Antineuroinflammatory and antiproliferative activities of constituents from Tilia amurensis

Article abstract of DOI:10.1248/cpb.c15-00393

As part of our ongoing search for bioactive constituents of natural Korean medicinal resources, we found in a preliminary study that the methanol (MeOH) extract from the trunks of Tilia amurensis RUPR. showed an inhibitory effect on nitric oxide (NO) production in an activated murine microglial cell line. A bioassayguided fractionation and chemical investigation of the MeOH extract resulted in the isolation and identification of a new isoflavonoid glycoside, orobol 4'-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside (1) and 16 known compounds (2-17). The structure of the new compound was determined by spectroscopic methods, i.e., one-dimensional (1D) and two-dimensional (2D)-NMR techniques and high resolution (HR)-MS, and chemical methods. The antineuroinflammatory activities of the isolated compounds were determined by measuring NO levels in the medium using murine microglial BV-2 cells. Among them, 12 compounds, including compound 1 (most active with an IC₅₀ value of 23.42 μm), inhibited NO production in lipopolysaccharidestimulated BV-2 cells. Moreover, compounds 1-4 showed moderate antiproliferative activities against the SK-MEL-2 cell line, with IC₅₀ values ranging from 12.31 to 19.67 μM.

Full text of DOI:10.1248/cpb.c15-00393

Vol. 63, No. 10
Chem. Pharm. Bull. 63, 837–842 (2015)
837
Note
Antineuroinflammatory and Antiproliferative Activities of Constituents
from Tilia amurensis
a
b
a
a
a
a
Ki Hyun Kim, Eunjung Moon, Joon Min Cha, Seulah Lee, Jae Sik Yu, Chung Sub Kim,
b
c
,a
Sun Yeou Kim, Sang Un Choi, and Kang Ro Lee*
a
b
School of Pharmacy, Sungkyunkwan University; Suwon 440–746, Republic of Korea: College of Pharmacy, Gachon
c
University; Incheon 406–799, Republic of Korea: and Korea Research Institute of Chemical Technology; Deajeon
305–600, Republic of Korea.
Received May 8, 2015; accepted July 3, 2015
As part of our ongoing search for bioactive constituents of natural Korean medicinal resources, we found
in a preliminary study that the methanol (MeOH) extract from the trunks of Tilia amurensis RUPR. showed
an inhibitory effect on nitric oxide (NO) production in an activated murine microglial cell line. A bioassay-
guided fractionation and chemical investigation of the MeOH extract resulted in the isolation and identifica-
tion of a new isoflavonoid glycoside, orobol 4ꢀ-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside (1) and 16
known compounds (2–17). The structure of the new compound was determined by spectroscopic methods,
i.e., one-dimensional (1D) and two-dimensional (2D)-NMR techniques and high resolution (HR)-MS, and
chemical methods. The antineuroinflammatory activities of the isolated compounds were determined by mea-
suring NO levels in the medium using murine microglial BV-2 cells. Among them, 12 compounds, including
compound 1 (most active with an IC50 value of 23.42µM), inhibited NO production in lipopolysaccharide-
stimulated BV-2 cells. Moreover, compounds 1–4 showed moderate antiproliferative activities against the SK-
MEL-2 cell line, with IC50 values ranging from 12.31 to 19.67µM.
Key words Tilia amurensis; Tiliaceae; isoflavonoid glycoside; nitric oxide; antineuroinflammation; antiprolif-
eration
Microglia are resident immune cells in the central nervous ry metabolites of T. amurensis trunk since its MeOH extract
system (CNS). These cells are rapidly activated by patho- showed anti-neuroinflammatory activity by inhibiting lipo-
genic stimuli and consequently produce various proinflam- polysaccharide (LPS)-stimulated NO production. A bioassay-
1,2)
matory mediators and cytokines.
In particular, excessive guided fractionation and chemical investigation of the MeOH
nitric oxide (NO) produced from activated microglia has been extract resulted in the isolation and identification of a new
known to induce neuronal cell death through many in vitro isoflavonoid glycoside, orobol 4′-O-β-D-apiofuranosyl-(1→6)-
3,4)
and in vivo studies. Therefore, discovering compounds that β-D-glucopyranoside (1) and 16 known compounds (2–17).
inhibit NO production in activated microglia is an important Here, we describe the isolation, structural elucidation, and the
strategy to prevent progressive neuronal damage. As part of anti-neuroinflammatory and antiproliferative activities of the
our ongoing search for bioactive constituents from natural isolated compounds from the T. amurensis trunk.
Korean medicinal resources, we found that the methanol
(
MeOH) extract from the trunks of Tilia amurensis RUPR. ex- Results and Discussion
hibited inhibitory effect on NO production using an activated
The MeOH extract of T. amurensis trunks was subjected
murine microglial cell line BV-2 in the screening study.
to liquid–liquid solvent-partitioning to yield n-hexane, chlo-
T. amurensis belongs to the family of Tiliaceae and is roform (CHCl ), ethyl acetate (EtOAc), and n-butyl alcohol
3
commonly known as bee tree. It is mostly found near Russia (n-BuOH) soluble factions. Among them, the active EtOAc-
and areas in East Asia, such as China, Korea, and Japan. Its soluble fraction with anti-neuroinflammatory effect was
leaves have been used as traditional Korean medicine to treat further separated by silica gel or Sephadex LH-20 column
5
)
cancer and rheumatoid arthritis. In addition, tea made from chromatography, and subsequent HPLC purification to obtain
the flowers of the plant have common medicinal uses, such one new isoflavonoid glycoside (1) and 16 known compounds
6
)
as antispasmodic, diaphoretic, and sedative. Earlier pharma- (2–17) (Fig. 1). To the best our knowledge, the presence
cological study on DNA topoisomerase inhibitory activity of of isoflavonoid glycoside is the second example from the
7
)
T. amurensis has indicated anticancer properties. Recently, genus Tilia. The first isoflavonoid glycoside, orobol 4′-O-β-
11)
neuroprotective compounds such as (−)-epicatechin, scopolec- glucopyranoside, was isolated from T. taquetii SCHNEIDER.
tin, nudiposide and lyoniside, were isolated from T. amurensis Compound 1 was isolated as a yellowish gum. The molecu-
8
)
and quality-evaluated. However, with the exception of our lar formula was established as C H O based on the positive
2
6
28 15,
+
previous study, not much research has been conducted on this ion peak at m/z 603.1323 [M+Na] in the high resolution-
plant. We have previously reported the anti-inflammatory and electrospray ionization (HR-ESI)-MS (Calcd for C H O Na,
antitumor activities of lignan constituents and a novel flavan- 603.1326) with C-NMR spectroscopy. The IR absorp-
-ol dimer in T. amurensis.
Our continuing interest in bioactive constituents from T. 1450 cm ), carbonyl (1656cm ), and hydroxyl (3354cm )
amurensis led us to further investigate anti-neuroinflammato- groups. The UV spectrum (λmax 258nm) of 1 was typical of
2
6
28 15
13
9,10)
3
tion spectrum suggested the presence of phenyl (2946 and
−1
−1
−1
*
To whom correspondence should be addressed. e-mail: krlee@skku.edu
©
2015 The Pharmaceutical Society of Japan

8
38
Chem. Pharm. Bull.
Vol. 63, No. 10 (2015)
Fig. 1. Chemical Structures of Compounds 1–17
1
13
a)
Table 1. H- and C-NMR Data of Compound 1 in CD OD
3
1
Position
δH
δC
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
8.08 s
154.0
123.0
180.6
162.6
99.3
6.21 brs
6.32 brs
164.8
93.7
1
1
Fig. 2. H– H COSY (Bold Lines) Correlations and Key HMBCs (Ar-
rows) of 1
158.6
104.8
126.7
116.9
147.2
145.6
117.6
120.6
102.9
73.8
1
′
′
′
′
′
′
signals comprising of 15 signals attributable to isoflavone skel-
eton and the other 11 signals from the two sugars, indicating
that compound 1 is isoflavonoid glycoside. The aglycone of
1 was determined as 5,7,3′,4′-tetrahydroxyisoflavone, known
as orobol by analysis of the H– H correlation spectroscopy
COSY), heteronuclear multiple quantum correlation (HMQC),
and heteronuclear multiple bond correlation (HMBC) spectra
Fig. 2). The H- and C-NMR spectra (Table 1) of 1 were
very similar to those of orobol 4′-O-β-glucopyranoside,
except for the signals of an additional sugar unit, apiose (δC
7.09 d (1.5)
1
1
7.25 d (8.5)
6.98 dd (8.5, 1.5)
4.83 d (7.5)
3.53 dd (9.0, 7.5)
3.48m
(
Glc 1″
2
3
4
5
6
″
″
″
″
″
1
13
(
76.4
11)
3.45m
70.5
3.37m
76.1
15)
1
09.9, 79.3, 76.9, 73.6, and 64.4). Acid hydrolysis of 1 yield-
ed glucose and apiose detected by co-TLC comparison with
authentic samples. A large coupling constant (J=7.5Hz) for
the anomeric proton (δH 4.83) of the glucose in the H-NMR
4.03 dd (11.5, 1.5)
68.0
3
.61 dd (11.5, 5.5)
Api 1‴
5.00 d (1.5)
3.93 d (1.5)
109.9
76.9
79.3
73.6
1
2
3
4
‴
‴
‴
spectrum suggested a β-configuration in glucose, and the
apiose unit was determined to have a β-configuration due to
a coupling constant (J=1.5Hz) of H-1‴ and the chemical shift
3.98 d (10.0)
3
.76 d (10.0)
13
15)
5
‴
3.59 s
64.4
of its anomeric carbon (δ 109.9) in the C-NMR spectrum.
C
1
13
The glucose C-2″ signal speared at δ 73.8, while that of C-6″
a) H- and C-NMR data were recorded at 500 and 125MHz, respectively. Cou-
C
pling constants (in Hz) are shown in parentheses.
appeared at δ 68.0, suggesting that the interglycosidic linkage
C
15)
is apiosyl-(1→6)-glucose, which was also confirmed by the
12)
compounds with an isoflavone skeleton, which was also sup- HMBC correlation between H-1‴ (δ 5.00) and C-6″ (δ 68.0).
H
C
ported by the characteristic resonance for H-2 of an isoflavone The glycosidation position was determined as C-4′ by the
1
13,14)
observed at δ 8.08 (1H, s) in the H-NMR spectrum.
The HMBC correlation between the glucosyl anomeric proton H-1″
H
13
C-NMR data (Table 1) of 1 showed a total of 26 carbon (δ 4.83) and C-4′ (δ 145.6) of A ring (Fig. 2). Furthermore,
H
C

Vol. 63, No. 10 (2015)
Chem. Pharm. Bull.
839
2
8,37)
the D-glucose and D-apiose were identified by gas chromatog- synthase expression in LPS-stimulated RAW264.7.
How-
raphy (GC) analysis of their chiral derivatives in the acidic hy- ever, anti-neuroinflammatory effects of these compounds in
1
6,17)
drolysate.
′-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside.
The known compounds were identified as pratensein-7-O-β- compounds. Excess production of NO by activated microglia
Thus, compound 1 was characterized as orobol microglial cells have not been reported yet. Our study is the
4
first to show the anti-neuroinflammatory properties of these
18)
18)
3,4)
D-glucoside (2), orobol 7-O-β-D-glucoside (3), orobol 4′-O- induces neuronal cell death,
β-glucopyranoside (4), kelampayoside A (5), osmanthuside various neurodegeneration in the CNS. Among isolates (1–17)
H (6), salidroside (7), dihydroconiferin (8), isotachioside from the active EtOAc-soluble fraction of T. amurensis, com-
9), tachioside (10), koaburside (11), 2-methoxyhydro- pounds 1–3, 6–7, 13 and 15 were determined to be the active
which consequently leads to
1
1)
19)
22)
2
0)
21)
2
3)
23)
24)
(
2
5)
26)
27)
quinone (12),
scopoletin (13),
scopolin (14),
fraxin ingredients responsible for anti-neuroinflammatory property
and adenosine of the EtOAc-soluble fraction. Particularly, compounds 2, 3
17) by comparing the spectroscopic data with previously and 15 (relatively high-yield isolates) are possible to be main
reported values.
contributors to the activity. The present study suggests that
2
8)
29)
(
(
15),
n-butyl β-D-glucopyranoside (16),
3
0)
The isolated compounds (1–17) were examined for their the anti-neuroinflammatory compounds isolated from T. amu-
anti-neuroinflammatory activities by measuring the NO lev- rensis have beneficial therapeutic potential against neurode-
els produced in LPS-activated BV-2 cells, a microglial cell generative diseases.
line. In this study, twelve compounds had an IC50 of less than
00µM. Among them, compounds 1–3, 6–7, 13 and 15 showed were additionally evaluated by determining their inhibitory
Next, the antiproliferative activities of the isolates 1–17
2
significant inhibitory effects on NO production. These com- effects on four human tumor cell lines, namely A549 (non-
pounds had no effect on cell viability in LPS-treated BV-2 small cell lung carcinoma), SK-OV-3 (ovary malignant as-
at their respective IC50 values (data not shown). The new iso- cites), SK-MEL-2 (skin melanoma), and HCT-15 (colon adeno-
38)
flavonoid glycoside, compound 1 showed the highest activity carcinoma) using the SRB bioassay. The results (Table 3)
with an IC50 value of 23.42µM (Table 2). showed that only compounds 1–4, which are isoflavonoid
Some previous studies reported anti-inflammatory activi- glycosides among the isolates, showed moderate antiprolifera-
31–33)
ties of flavonoid glycosides.
Isoflavonoid glycosides (1–3) tive activities against SK-MEL-2 cell line with IC50 values
isolated from T. amurensis also exhibited inhibitory activi- ranging from 12.31 to 19.67µM. In particular, compound 3
ties on inflammation in activated microglial cells. However, showed antiproliferative activities against all the tumor cell
orobol 4′-O-β-glucopyranoside (4) does not have influence lines, A549, SK-OV-3, SK-MEL-2, and HCT-15 with IC50 val-
NO production in LPS-treated BV-2, unlike orobol 4′-O-β-D- ues of 8.41, 22.56, 13.93, and 28.28µM, respectively (Table 3).
apiofuranosyl-(1→6)-β-D-glucopyranoside (1), the most active
compound. These results suggested that the addition of β-D-
Table 3. Antiproliferative Activities of Compounds 1–4 from T. amu-
apiofuranosyl group to glycosylation in C-4′ hydroxyl group rensis
might play a role on NO inhibition in BV-2. In this study,
compounds 6 and 7 also significantly inhibited NO production
in LPS-stimulated murine microglial cell line. The inhibitory
a)
IC50 (µM)
Compounds
A549
SK-OV-3
SK-MEL-2
HCT-15
effect of salidroside (7) on NO production was also reported
in murine macrophage RAW264.7. Both osmanthuside H
1
>30.0
>30.0
8.41
>30.0
>30.0
22.56
>30.0
0.003
19.67
18.24
13.93
12.31
0.002
>30.0
>30.0
28.28
>30.0
0.081
3
4)
2
(
6) and salidroside (7) are glucosides of tyrosol. The inhibi-
3
4
35,36)
tory effect of tyrosol on NO synthesis is already known.
>30.0
0.001
Therefore, it is possible that the tyrosol skeleton structure may
be important to anti-neuroinflammatory efficacy. According
to previous studies, scopoletin (13) and fraxin (15) can also
b)
Doxorubicin
a) IC50 value of compounds against each cancer cell line defined as the concentra-
tion (µM) that caused 50% inhibition of cell growth in vitro; Values are means of
suppress NO production by inhibiting inducible nitric oxide triplicate determinations. b) Doxorubicin as a positive control.
Table 2. Inhibitory Effects on NO Production of Fractions and Compounds 1–17 in LPS-Activated BV-2 Cells
a)
a)
Compounds
IC50 (µg/mL or µM)
Compounds
IC50 (µM)
Crude MeOH extract
37.81
>200
84.92
31.03
172.38
23.42
32.23
31.85
>200
>200
25.99
35.64
8
9
158.49
n-Hexane fraction
50.29
CHCl fraction
10
11
12
13
14
15
16
17
116.66
3
EtOAc fraction
>200
n-BuOH fraction
58.87
1
2
3
4
5
6
7
37.69
148.92
30.02
>200
>200
14.77
b)
NMMA
a) IC50 value of extract and fractions was defined as the concentration (µg/mL) that caused 50% inhibition of NO production in LPS-activated BV-2 cells, while that of com-
G
pounds was defined as the concentration (µM). b) NMMA (N -monomethyl L-arginine, nitric oxide synthase inhibitor) as a positive control.

8
40
Chem. Pharm. Bull.
Vol. 63, No. 10 (2015)
All the other compounds were inactive (IC >30µM) in all the (flow rate; 2mL/min, 50% MeOH) to yield compound 8
5
0
cell lines.
(4mg). Fraction PNM5 was separated with Sephadex LH-20
with 100% MeOH to yield 7 fractions (PNM51–57). Fraction
PNM51 was separated by the purification of semi-preparative
Experimental
General Experimental Procedures Optical rotations reverse-phase HPLC using 50% MeOH to furnish compound
were measured on a Jasco P-1020 polarimeter (Jasco, Easton, 16 (4mg). Fraction PNM52 was applied to C18 Waters Sep-
MD, U.S.A.). IR spectra were recorded on a Bruker IFS-66/S Pak Vac 6 cc with 40% MeOH and purified by preparative
FT-IR spectrometer (Bruker, Karlsruhe, Germany). ESI and reverse-phase HPLC using 38% MeOH to yield compounds
HR-ESI mass spectra were recorded on a SI-2/LCQ DecaXP 7 (4mg) and 11 (4mg). Fraction PNM53 was separated by
Liquid chromatography (LC)-mass spectrometer (Thermo chromatography on LiChroprep Lobar-A RP-18 column with
Scientific, West Palm Beach, PL, U.S.A.). NMR spectra were 40% MeOH to give five subfractions (PNM531–535). From
recorded on a Varian UNITY INOVA 500 NMR spectrometer those subfractions, subtraction PNM532 was further purified
1
(
Varian, Palo Alto, CA, U.S.A.) operating at 500MHz ( H) by semi-preparative reverse-phase HPLC (flow rate; 2mL/min,
13
and 125MHz ( C), with chemical shifts given in ppm (δ). 25% MeOH) to yield compounds 9 (7mg) and 10 (6mg). Sub-
Preparative HPLC used a Gilson 306 pump (Gilson, Middle- fraction PNM533 was further purified by semi-preparative
ton, WI, U.S.A.) with a Shodex refractive index detector reverse-phase HPLC (flow rate; 2mL/min, 40% MeOH) to
(Shodex, New York, NY, U.S.A.). Low-pressure liquid chro- obtain compounds 14 (12mg) and 15 (19mg). Fraction PNM1
matography (LPLC) was carried out over a LiChroprep Lobar- and PNM2 were consolidated and separated by RP-C18 silica
A Si 60 column (240mm ×10 mm i.d.; Merck, Darmstadt, gel column chromatography with MeOH to H O gradient in-
2
Germany) with a FMI QSY-0 pump (Teledyne Isco, Lincoln, creasing from 80 to 100% to give three fractions (PNM21–23).
NE, U.S.A.). Column chromatography was performed with a Fraction PNM21 was subjected to fractionation with C18 Wa-
silica gel 60 (Merck, 230–400 mesh). The packing material ters Sep-Pak Vac 6 cc with 100% MeOH to yield subfractions
for molecular sieve column chromatography was Sephadex PNM211 and PNM212. Subfraction PNM211 was purified by
LH-20 (Pharmacia, Uppsala, Sweden). Merck precoated silica preparative reverse-phase HPLC with a solvent system of 45%
gel F254 plates and reversed-phase (RP)-18F254s plates (Merck, MeOH (flow rate; 2mL/min) to give compounds 12 (4mg) and
Darmstadt, Germany) were used for thin-layer chromatogra- 13 (4mg).
phy (TLC). Spots were detected on TLC under UV light or by
heating after spraying with anisaldehyde–sulfuric acid.
Orobol 4′-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside
25
(1): Yellowish gum; [α] +20.6 (c=0.30, MeOH); UV (MeOH)
D
Plant Materials The trunk of T. amurensis was collected λmax (logε) 288 (1.2), 258 (1.9), 218 (3.8) nm; IR (KBr)
from Hwacheon-Myun, Hongcheon city, Gangwon-do, Korea, νmax 3354, 2946, 2832, 1656, 1508, 1450, 1365, 1177, 1055,
−
1
1
13
in March 2010. Samples of plant material were identified by 998cm ; H- (500MHz) and C- (125MHz) NMR data, see
+
one of the authors (K. R. Lee). A voucher specimen (SKKU Table 1; ESI-MS (positive-ion mode) m/z: 603 [M+Na] . HR-
+
2
010–03) was deposited in the herbarium of the School of ESI-MS (positive-ion mode) m/z: 603.1323 [M+Na] (calcd for
Pharmacy, Sungkyunkwan University, Suwon, Korea.
C H O Na, 603.1326).
26 28 15
Extraction and Isolation The air-dried T. amurensis
Acid Hydrolysis of 1 and Sugar Analysis Compound 1
trunks (4kg) were extracted twice with 80% aqueous MeOH (2mg) was hydrolyzed by 1N HCl (dioxane–H O, 1:1, 5mL)
2
(
2×4h) under reflux. The extract was filtered and then concen- under reflux conditions for 3h. After cooling, the reaction
trated under vacuum to afford a crude MeOH extract (360g). mixture was diluted with H O and extracted with CHCl .
2
3
The extract was then partitioned with n-hexane, CHCl3, A sample of the aqueous layer was neutralized by passage
EtOAc, and n-BuOH to yield 11.8, 38.5, 10.2, and 72.5g of through an Amberlite IRA-67 column and repeatedly evapo-
residues, respectively. Each fraction was evaluated for its anti- rated under reduced pressure to give the sugar fraction. The
neuroinflammatory effect in an activated murine microglial sugars in the fraction were analyzed by silica gel TLC by
cell line. Among the four fractions, the EtOAc-soluble frac- comparison with authentic samples. The solvent system was
tion showed the most significant activity (Table 2). Thus, the CHCl –MeOH–H O (8:5:1). Spots were visualized by spray-
3
2
EtOAc-soluble fraction was separated with silica gel column ing with 95% EtOH–H SO –anisaldehyde (9:0.5:0.5), then
2
4
chromatography using a gradient of MeOH to CHCl from heated at 120°C for 3min. The Rf of glucose and apiose were
3
10 to 50% and this yielded 9 fractions (PNM1–9). Fraction 0.30 and 0.45, respectively for sugars of 1. For GC analysis,
PNM7 was subjected to Sephadex LH-20 with 100% MeOH each sugar fraction was dissolved in anhydrous pyridine
to give 8 fractions (PNM71–78). Fraction PNM76 and PNM77 (100 µL), and 0.1M L-cysteine methyl ester hydrochloride in
16,17)
was consolidated and subjected to fractionation with silica anhydrous pyridine (200µL) was added.
The mixture was
gel column chromatography (CHCl –MeOH–H O, 9:3:0.5) stirred at 60°C for 1h. Then 150µL of HMDS/TMCS (hexa-
3
2
to yield 4 subfractions (PNM771–774). Subfraction PNM772 methyldisilazane–trimethychlorosilane–pyridine, 3:1:10) was
was further purified by semi-preparative reverse-phase HPLC added, and the mixture was stirred at 60°C for another 30min.
(flow rate; 2mL/min, 55% MeOH) to give compounds 1 (6mg) The precipitate was centrifuged off, and the supernatant was
and 4 (3mg). Subfraction PNM773 was further purified by concentrated under an N stream. The residue was partitioned
2
semi-preparative reverse-phase HPLC (flow rate; 2mL/min, between n-hexane and H O (0.1mL each), and the hexane
2
35% MeOH) to give compounds 2 (12mg), 3 (10mg), and 17 layer (1µL) was analyzed by GC experiment. D-Glucose and
(
5mg). Fraction PNM73 was also purified by semi-preparative D-apiose were detected by co-injection of the hydrolysate with
reverse-phase HPLC (flow rate; 2mL/min, 45% MeOH) to standard silylated samples (D-glucose: 11.38min; L-glucose:
yield compounds 5 (9mg) and 6 (9mg). Fraction PNM72 was 12.62min; D-apiose: 5.08; L-apiose: 5.65). The retention
further purified by semi-preparative reverse-phase HPLC times of sugars obtained by acid hydrolysis were D-glucose

Vol. 63, No. 10 (2015)
Chem. Pharm. Bull.
841
(
11.41min) and D-apiose (5.05min) for 1. The standards of References
sugars, D-glucose, L-glucose, and D-apiose were obtained from
Sigma-Aldrich, U.S.A, and L-apiose was from Santa Cruz Bio-
technology, Inc., U.S.A.
Cell Culture BV2 (microglia from murine) was gener-
ously provided by Dr. E. Choi from Korea University (Seoul,
Korea). It was maintained in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 5% FBS, 100units/mL
penicillin and 100µg/mL streptomycin. All tumor cell cul-
tures were maintained using RPMI1640 cell growth medium
1) Hanisch U. K., Glia, 40, 140–155 (2002).
2)
Minghetti L., Levi G., Prog. Neurobiol., 54, 99–125 (1998).
3) Boje K. M., Arora P. K., Brain Res., 587, 250–256 (1992).
4
)
Takeuchi A., Isobe K. I., Miyaishi O., Sawada M., Fan Z. H., Naka-
shima I., Kiuchi K., Eur. J. Neurosci., 10, 1613–1620 (1998).
Ahn D. K., “Illustrated Book of Korean Medicinal Herbs,” Kyo-
haksa, Seoul, 2003.
5
)
6
)
Lauriault J., “Identification Guide to the Trees of Canada,” Fitzhen-
ry and Whiteside, Ontario, 1989.
7) Choi J. Y., Seo C. S., Zheng M. S., Lee C. S., Son J. K., Arch.
Pharm. Res., 31, 1413–1418 (2008).
8) Lee B., Weon J. B., Yun B. R., Lee J., Eom M. R., Ma C. J., Phar-
macogn. Mag., 10, 195–199 (2014).
(
Gibco, Carlsbad, CA, U.S.A.), supplemented with 5% fetal
bovine serum (FBS) (Gibco), 100 units/mL penicillin and
00 µg/mL streptomycin. Human tumor cell lines such as
1
9)
Kim K. H., Moon E., Kim S. Y., Choi S. U., Lee K. R., Food Chem.
Toxicol., 50, 3680–3686 (2012).
A549 (non-small cell lung carcinoma), SK-OV-3 (ovary malig-
nant ascites), SK-MEL-2 (skin melanoma), and HCT-15 (colon
adenocarcinoma) were provided by the National Cancer Insti-
tute (NCI). All cells were incubated at 37°C in a humidified
incubator with 5% CO2.
1
0) Kim K. H., Moon E., Kim S. Y., Choi S. U., Lee K. R., Bull. Ko-
rean Chem. Soc., 36, 367–369 (2015).
1) Kang Y. M., Lee N. H., Bull. Korean Chem. Soc., 32, 1048–1050
2011).
2) Watanabe K., Kinjo J., Nohara T., Chem. Pharm. Bull., 41, 394–396
1993).
1
(
Measurement of Nitric Oxide Production and Cell Vi-
1
4
ability BV-2 cells were plated into a 96-well plate (3×10
(
cells/well). After 24h, cells were pretreated with com- 13) Lee J. W., Lee C., Jin Q., Lee M. S., Kim Y., Hong J. T., Lee M. K.,
pounds 1–17 (in dimethyl sulfoxide (DMSO)) for 30min,
Hwang B. Y., Arch. Pharm. Res., 38, 991–997 (2015).
and then stimulated with 100ng/mL of microglial activator 14) Murthy M. S. R., Rao E. V., Ward R. S., Magn. Reson. Chem., 24,
3
9)
2
25–230 (1986).
5) Kim K. H., Ha S. K., Choi S. U., Kim S. Y., Lee K. R., Chem.
Pharm. Bull., 59, 1425–1429 (2011).
6) Bognár R., Somogyi L., Györgydeák Z., Justus Liebigs Ann. Chem.,
38, 68–78 (1970).
7) Hara S., Okabe H., Mihashi K., Chem. Pharm. Bull., 35, 501–506
1987).
8) Anhut S., Dietmar Zinsmeister H., Mues R., Barz W., Mackenbrock
LPS for another 24h. Nitrite, a soluble oxidation prod-
uct of NO, was measured in the culture media using the
Griess reaction. The supernatant (50µL) was harvested
and mixed with an equal volume of Griess reagent (1%
sulfanilamide, 0.1% N-1-naphtylethylenediamine dihydro-
chloride in 5% phosphoric acid). After 10min, the absor-
bance at 570nm was measured using a microplate reader.
Sodium nitrite was used as a standard to calculate the
1
1
7
1
(
1
K., Köster J., Markham K. R., Phytochemistry, 23, 1073–1075
(1984).
−
NO2 concentration. Cell viability was assessed by a 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) 19) Kanchanapoom T., Kasai R., Yamasaki K., Phytochemistry, 59,
G
5
51–556 (2002).
assay. N -monomethyl-L-arginine (L-NMMA, Sigma, St.
2
0) Lei Y., Wu L. J., Shi H. M., Tu P. F., Helv. Chim. Acta, 91, 495–500
Louis, MO, U.S.A.), a well-known nitric oxide synthase
NOS) inhibitor, was tested as a positive control.
Antiproliferative Effect Assessment Antiproliferative
effect of the isolated compounds against cultured human
tumor cell lines was evaluated by the sulforhodamine B
SRB) method. Each tumor cell line plated on standard 96-
4
0)
(2008).
(
2
2
2
1) Chu Y. H., Chen C. J., Wu S. H., Hsieh J. F., J. Agric. Food Chem.,
62, 3742–3749 (2014).
2) Mazur M., Hope-Ross K., Kadla J. F., Sederoff R., Chang H. M.,
J. Wood Chem. Technol., 27, 1–8 (2007).
3) Inoshiri S., Sasaki M., Kohda H., Otsuka H., Yamasaki K., Phyto-
chemistry, 26, 2811–2814 (1987).
38)
(
well flat-bottom microplates was incubated for 24h at 37°C
in a humidified atmosphere of 5% CO . The attached cells 24) Jin H. G., Kim A. R., Ko H. J., Woo E. R., Arch. Pharm. Res., 38,
2
were then incubated with the serially diluted compounds (in
591–597 (2015).
DMSO). After continuous exposure to the compounds for 25) Ferreira J. A., Nel J. W., Vincent Brandt E., Bezuidenhoudt B. C.
B., Ferreira D., J. Chem. Soc., Perkin Trans. 1, 1995, 1049–1056
4
8h, the culture medium was removed from each well and the
(
1995).
6) Cassady J. M., Ojima N., Chang C., Mclaughlin J. L., J. Nat. Prod.,
2, 274–278 (1979).
cells were fixed with 10% cold trichloroacetic acid at 4°C for
2
2
2
1
0
h. After washing with tap water, the cells were stained with
.4% SRB dye and incubated for 30min at room temperature.
4
7) Kim A. R., Ko H. J., Chowdhury M. A., Chang Y. S., Woo E. R.,
Arch. Pharm. Res., 38, 1059–1065 (2015).
8) Kwon J. H., Kim S. B., Park K. H., Lee M. W., Arch. Pharm. Res.,
The cells were washed again and then solubilized with 10mM
unbuffered Tris base solution (pH 10.5). The absorbance was
measured spectrophotometrically at 520nm with a microtiter
plate reader.
3
4, 1459–1466 (2011).
29) Antkowiak R., Antkowiak W. Z., Banczyk I., Mikolajcyk I., Can. J.
Chem., 81, 118–124 (2003).
Acknowledgment This work was supported by the Basic 30) Patching S. G., Baldwin S. A., Baldwin A. D., Young J. D., Gal-
lagher M. P., Henderson P. J. F., Herbert R. B., Org. Biomol. Chem.,
Science Research Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Educa-
tion, Science and Technology (2013R1A1A2A10005315).
3, 462–470 (2005).
3
1) Senthamilselvi M. M., Kesavan D., Sulochana N., Org. Med. Chem.
Lett., 2, 19 (2012).
3
2) González Mosquera D. M., Ortega Y. H., Kilonda A., Dehaen W.,
Conflict of Interest The authors declare no conflict of
interest.
Pieters L., Apers S., Phytochem. Lett., 4, 231–234 (2011).
33) Antunes-Ricardo M., Gutiérrez-Uribe J. A., Martínez-Vitela C.,

8
42
Chem. Pharm. Bull.
Serna-Saldivar S. O., Biomed. Res. Int., 2015, 847320 (2015).
400–403 (1999).
Vol. 63, No. 10 (2015)
3
4) Song B., Huang G., Xiong Y., Liu J., Xu L., Wang Z., Li G., Lu J.,
38) Song J. H., Choi H. J., Song H. H., Hong E. H., Lee B. R., Oh S. R.,
Choi K., Yeo S. G., Lee Y. P., Cho S., Ko H. J., J. Ginseng Res., 38,
173–179 (2014).
Guan S., J. Med. Food, 16, 997–1003 (2013).
35) De Stefano D., Maiuri M. C., Simeon V., Grassia G., Soscia A., Ci-
nelli M. P., Carnuccio R., Eur. J. Pharm., 566, 192–199 (2007).
6) Wang P., Zhu Q., Wu N., Siow Y. L., Aukema H., O K., J. Agric.
Food Chem., 61, 3669–3675 (2013).
7) Kang T. H., Pae H. O., Jeong S. J., Yoo J. C., Choi B. M., Jun C. D.,
Chung H. T., Miyamoto T., Higuchi R., Kim Y. C., Planta Med., 65,
39) Weinstein J. R., Swarts S., Bishop C., Hanisch U. K., Möller T.,
Glia, 56, 16–26 (2008).
40) Reif D. W., McCreedy S. A., Arch. Biochem. Biophys., 320, 170–176
(1995).
3
3