Antineuroinflammatory constituents from the root extract of Paris verticillata

Article abstract of DOI:10.1139/V10-166

Two new cyclopropanoic fatty acid glycosides, named parisveroside A (1) and parisveroside B (2), were isolated from a MeOH extract of the roots of Paris verticillata (Liliaceae) together with three other known compounds, salicin (3), 3-(-d-glucopyranosyloxymethyl)-2-(4-hydroxy-3-methoxyphenyl)-5-(3- hydroxypropyl)-7-methoxy-(2R,3S)-dihydrobenzofuran (4), and allantoin (5). Their structures were elucidated on the basis of spectroscopic data, including 1D and 2D NMR, HR-FAB-MS, and chemical evidence. To investigate the antineuroinflammatory effects of the isolated compounds (1-5), nitric oxide (NO) production was evaluated in the lipopolysaccharide-activated microglia cell line, BV-2. Compounds 2 and 4 significantly inhibited NO production with IC ₅₀ values of 74.8 and 60.5 mol/L, respectively.

Full text of DOI:10.1139/V10-166

441
Antineuroinflammatory constituents from the root
extract of Paris verticillata
Ki Hyun Kim, Kyu Ha Lee, Ho Kyung Kim, Eunjung Moon, Sung-Hoon Kim,
Sun Yeou Kim, Kyung Ran Kim, and Kang Ro Lee
Abstract: Two new cyclopropanoic fatty acid glycosides, named parisveroside A (1) and parisveroside B (2), were iso-
lated from a MeOH extract of the roots of Paris verticillata (Liliaceae) together with three other known compounds, sali-
cin (3), 3-(b-D-glucopyranosyloxymethyl)-2-(4-hydroxy-3-methoxyphenyl)-5-(3-hydroxypropyl)-7-methoxy-(2R,3S)-
dihydrobenzofuran (4), and allantoin (5). Their structures were elucidated on the basis of spectroscopic data, including 1D
and 2D NMR, HR-FAB-MS, and chemical evidence. To investigate the antineuroinflammatory effects of the isolated com-
pounds (1–5), nitric oxide (NO) production was evaluated in the lipopolysaccharide-activated microglia cell line, BV-2.
Compounds 2 and 4 significantly inhibited NO production with IC50 values of 74.8 and 60.5 mmol/L, respectively.
Key words: Paris verticillata, Liliaceae, fatty acid glycosides, parisveroside A, parisveroside B, neuroinflammation.
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Resume : Utilisant comme produit de depart le melange obtenu par extraction des racines de Paris verticillata (Liliacee) a
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l’aide de methanol, on a isole deux nouveaux glycosides derives de l’acide gras cyclopropanoıque, soit le parisveroside A
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(1) et le parisveroside B (2), ainsi que trois autres produits connus, la salicine (3), le 3-(b-D-glucopyranosyloxymethyl)-2-
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(4-hydroxy-3-methoxyphenyl)-5-(3-hydroxypropyl)-7-methoxy-(2R,3S)-dihydrobenzofurane (4) et l’allantoıne (5). On a elu-
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cide les structures en se basant sur des preuves chimiques et des donnees spectroscopiques, dont celles des RMN 1D et 2D
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et de la spectrometrie de masse a haute resolution avec bombardement avec des atomes rapides (SM-HR-BAR). Pour etu-
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dier les effets anti-neuroinflammatoires des composes isoles, on a evalue la production d’oxyde d’azote (NO) dans des cel-
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lules BV-2 de la lignee cellulaire de microglie activee par un lipopolysaccharide. Les composes 2 et 4 inhibent d’une
fac¸on significative la production de NO avec des valeurs de IC50 de 74,8 et 60,5 mmol/L respectivement.
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Mots-cles : Paris verticillata, Liliacee, glycosides d’acides gras, parisveroside A, parisveroside B, neuroinflammation.
stituents from the roots of P. verticillata, we further isolated
Introduction
two new cyclopropanoic fatty acid glycosides, named paris-
veroside A (1) and parisveroside B (2), together with three
previously reported compounds (3–5). Compounds 3–5 were
characterized for the first time from this plant. Here, we also
investigated the antineuroinflammatory effects of isolated
compounds (1–5) in lipopolysaccharide (LPS)-activated BV-
2 cells, a microglia cell line.
The genus Paris consists of less than two dozen herba-
ceous plants that have 4- to 11-merous flowers.1 Some Paris
species are used in traditional Chinese medicine for their an-
algesic and anticoagulant properties, most notably as an in-
gredient of Yunnan Baiyao.2 In Korea, the perennial herb
Paris verticillata (Liliaceae) is widely distributed in valleys
and its root has been used as a Korean traditional medicine
against asthma, boils, and chronic bronchitis.3 Few phyto-
chemical and biological investigations on this plant have
been previously conducted. The presence of several constit-
uents, including saponins, sterols, and flavonoids, was re-
ported from the aerial parts of P. verticillata.4 In our
continuing search for bioactive constituents from Korean
medicinal plants, we investigated a MeOH extract from the
roots of P. verticillata, which is in use as traditional medi-
cine. The investigation led to the isolation of a phenolic
amide, phenolics, and pyrrolizidine alkaloids with cytotoxic
activity.5,6 In our continuing investigation of bioactive con-
Results and discussion
The 80% MeOH extract of the roots of P. verticillata was
subjected to repeated column chromatography on silica gel
to afford two new cyclopropanoic fatty acid glycosides (1–
2), together with three known compounds (3–5) (Fig. 1).
Their structures were elucidated by 1D and 2D NMR and
HR-FAB-MS and compared with published data.
Parisveroside A (1) was isolated as a colorless gum with a
molecular formula of C17H30O8 on the basis of the [M + H]+
peak at m/z 363.2021 (calcd. for C17H31O8: 363.2019) in the
Received 24 August 2010. Accepted 18 November 2010. Published on the NRC Research Press Web site at canjchem.nrc.ca on
16 February 2011.
K.H. Kim, K.H. Lee, H.K. Kim, K.R. Kim, and K.R. Lee.1 Natural Products Laboratory, School of Pharmacy, Sungkyunkwan
University, Suwon 440-746, Korea.
E. Moon and S.Y. Kim. Graduate School of East-West Medical Science, Kyung Hee University, No. 1 Seocheon-dong, Gihung-gu,
Yongin-city, Gyeonggi-do 446-701, Korea.
S.-H. Kim. Cancer preventive Material Development Research center, Kyung Hee University, Seoul 151-742, Korea.
1Corresponding author (e-mail: krlee@skku.ac.kr).
Can. J. Chem. 89: 441–445 (2011)
doi:10.1139/V10-166
Published by NRC Research Press

442
Can. J. Chem. Vol. 89, 2011
1
Fig. 1. Chemical structures of compounds 1–5.
Fig. 2. Key H–1H COSY and HMBC correlations of 1.
is located at C-9. The stereochemistry, including the abso-
lute configuration at C-9, was established by a combination
of analyses, including correlations in the NOESY experi-
ment and the modified Mosher’s method.9 The proton sig-
nals for cyclopropylmethylene at d 0.73 and –0.11 revealed
the presence of a cis-1,2-disubstituted cyclopropane ring.7,8
In the trans form, there are no signals for the cyclopropane
proton above d 0. The cis-cyclopropylmethine protons were
also confirmed by the NOESY correlation H-3/H-5. On en-
zymatic hydrolysis of 1 with b-glucosidase, a new fatty acid,
9-hydroxy-cis-cascarillic acid (1a) was obtained. Compound
1a was treated with (+)- and (–)-a-methoxy-a-trifluorome-
thylphenyl acetyl (MTPA) chlorides in NMR tubes to afford
(R)-MTPA ester (1r) and (S)-MTPA ester (1s), respectively.9
1
Diagnostic H NMR chemical-shift differences between the
MTPA esters of 1 (Dd = dS – dR) (Fig. 3) indicated the 9R
configuration of 1. Thus, the structure of 1 was determined
as 9R-(b-D-glucopyranosyloxy)-cis-cascarillic acid, and it
was named parisveroside A (Fig. 1).
HR-FAB-MS. The IR spectrum of 1 indicated the presence
Parisveroside B (2) was isolated as a colorless gum. Its
molecular formula was determined to be C18H32O8 from the
[M + Na]+ peak at m/z 399.2012 (calcd. for C18H32NaO8:
399.1995) in the HR-FAB-MS spectrum. Its IR spectrum re-
of hydroxyl groups (3390 cm–1) and a carbonyl group
1
(1698 cm–1). The H NMR spectrum of 1 showed an anome-
ric proton at d 4.34 (1H, d, J = 8.0 Hz, H-1’), which is a
characteristic signal for a b-glucopyranosyl unit, and six
oxygenated protons attributed to a sugar at d 3.86–3.26. It
also displayed signals for one methyl at d 0.93 (3H, t, J =
6.5 Hz, H-11), 10 methylenes at d 2.34–2.24 (2H, m, H-2),
1.62–1.33 (8H, m, H-6, 7, 8, 10), and one oxygenated me-
thine at d 3.75 (1H, m, H-9), as well as a cyclopropane ring
at d 1.08 (1H, m, H-3), 0.82 (1H, m, H-5), 0.73 (1H, m, H-
4a), and –0.11 (1H, q, J = 5.5 Hz, H-4b). The 13C NMR and
DEPT spectra of 1 showed signals of 11 carbons of an agly-
cone attributable to one methyl, six methylenes, three me-
thines (one oxygenated), and one quaternary carbon
(carboxyl), along with signals for a b-glucopyranosyl unit (d
102.3, 77.0, 76.5, 74.1, 70.6, and 61.7). These data sug-
gested that compound 1 was a glycosidic fatty acid with a
cyclopropane ring.7,8 The glucose unit obtained after hydrol-
1
vealed similar absorption bands to 1. The H and 13C NMR
spectra of 2 were almost identical to those of 1, except for
the resonance of one additional methoxy signal. On the basis
1
of the H–1H COSY and HSQC spectra, all protons and car-
bons could be assigned. The HMBC correlation between the
methoxy proton at d 3.69 and C-1 at d 174.9 indicated that
the methoxy group was located at C-1. Enzymatic hydrolysis
of 2 yielded a new fatty acid, methyl-9-hydroxy-cis-cascaril-
late (2a) and glucose as the sugar component, having a b-D-
1
configuration from the H and 13C NMR data and optical ro-
tation analysis. The glycosidic linkage was determined from
the HMBC correlation H-1’/C-9. The stereochemistry of 2
was assigned to be the same as that of 1 on the basis of the
comparison of the NMR data and optical rotation values:
25
½aꢀD +26.6 (c 0.10, MeOH) of 2 with those of 1. Thus, the
25
ysis gave a positive optical rotation, ½aꢀD +48.6 (c 0.05,
structure of 2 was determined as methyl-9R-(b-D-glucopyra-
nosyloxy)-cis-cascarillate, and it was named parisveroside B
(Fig. 1).
H2O), indicating that it was D-glucose.
The proton and protonated carbon NMR signals of 1 were
assigned unambiguously by the HSQC experiment. The cy-
Three other known compounds obtained in this investiga-
tion were identified as salicin (3),10 3-(b-D-glucopyranosy-
loxymethyl)-2-(4-hydroxy-3-methoxyphenyl)-5-(3-hydroxypropyl)-
7-methoxy-(2R,3S)-dihydrobenzofuran (4),11 and allantoin (5)12
by comparison of their spectroscopic data with those pub-
lished in the literature (Fig. 1). The known compounds 3–5
are reported from this plant source for the first time.
1
clopropanoic fatty acid was further confirmed by the H–1H
COSY correlations from H-2 to H-11 and the HMBC corre-
lations H-2/C-5, H-3/C-1, H-4/C-2, 6, H-9/C-7, and H-11/C-9
(Fig. 2), together with their chemical shift values. The
HMBC correlation of the anomeric proton H-1’ (d 4.34)
with C-9 (d 80.7) indicated that the b-D-glucopyranosyl unit
Published by NRC Research Press

Kim et al.
443
Fig. 3. Dd Values (dS – dR) in ppm of the two a-methoxy-a-tri-
fluoromethylphenyl acetyl (MTPA) esters derived from 1a.
Plant material
The roots of P. verticillata were collected at Mount O-
Dae, Gangwon Province, Korea, in August 2002, and the
plants were identified by one of the authors (K.R.L.). A
voucher specimen (SKKU 2002-08) was deposited in the
herbarium of the School of Pharmacy, Sungkyunkwan Uni-
versity, Suwon, Korea.
Microglia, the unique population of central nervous sys-
tem resident immune cells, have been implicated in the
pathogenesis of a variety of neurodegenerative diseases in-
cluding Parkinson’s disease and Alzheimer’s disease.13,14
Excessive production of nitric oxide (NO) and proinflamma-
tory cytokines from activated microglia play an important
role in neurodegenerative disorders. Therefore, regulation of
the microglial activation will be an important strategy to
prevent the progressive damage of the neurodegenerative
diseases. Here, the antineuroinflammatory activities of the
isolated compounds (1–5) were evaluated by assessing NO
production in the BV-2 cells. BV-2 cells were pretreated
with or without compounds 1–5 for 30 min prior to the ad-
dition of LPS (100 ng/mL). As shown in Fig. 4, compounds
2 and 4 significantly inhibited the NO production in LPS-
stimulated BV-2 cells. Among tested compounds, compound
4 was the most potent. Pretreatment of BV-2 cells with com-
pound 4 inhibited LPS-induced NO production in a dose-
dependent manner with an IC50 value of 60.5 mmol/L. It re-
vealed effective ratios of 23.9% 5.7%, 30.2% 6.2%, and
44.9% 3.5% at concentrations of 10, 20, and 50 mmol/L,
respectively, in comparison with the control. The IC50 value
of compound 2 was also 74.8 mmol/L. On the other hand,
none of the compounds showed any cytotoxicity at concen-
trations used in the present study (data not shown).
Extraction and isolation
The roots of P. verticillata (1.5 kg) were extracted using
80% aq MeOH at room temperature over a period of 3 days
three times. The solvent was evaporated under reduced pres-
sure to give a crude extract (180 g). The crude extract was
dissolved in distilled water (800 mL) and successively ex-
tracted with n-hexane, CH2Cl2, EtOAc, and n-BuOH to pro-
vide n-hexane (5.2 g), CH2Cl2 (3.5 g), EtOAc (4.4 g), and n-
BuOH extracts (20 g). The EtOAc-soluble fraction (4.4 g)
was subjected to normal-phase CC over a silica gel using
CHCl3–MeOH of increasing polarity (5:1–1:1) to give five
fractions (E1–E5). Fraction E3 (1.2 g) was subjected to re-
versed-phase CC over a RP-C18 silica gel using MeOH–
H2O (2:3) to provide four subfractions (E31–E34). Subfrac-
tion E33 (120 mg) was separated by preparative normal-
phase HPLC using a solvent system of n-hexane–CHCl3–
MeOH (3:5:1) over 30 min at a flow rate of 2.0 mL/min
(Apollo silica 5 m column; Shodex refractive index detector)
to obtain parisveroside B (2, 7 mg). The n-BuOH-soluble
fraction (10 g) was subjected to CC over HP-20 resin using
100% H2O and 100% MeOH to give two fractions (B1,
100% H2O, and B2, 100% MeOH). The B1 fraction (2 g)
was washed using MeOH and recrystallized to give allantoin
(5, 200 mg). The B2 fraction (3 g) was subjected to normal-
phase CC over silica gel using CHCl3–MeOH–H2O
(10:10:0.5) to give six fractions (B21–B26). The B21 frac-
tion (180 mg) was further separated using silica gel CC
with CHCl3–MeOH–H2O (10:4:0.5) and purified by prepara-
tive normal-phase HPLC using a solvent system of CHCl3–
MeOH–H2O (9:4:0.5) to afford salicin (3, 6 mg) and 3-(b-D-
glucopyranosyloxymethyl)-2-(4-hydroxy-3-methoxyphenyl)-
5-(3-hydroxypropyl)-7-methoxy-dihydrobenzofuran (4, 4 mg).
The B22 fraction (70 mg) was purified using silica gel CC
with CHCl3–MeOH–H2O (9:4:0.5) to yield parisveroside B
(1, 5 mg).
Microglial cell activation plays a key role in mediating in-
flammatory processes in the CNS, which are associated with
various neurodegenerative diseases. NO is one of the main
proinflammatory mediators produced by activated micro-
glia.15 Therefore, compounds 2 and 4 from P. verticillata
might be of therapeutic interest with respect to the treatment
of neurodegenerative disease mediated by neuroinflamma-
tion.
Experimental
General experimental procedures
Optical rotations were measured on a Jasco P-1020 polar-
imeter in MeOH or CHCl3. IR spectra were recorded on a
Bruker IFS-66/S FT-IR spectrometer. FAB and HR-FAB
mass spectra were obtained on a JEOL JMS700 mass spec-
trometer. NMR spectra, including 1H–1H COSY, HSQC,
HMBC, and NOESY experiments, were recorded on a Var-
ian UNITY INOVA 500 NMR spectrometer operating at
500 MHz (1H) and 125 MHz (13C) with chemical shifts
given in ppm (d). Preparative HPLC was conducted using a
Gilson 306 pump with a Shodex refractive index detector
and an Apollo silica 5 m column (250 mm ꢁ 10 mm). Silica
gel 60 (Merck, 70–230 mesh and 230–400 mesh) and RP-
C18 silica gel (Merck, 230–400 mesh) were used for column
chromatography (CC). Merck precoated silica gel F254 plates
and RP-18 F254s plates were used for TLC. Spots were de-
tected on TLC under UV light or by heating after spraying
with 10% H2SO4 in C2H5OH (v/v).
Parisveroside A (1)
Colorless gum; ½aꢀ
25
+36.5 (c 0.05, MeOH). IR
(KBr, cm–1) nmax: 3390,D2949, 1698, 1522, 1450, 1026, 674.
1H NMR (CD3OD, 500 MHz) d: 4.34 (1H, d, J = 8.0 Hz, H-
1’), 3.86 (1H, dd, J = 11.0, 1.0 Hz, H-6’a), 3.77 (1H, dd, J =
11.0, 3.5 Hz, H-6’b), 3.75 (1H, m, H-9), 3.46–3.35 (3H, m,
H-2’, 3’, 4’), 3.26 (1H, m, H-5’), 2.34–2.24 (2H, m, H-2),
1.62–1.58 (4H, m, H-8, 10), 1.43–1.33 (4H, m, H-6, 7),
1.08 (1H, m, H-3), 0.93 (3H, t, J = 6.5 Hz, H-11), 0.82
(1H, m, H-5), 0.73 (1H, m, H-4a), –0.11 (1H, q, J =
5.5 Hz, H-4b). 13C NMR (CD3OD, 125 MHz) d: 177.5 (C-
1), 102.3 (C-1’), 80.7 (C-9), 77.0 (C-3’), 76.5 (C-5’), 74.1
(C-2’), 70.6 (C-4’), 61.7 (C-6’), 34.1 (C-2), 33.1 (C-8), 28.8
(C-6), 27.4 (C-10), 25.2 (C-7), 15.4 (C-5), 11.7 (C-3), 10.2
(C-4), 8.8 (C-11). HR-FAB-MS m/z 363.2021 [M + H]+
(calcd. for C17H31O8: 336.2019).
Published by NRC Research Press

444
Can. J. Chem. Vol. 89, 2011
Fig. 4. Inhibitory effects of compounds 1–5 on nitric oxide (NO) production in lipopolysaccharide (LPS)-activatied BV-2 cells.
Parisveroside B (2)
Colorless gum; ½aꢀ
(1H, m, H-3), 1.05 (3H, t, J = 6.5 Hz, H-11), 0.87 (1H, m, H-
5), 0.77 (1H, m, H-4a), –0.01 (1H, q, J = 5.5 Hz, H-4b). HR-
FAB-MS m/z 215.1652 [M + H]+ (calcd. for C12H23O3:
215.1647).
25
+26.6 (c 0.10, MeOH). IR
(KBr, cm–1) nmax: 3395,D2948, 1702, 1515, 1455, 1026, 674.
1H NMR (CD3OD, 500 MHz) d: 4.33 (1H, d, J = 8.0 Hz, H-
1’), 3.86 (1H, dd, J = 11.5, 1.5 Hz, H-6’a), 3.69 (3H, s,
OCH3), 3.68 (1H, dd, J = 11.5, 3.5 Hz, H-6’b), 3.65 (1H,
m, H-9), 3.38–3.33 (3H, m, H-2’, 3’, 4’), 3.25 (1H, m, H-5’),
2.39–2.30 (2H, m, H-2), 1.64–1.56 (4H, m, H-8, 10), 1.50–
1.35 (4H, m, H-6, 7), 1.08 (1H, m, H-3), 0.93 (3H, t, J =
6.5 Hz, H-11), 0.83 (1H, m, H-5), 0.73 (1H, m, H-4a), –0.10
(1H, q, J = 5.5 Hz, H-4b). 13C NMR (CD3OD, 125 MHz) d:
174.9 (C-1), 102.4 (C-1’), 80.7 (C-9), 77.1 (C-3’), 76.6 (C-
5’), 74.2 (C-2’), 70.7 (C-4’), 61.8 (C-6’), 50.9 (OCH3), 33.3
(C-2), 33.1 (C-8), 28.8 (C-6), 27.5 (C-10), 25.1 (C-7), 15.4
(C-5), 11.4 (C-3), 10.2 (C-4), 8.9 (C-11). HR-FAB-MS m/z
399.2012 [M + Na]+ (calcd. for C18H32NaO8: 399.1995).
Preparation of the Mosher ester derivatives of 1a
Compound 1a (0.7 mg) was transferred to a clean and
completely dry NMR tube. Deuterated pyridine (0.7 mL) and
(S)-(+)-a-methoxy-a-(trifluoromethyl)phenylacetyl chloride,
(S)-MTPA-Cl, (5 mL) were added immediately to the NMR
tube and then the NMR tube was shaken carefully to mix
the sample and (S)-MTPA-Cl evenly. The reaction NMR
tube was then permitted to stand at room temperature over-
night. The completed reaction afforded the (R)-MTPA ester
derivative (1r) of 1a. The (S)-MTPA ester derivative (1s) of
1a was obtained in the same manner as described for 1r.
1
The H NMR spectra for 1r and 1s were directly measured
with the reaction NMR tubes. 1s: 1H NMR (C5D5N,
500 MHz) d: 5.184 (1H, m, H-9), 2.537 (1H, m, H-2a),
2.485 (1H, m, H-2b), 1.762–1.559 (4H, m, H-8, 10), 1.257
(2H, m, H-7), 1.200 (2H, m, H-6), 1.156 (1H, m, H-3),
1.042 (3H, t, J = 6.5 Hz, H-11), 0.801 (1H, m, H-5), 0.686
Enzymatic hydrolysis of 1 and 2
Compounds 1 and 2 (3 mg each) were dissolved in H2O
(3 mL) and b-glucosidase (emulsin, 9 mg) was added to the
solution and kept at 37 8C for 3 days, in each case. The re-
action mixtures were extracted with CHCl3, and the aqueous
phases compared with an authentic sugar sample using co-
TLC (MeCN–H2O, 6:1, Rf 0.33 for glucose). Identification
of D-glucose in the aqueous layer was carried out by com-
paring the optical rotation of the liberated glucose
1
(1H, m, H-4a), –0.078 (1H, q, J = 5.5 Hz, H-4b). 1r: H
NMR (C5D5N, 500 MHz) d: 5.192 (1H, m, H-9), 2.540 (1H,
m, H-2a), 2.490 (1H, m, H-2b), 1.772–1.560 (4H, m, H-8,
10), 1.278 (2H, m, H-7), 1.214 (2H, m, H-6), 1.168 (1H, m,
H-3), 1.014 (3H, t, J = 6.5 Hz, H-11), 0.817 (1H, m, H-5),
0.696 (1H, m, H-4a), –0.065 (1H, q, J = 5.5 Hz, H-4b).
25
25
(½aꢀD +48.6 (c 0.05, H2O) from 1 and ½aꢀD +45.3 (c 0.05,
H2O) from 2) with that of an authentic sample of D-glucose.16
The CHCl3 layers of 1 and 2 were followed by purification
on a silica gel Waters Sep-Pak Vac 6cc (CHCl3–MeOH,
15:1) to afford new compounds 1a (1.4 mg) and 2a (1.1 mg).
Measurement of NO production and cell viability
The BV-2 mouse microglial cell line was maintained in
Dulbecco’s modified Eagle medium (DMEM) supplemented
with 5% fetal bovine serum (FBS) and 1% penicillin–
streptomycin (PS). To measure NO production, BV-2 cells
were plated into a 96 well plate (3 ꢁ 104 cells/well) and
treated with 100 ng/mL LPS in the presence or absence of
chrysin for 24 h. Nitrite, a soluble oxidation product of NO,
was measured in the culture media using the Griess reaction.
The supernatant (50 mL) was harvested and mixed with an
equal volume of Griess reagent (1% sulfanilamide and 0.1%
N-1-napthylethylenediamine dihydrochloride in 5% phos-
25
1
1a: colorless gum; ½aꢀD –12.5 (c 0.04, CHCl3). H NMR
(C5D5N, 500 MHz) d: 3.95 (1H, m, H-9), 2.62–2.48 (2H, m,
H-2), 1.64–1.57 (4H, m, H-8, 10), 1.30 (2H, m, H-7), 1.24
(2H, m, H-6), 1.17 (1H, m, H-3), 1.06 (3H, t, J = 6.5 Hz, H-
11), 0.85 (1H, m, H-5), 0.76 (1H, m, H-4a), –0.03 (1H, q, J =
5.5 Hz, H-4b). HR-FAB-MS m/z 201.1485 [M + H]+ (calcd.
25
for C11H21O3: 201.1491). 2a: colorless gum; ½aꢀD –20.7 (c
1
0.03, CHCl3). H NMR (C5D5N, 500 MHz) d: 3.91 (1H, m,
H-9), 3.70 (3H, s, OCH3), 2.65–2.47 (2H, m, H-2), 1.65–1.58
(4H, m, H-8, 10), 1.28 (2H, m, H-7), 1.23 (2H, m, H-6), 1.19
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Kim et al.
445
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phoric acid). After 10 min, the absorbance at 540 nm was
measured using a microplate reader. Sodium nitrite was
used as a standard to calculate the NOꢂ2 concentration. Cell
viability was assessed by a 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyl-tetrazolium bromide (MTT) assay.17 NG-Mono-
methyl-L-arginine (L-NMMA, Sigma-Aldrich), a well-known
NOS inhibitor, was tested as a positive control. The IC50
value for the positive control, L-NMMA, was 18.0 mmol/L.
Acknowledgements
The authors would like to thank Mr. Do Kyun Kim, Dr.
Eun Jung Bang, and Dr. Jung Ju Seo at the Korea Basic Sci-
ence Institute for the NMR and MS spectra measurements.
This work was supported by a Korea Science and Engineer-
ing Foundation (KOSEF) Grant funded by the Korean Gov-
ernment Ministry of Education, Science, and Technology
(MEST) (No. 2009-0063466).
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