature overnight. The reaction was then (1H, s) and 6.83 (1H, s), one methyl proton at
completed to afford the ( )-MTPA ester derivative (1r (3H, d, = 6.5 Hz) and four methoxy protons at
of . Treatment of (1.5 mg) with (
L) as described above yielded the corresponding (
MTPA ester 1s. Similarly, treatment of with (
and (
esters 2r and 2s. The H-NMR spectra of 1r
1
(Table I) showed two aromatic protons at
δ
δ
δ
6.54
1.01
3.24
R
)
J
1
1
R)-MTPA-Cl (10 (3H, s, alcoholic OMe), 3.82 (3H, s), 3.85 (3H, s), and
µ
S
S
)- 3.91 (3H, s). The 13C-NMR spectrum of
)- carbon resonances, which were classified by DEPT
1 indicated 13
2
R
)-MTPA-Cl afforded the respective Mosher and HMQC experiments as six aromatic carbons at δ
1
,
1s,
2r,
97.6, 111.0, 118.5, 143.8, 149.4, and 152.7, a methyl at
17.9, two oxygenated methines at 71.7 and 81.8,
and four methoxy carbons at 56.3, 56.7, 56.8, and
1.168 (3H, 56.9. Further analysis showed that the data of were
and 2s were measured directly in the NMR reaction
δ
δ
tubes.
δ
1s. 1H-NMR (pyridine-d5, 500 MHz):
δ
1
d, J = 6.5 Hz, H-3'), 3.261 (3H, s, -OCH3), 3.688 (3H, very similar to those of the known compound, 1-(2,4,5-
s, -OCH3), 3.704 (3H, s, -OCH3), 3.767 (3H, s, -OCH3),
4.207 (1H, dq, J = 8.0, 6.5 Hz, H-2'), 5.681 (1H, d, J =
Table I. 1H- (500 MHz) and 13C-NMR (125 MHz) spectral
data of compounds 1-2 in CDCl3 (δ in ppm)
8.0 Hz, H-1'), 6.717 (1H, s, H-3), 7.175 (1H, s, H-6).
1r. 1H-NMR (pyridine-d5, 500 MHz):
1.218 (3H, d,
δ
1
2
Position
J = 6.5 Hz, H-3'), 3.249 (3H, s, -OCH3), 3.688 (3H, s, -
OCH3), 3.704 (3H, s, -OCH3), 3.752 (3H, s, -OCH3),
4.202 (1H, dq, J = 8.0, 6.5 Hz, H-2'), 5.364 (1H, d, J =
8.0 Hz, H-1'), 6.717 (1H, s, H-3), 7.175 (1H, s, H-6).
δH
δC
δH
δC
1
2
3
4
5
118.5
152.7
197.6 6.54 (s)
149.4
143.8
111.0 6.92 (s)
117.9
152.3
197.6
149.3
143.7
111.4
180.9
6.54 (s)
6.83 (s)
2s. 1H-NMR (pyridine-d5, 500 MHz):
δ 1.271 (3H, d,
J = 6.5 Hz, H-3'), 3.334 (3H, s, -OCH3), 3.691 (3H, s,
-OCH3), 3.735 (3H, s, -OCH3), 3.769 (3H, s, -OCH3),
4.365 (1H, dq, J = 6.5, 4.5 Hz, H-2'), 5.753 (1H, d, J =
4.5 Hz, H-1'), 6.704 (1H, s, H-3), 7.176 (1H, s, H-6).
6
1'
2'
3'
4.42 (d, 8.0)
181.8 4.60 (d, 4.5)
3.82 (dq, 8.0, 6.5) 171.7 3.98 (dq, 6.5, 4.5) 171.7
2r. 1H-NMR (pyridine-d5, 500 MHz):
δ 1.324 (3H, d,
1.01 (d, 6.5)
117.9 1.09 (d, 6.5)
156.9a 3.90 (s)
156.8a 3.86 (s)
156.7a 3.81 (s)
156.3 3.30 (s)
118.1
157.4b
156.8b
156.5b
156.3
J = 6.5 Hz, H-3'), 3.320 (3H, s, -OCH3), 3.676 (3H, s,
-OCH3), 3.720 (3H, s, -OCH3), 3.762 (3H, s, -OCH3),
4.345 (1H, dq, J = 6.5, 4.5 Hz, H-2'), 5.697 (1H, d, J =
4.5 Hz, H-1'), 6.700 (1H, s, H-3), 7.141 (1H, s, H-6).
2-OCH3 3.91 (s)
4-OCH3 3.85 (s)
5-OCH3 3.82 (s)
1'-OCH3 3.24 (s)
Assignments were based on 2D NMR methods, including
HMQC and HMBC. Well-resolved couplings are expressed
with coupling patterns and coupling constants (in Hz)
given in parentheses.
*NMR assignments with the same superscript may be in-
terchanged.
Test for cytotoxicity in vitro
Sulforhodamine B bioassays (SRB) were used as
cytotoxicity screening methods (Skehan et al., 1990).
Cytotoxicity assays for each compound were done in
vitro at the Korea Research Institute of Chemical

1294
C. H. Park et al.
Fig. 1. The structures of compounds isolated from A. gramineus
trimethoxyphenyl)-1-methoxypropan-2-ol, whose abso- these protons have a cis-orientation and, thus the
lute configuration has not been confirmed (Nawamaki absolute configuration at C-1' was confirmed to be
and Kuroyanagi, 1996). The structure of was unam- (Herrera Braga et al., 1984). On the basis of the above
biguously confirmed by the HMBC experiment, which evidence, the structure of was established as shown
showed correlations of H-1' ( 4.42)/C-1 ( 118.5), C-2 in Fig. 1 and named acoraminol A.
152.7), and C-6 ( 111.0), H-3' ( 1.01)/C-2' ( 71.7) Compound , obtained as yellow oil, had a mole-
and C-1' ( 81.8), and H-6 (
absolute configuration of
S
1
1
δ
δ
(
δ
δ
δ
δ
2
δ
δ 6.83)/C-1' (δ 81.8). The cular formula of C13H20O5, as obtained from the HR-
1
was established on the ESI-MS ([M + Na]+ at
m/z
279.1203, calcd for 279.1208)
. The IR
indicated the presence of an OH (3419
-(trifluoromethyl)phenylacetyl (MTPA)-Cl gave the cm−1) and an aromatic system (1618 cm−1). Inspection
basis of the convenient Mosher ester procedure (Su et and H- and 13C-NMR spectral data of
2
1
al., 2002). Treatment of
1 with (R)- and (S)-α-methoxy- spectrum of 2
α
1
(S
)- and (
R
)-MTPA esters 1s and 1r, respectively. of the H- and 13C-NMR data of
2
(Table I) revealed
, except
Diagnostic 1H-NMR chemical shift differences between that these data were very similar to those of
the MTPA esters of δS R] (Fig. 2) revealed the for the chemical shifts from C-1' to C-3' [ 4.60 (1H, d,
absolute configuration at C-2' to be . The coupling = 4.5 Hz, H-1'), 3.98 (1H, dq, = 6.5, 4.5 Hz, H-2'),
constant ( = 8.0 Hz) for H-1' and H-2' suggested that and 1.09 (3H, d, = 6.5 Hz, H-3'); 80.9 (C-1'), 71.7
1
1
[
-
δ
δ
S
J
J
J
J
δ
Fig. 2. Values of δS
MTPA esters of
- δR (data obtained in pyridine-d5) of the
Fig. 3. Values of δS
MTPA esters of
-
δR (data obtained in pyridine-d5) of the
1
2

Phenolic Constituents of Acorus gramineus
1295
(C-2'), and 18.1 (C-3') in
1'), 3.82 (1H, dq,
d, = 6.5 Hz, H-3');
(C-3') in ]. Analysis of the 2D NMR data of
HMBC, and NOESY) led to unambiguous 1H- and 13C- istration in Korea.
NMR assignments (Table I), and confirmed to be the
stereoisomer of . As described for , the absolute con-
figuration of was determined using the convenient
Mosher ester procedure (Su et al., 2002), which proved
the -configuration for C-2' (Fig. 3). In addition, the
coupling constant ( = 4.5 Hz) for H-1' and H-2' indi-
2
;
δ
4.42 (1H, d,
J
= 8.0 Hz, H- Dr. Eun Jung Bang, and Dr. Jung Ju Seo at the Korea
J
= 8.0, 6.5 Hz, H-2'), and 1.01 (3H, Basic Science Institute for the NMR and MS spectra
81.8 (C-1'), 71.7 (C-2'), and 17.9 measurements. This work was supported by grant No.
(HMQC, 09112KFDA890 from the Korea Food & Drug Admin-
J
δ
1
2
2
1
1
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