Phenolic derivatives from the stems of Lagerstroemia indica and their biological activity

Article abstract of DOI:10.3987/COM-15-13328

The purification of a MeOH extract from the stems of Lagerstroemia indica (Lythraceae) using repeated column chromatography afforded three new phenolic glycosides (1-3), and a new flavone derivative (4) along with fifteen known compounds (5-19). The structures of new compounds were determined through spectral analysis, including 1D, 2D-NMR and MS data. Compounds 1-19 were evaluated for their inhibitory activities on nitric oxide (NO) production in an activated murine microglial cell line and cytotoxic activities against four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15).

Full text of DOI:10.3987/COM-15-13328

HETEROCYCLES, Vol. 91, No. 12, 2015
2355
HETEROCYCLES, Vol. 91, No. 12, 2015, pp. 2355 - 2366. © 2015 The Japan Institute of Heterocyclic Chemistry
Received, 17th September, 2015, Accepted, 2nd November, 2015, Published online, 24th November, 2015
DOI: 10.3987/COM-15-13328
PHENOLIC DERIVATIVES FROM THE STEMS OF LAGERSTROEMIA
INDICA AND THEIR BIOLOGICALACTIVITY
Kyeong Wan Woo,a Won Se Suh, a Lalita, Subedi,b Sun Yeou Kim,b,c Sang Un
Choi,d Ki Hyun Kim,a and Kang Ro Lee*,a
aNatural Products Laboratory, School of Pharmacy, Sungkyunkwan University;
b
2066 Seobu-Ro, Jangan-ku, Suwon, Gyeonggi-do, Republic of Korea. Gachon
Institute of Pharmaceutical Science and cCollege of Pharmacy, Gachon University,
d
Incheon 406-799, Republic of Korea. Korea Research Institute of Chemical
Technology, Deajeon 305-600, Korea, E-mail : krlee@skku.edu
Abstract - The purification of a MeOH extract from the stems of Lagerstroemia
indica (Lythraceae) using repeated column chromatography afforded three new
phenolic glycosides (1-3), and a new flavone derivative (4) along with fifteen
known compounds (5-19). The structures of new compounds were determined
through spectral analysis, including 1D, 2D-NMR and MS data. Compounds 1-19
were evaluated for their inhibitory activities on nitric oxide (NO) production in an
activated murine microglial cell line and cytotoxic activities against four human
tumor cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15).
Lagerstroemia indica (Lythraceae) is a decorative shrub, widely distributed in Korea, Japan, and China.1
The root of this plant has been used as an astringent, detoxicant and diuretic in traditional Indian
medicine.2 Previous phytochemical investigations of L. indica have reported the isolation of alkaloids,
triterpenes, and flavonoids.2-5 Recent studies have reported that the active compound, cytisoside show
antimicrobial activity against five pathogenic microorganisms.6 Antioxidant and aldose reductase
inhibitory activity of the its MeOH extract was also reported.7,8 In the course of continuing to search for
biologically active compounds from Korean medicinal plant sources, we performed a phytochemical
investigation on the stems of L. indica. The CHCl3, EtOAc, and n-BuOH fractions were subjected to
repeated column chromatography on silica gel, sephadex LH-20, and semi-preparative HPLC separation
to yield three new phenolic glycosides (1–3), named strosides A–C, and a new flavone derivative (4),
lagerindiol, together with fifteen known compounds (5–19) (Figure 1). The structures of these new

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HETEROCYCLES, Vol. 91, No. 12, 2015
compounds were determined by spectroscopic methods including 1D, 2D-NMR (COSY, HMQC, and
HMBC) and enzyme hydrolysis. The isolated compounds (1-19) were evaluated for their inhibitory
effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated BV-2 cells, a microglial
cell line and their cytotoxicity against four human cancer cell lines in vitro using a sulforhodamine B
bioassay.
HO
HO
OMe
3
O
OH1''
O
4
OH
HO
9
OH
8
HO
4
1
OH
1
3'
7
1'
OMe
OH
HO
O
8
3
MeO
O
7
9
OH
OH
1'
4'
O
1
2
OH
3'
1''
4'
O
HO
OH
HO
O
4
B
D
1'
O
1
9
OH
HO
O
C
OH
1'
3
MeO
7''
7
7
O
O
A
8''
9''
8
HO
OH
4
1''
5
OH
O
OH
HO
OH
4
3
OH
Figure 1. Structures of Compounds 1-4
Compound 1 was obtained as a brown gum. In its HR-FAB-MS spectrum, the molecular ion [M + H]+
was observed at m/z 481.1710 [M + H]+ (calcd. for C23H29O11: 481.1710), suggesting the molecular
1
formula was C23H28O11 of 1. The H-NMR spectrum showed the presence of two 1,3,4-trisubstitued
aromatic rings [δH 7.69 (1H, dd, J = 8.5, 2.0 Hz, H-6), 7.62 (1H, d, J = 2.0 Hz, H-2), 7.18 (1H, d, J = 8.5
Hz, H-5), 6.90 (1H, d, J = 2.0 Hz, H-2'), 6.76 (1H, dd, J = 8.5, 2.0 Hz, H-6'), 6.74 (1H, d, J = 8.5 Hz,
H-5')], one oxymethylene [δH 4.27 (1H, dd, J = 10.5, 9.0 Hz, H-9a) and 3.73 (1H, dd, J = 10.5, 5.0 Hz,
H-9b)], and one methine [δH 4.78 (1H, dd, J = 8.5, 5.0 Hz, H-8)], and two methoxy protons [δH 3.89 (3H,
s, 3-OCH3) and 3.84 (3H, s, 3'-OCH3)]. In the 13C-NMR spectrum, 17 carbon signals, including 12
aromatic carbons, one ketone [δC 199.3, (C-7)], one oxymethylene [δC 65.5, (C-9)], one methine [δC 56.7
(C-8)], and two methoxy groups [δC 56.7, (3-OCH3) and 56.5 (3'-OCH3)] exhibited, together with a
glucose unit [δC 101.9 (C-1''), 78.4 (C-3''), 78.0 (C-5''), 74.8 (C-2''), 71.4 (C-4''), and 62.6 (C-6'') in the
13C-NMR].9 The NMR data were very similar to those of evofolin-B (18),10 except for the additional
glucose moiety. Enzyme hydrolysis of 1 afforded the aglycone and sugar residue. The aglycone was

HETEROCYCLES, Vol. 91, No. 12, 2015
2357
1
confirmed to be evofolin-B (1a) by comparison of H-NMR and FAB-MS data.10 The D-glucose was
detected by co-TLC comparison and identified by the sign of its specific rotation value.11 The coupling
constant (J = 7.5 Hz) of the anomeric proton at δH 5.01 indicated the β-orientation of glucose.9 The
position of glucose was established by an HMBC experiment, in which a long-range correlation was
identified between the H-1'' (δH 5.01) and the C-4 (δC 152.3) (Figure 2). The absolute configuration of 1
was assigned as 8S by the analysis of circular dichroism (CD) spectrum of 1 showing the negative Cotton
effects at 222 and 310 nm and positive Cotton effects 208 and 238 nm.12 Thus, the structure of compound
1 was determined as shown in Figure 1 and named stroside A.
HO
HO
OMe
O
O
OH
HO
OH
HO
OH
OMe
OH
OH
O
HO
MeO
O
OH
OH
O
2
1
OH
HO
O
OH
O
HO
O
OH
HO
O
O
OH
OH
MeO
O
O
OH
HO
OH
4
HO
OH
3
OH
Figure 2. Key HMBC (arrow) and COSY (bold line) correlations of 1-4
Compound 2 was obtained as a brown gum. The molecular formula C16H24O9 was determined by the
HR-FAB-MS m/z 361.1499 [M+H]+ (calcd. 361.1498). The 1H- and 13C- NMR spectra were very similar
to those of xylocoside A isolated from Xylosma controversum.13 A major difference between them was
found to be the location of glucose. The glucose moiety of 2 was confirmed to be located at C-9 by the
HMBC correlation between H-1' (δH 4.28) and C-9 (δC 74.7) (Fig. 2). The J values (J = 7.5 Hz) of the
anomeric proton at δH 4.46 indicated the presence of a β-glucopyranosyl unit.9 Enzyme hydrolysis of 2
1
afforded the aglycone and sugar unit, of which the former was identified by comparison of the H-NMR
data13 and the latter by comparison of co-TLC and optical rotation.11,14 Thus, the structure of compound 2
was determined as shown in Figure 1 and named stroside B.
Compound 3 was obtained as a brown gum. The molecular formula was determined to be C23H28O13 from

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HETEROCYCLES, Vol. 91, No. 12, 2015
1
the [M + H]+ peak at m/z 513.1602 (calcd. for C23H29O13: 513.1608) in the HR-FAB-MS. The H- and
13C-NMR spectra of 3 were very close to those of 1-β-D-glucosyloxy-2-(3-methoxy-4-hydroxyphenyl)-
propane-1,3-diol, which was isolated from Juniperus phcenicea,15 expect for the additional gallic acid
1
moiety [δH 7.11 (2H, s, H-2'', 6'') in the H-NMR: δC 168.5 (C-7''), 146.7 (C-3'', 5''), 140.0 (C-4''), 121.5
13
(C-1''), 110.3 (C-2'', 6'') in the C-NMR]. Its position was assigned as C-6' by the HMBC experiment,
showing correlation between H-6' [δH 4.56 (1H, dd, J = 12.0, 2.0 Hz) and 4.42 (1H, dd, J = 12.0, 5.0 Hz)]
and C-7'' (δC 168.5) (Figure 2). Thus, the structure of compound 3 was determined as shown in Figure
1 and named stroside C.
Compound 4 was obtained as a yellow gum. An HR-ESI-MS analysis indicated that the molecular
formula of 4 was C24H20O9 ([M+H-H2O]+ m/z 435. 1076, calcd 435.1080). The characteristic signals of
luteolin16 were observed at δH 7.42 (1H, d, J = 1.5 Hz, H-2'), 7.34 (1H, dd, J = 8.0, 1.5 Hz, H-6'), 6.88
1
(1H, d, J = 8.0 Hz, H-5'), 6.46 (1H, s, H-3), 6.30 (1H, brs, H-8), 6.08 (1H, brs, H-6) in the H-NMR
spectrum; at δC 182.3 (C-4), 164.9 (C-7), 163.9 (C-2), 161.8 (C-5), 158.0 (C-9), 147.5 (C-4'), 143.9 (C-3'),
123.9 (C-1′), 119.5 (C-6'), 117.4 (C-5'), 114.7 (C-2'), 103.9 (C-10), 103.3 (C-3), 98.9 (C-6), 93.7 (C-8) in
the 13C-NMR spectrum, and of p-hydroxylphenylglycerol moiety observed in the NMR data [δH 7.18 (2H,
d, J = 8.0 Hz, H-3'', 5''), 6.74 (2H, d, J = 8.0 Hz, H-2'', 6''), 4.84 (1H, d, J = 8.5 Hz, H-7''), 3.96 (1H, brs,
H-8''), 3.62 (1H, d, J = 12.0 Hz, H-9''a), 3.39 (1H, td, J = 12.0, 6.0 Hz, H-9''b); δC 158.0 (C-1''), 128.7
(C-3'', 5''), 126.8 (C-4''), 115.1 (C-2'', 6''), 78.6 (C-8''), 76.6 (C-7''), 60.5 (C-9'')]. Each partial structures
were determined by 2D-NMR spectral data (1H-1H COSY, HMQC, and HMBC data) (Figure 2). The
carbon chemical shift at C-4′ in 4 (δC 147.5) was upfield shifted comparing to that of luteolin (δC 149.8),16
suggesting that the p-hydroxylphenylglycerol moiety was located at the C-4′ position of the luteolin in 4.
The ΔδC8′′-C7′′ value of 4 in CD3OD was 2.0 ppm, which indicated that the glycerol moiety of 4 possesses
a threo relative configuration [erythro type : (ΔδC8′′-C7′′ < 1.0 ppm]. The positive optical rotation ([α]D25
+7.0) of 4 supported that its configuration is 7''S and 8''S.17-19 Thus, the structure of compound 4 was
determined as shown in Figure 1 and named lagerindiol.
The known compounds were identified as 9,9'-dihydroxy-3,4-methoxylenedioxy-3'-methoxy[7-O-4'-8-5']-
neolignan (5),20 pterospermin A (6),21 (2R,3S)-dihydrodehydroconiferyl alcohol (7),22 gochidioboside
(8),23 7S,8R-dihydrodehydrodiconiferyl alcohol 4-O-β-D-glucopyranoside (9),24 hovetrichoside A (10),25
hovetrichoside B (11),25 (1'S,2'R)-guaiacyl glycerol (12),26 carthamoside B5 (13),27 (+)-(7S,8S)-guaiacyl-
glycerol 8-O-β-D-glucopyranoside (14),19 D-threo-guaiacylglycerol 8-O-β-D-(6'-O-galloyl)glycopyrano-
side (15),28 alatusol A (16),29 ficusol (17),30 evofolin-B (18),10 and marphenol C (19)31 by comparison of
their spectroscopic data with reported data.

HETEROCYCLES, Vol. 91, No. 12, 2015
2359
Table 1. Inhibitory Effect on NO Production of Compounds 6, 7 and 16-19 in LPS-Activated BV-2 Cells
Compounds IC50 (μM)a
Cell Viability (%)b
21.4
14.6
35.4
36.0
22.0
44.9
18.35
6
7
16
17
18
155.66s6.3
144.59s2.2
137.8s4.1
110.75s5.4
106.4s6.9
111.3s6.8
98.2±4.5
19
L-NMMAc
a IC50 value of each compound was defined as the concentration (μM) that caused 50% inhibition of NO
production in LPS-activated BV-2 cells.
b Cell viability after treatment with 20 μM of each compound is expressed as a percentage (%) of the LPS
only treatment group. Results are averages of three independent experiments, and the data are expressed
as mean ± SD. Statistical comparisons were performed using a one-way ANOVA test with Student’s
t-test. Only *p-value < 0.05 was indicated as statistically significant.
c NMMA as a positive control.
Then, we evaluated the anti-inflammatory activities of the isolates (1–19) through the measurement of
produced NO levels in murine microglia BV2 cells stimulated by bacterial pathogen, LPS.32 Among the
tested compounds, compound 7 significantly inhibited LPS-stimulated NO production with IC50 values of
14.6 μM, which displayed more activity than L-NMMA, a well-known NOS inhibitor. Compound 18
showed the inhibitory activity with an IC50 of 22.0 μM in BV-2 cells without cell toxicity (Table 1). The
other (6, 16, 17, and 19) exhibited week NO production activity in the murine microglia BV-2 cell line.
The rest of the compounds did not show any significant inhibitory effects on NO production.
Table 2. Cytotoxicity of compounds 4 and 6 against four cultured human cancer cell lines using the SRB
assay in vitro
IC50 (μM)a
Compounds
A549
16.59
6.51
SK-OV-3
16.64
SK-MEL-2
17.26
HCT-15
8.83
5.87
4
6
9.13
11.38
Doxorubicinb
0.026 ± 0.005 0.067 ± 0.003 0.006 ± 0.001 0.013 ± 0.017
a IC50 value of compounds against cancer cell lines, defined as the concentration (μM) that caused 50%
inhibition of cell growth in vitro.
b Doxorubicin as positive control.
The isolated compounds (1-19) were also performed for cytotoxicity against the A549 (non-small cell
lung carcinoma), SK-OV-3 (ovary malignant ascites), SK-MEL-2 (skin melanoma), and HCT-15 (colon
adenocarcinoma) human tumor cell lines in vitro using the sulforhodamine B assay.33 Compounds 4 and 6

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HETEROCYCLES, Vol. 91, No. 12, 2015
(Table 2), which has flavones skeleton exhibited moderate cytotoxic activity against four human cell lines
(IC50 (4): 16.59, 16.64, 17.26, and 8.83 μM, and IC50 (6): 6.51, 9.13, 11.38, and 5.87 μM, respectively).
The other compounds were inactive (IC50 > 30.0 μM).
EXPERIMENTAL
General. Optical rotations were measured on a Jasco P-1020 polarimeter. IR spectra were recorded on a
Bruker IFS-66/S FT-IR spectrometer using methanol as a solvent. UV spectra were recorded with a
Shimadzu UV-1601 UV-Visible spectrophotometer using methanol as a solvent. HR-FAB mass spectra
were obtained on a JEOL JMS700 mass spectrometer. HR-ESI mass spectra were recorded on a
SI-2/LCQ DecaXP liquid chromatography (LC)-mass spectrometer (Thermo Scientific, West Palm Beach,
1
FL, USA). NMR spectra, including H-1H COSY, HMQC, HMBC, NOESY experiments, were recorded
on a Varian UNITY INOVA 700 NMR spectrometer operating at 700 MHz (1H) and 175 MHz (13C), with
chemical shifts given in ppm (δ). Semi-preparative HPLC used a Gilson 306 pump with a Shodex
refractive index detector. Silica gel 60 (Merck, 230-400 mesh) and RP-C18 silica gel (Merck, 230-400
mesh) were used for column chromatography. Low-pressure LC was performed over a LiChroprep
Lobar-A RP-18 (240 × 10 mm i.d.) column with a FMI QSY-O pump (ISCO). The packing material for
molecular sieve column chromatography was Sephadex LH-20 (Pharmacia Co. Ltd). Merck precoated
silica gel F254 plates and RP-18 F254s plates were used for TLC. Spots were detected on TLC under UV
light or by heating after spraying with anisaldehyde-sulfuric acid. Lipopolysaccharide (LPS) was obtained
from Sigma Chemical Company (St. Louis, USA).
Plant material. L. indica stems (5 kg) were collected from Goesan in Chungcheongbuk-do, Korea, in
May 2012. The plant was authenticated by one of the authors (K. R. Lee). A voucher specimen
(SKKU-NPL-1203) of the plant was deposited in the herbarium of the School of Pharmacy,
Sungkyunkwan University, Suwon, Korea.
Extraction and isolation. The air dried plant material (5 kg) were extracted with 80% MeOH three times
under reflux. The filtrate was evaporated under reduced pressure to yield a MeOH extract (300 g), which
was suspended in water (800 mL) and solvent-partitioned to afforded n-hexane (17.3 g, yield 5.73%),
CHCl3 (9.1 g, yield 3.03%), EtOAc (8.9 g, yield 2.96%), and BuOH (55.0 g, yield 18.33%) fractions. The
CHCl3 (9.1 g) fraction was chromatographed on a silica gel (230–400 mesh, 300 g) column eluted with
CHCl3-MeOH (30:1, 20:1, 10:1, and 1:1) to yield four fractions (Fr. C1 – Fr. C4). Fr. C1 (2.2 g) was
further chromatographed on a silica gel (230–400 mesh, 150 g) column eluted with CHCl3-MeOH (60:1)
to yield four fraction (Fr. C11 – Fr. C14). Fr. C12 (1.0 g) was separated over an RP-C18 silica gel

HETEROCYCLES, Vol. 91, No. 12, 2015
2361
(230–400 mesh) column (150 g, 1.5 × 30 cm) using a solvent system of 100% MeOH to give seven
subfractions (Fr. C121 – Fr. C127). Fr. C121 (200 mg) was purified by semi-preparative reversed-phase
HPLC using a 250 mm × 10 mm i.d., 10 μm, Econosil RP-18 column (Alltech) with a solvent system of
35% MeCN (1 L, flow rate; 2 mL/min) to give compounds 16 (3 mg), 17 (9 mg), and 19 (3 mg). Fr. C13
(200 mg) was separated over an RP-C18 silica gel (230–400 mesh) column (100 g, 1.5 × 30 cm) using a
solvent system of 60% MeOH to give seven subfractions (Fr. C131 – Fr. C137). Fr. C131 (50 mg) was
purified by semi-preparative reversed-phase HPLC with a solvent system of 40% MeCN to yield
compound 18 (9 mg). Fr. C14 (900 mg) was separated over an RP-C18 silica gel (230–400 mesh) column
(100 g, 1.5 × 30 cm) using a solvent system of 90~100% MeOH to give four subfractions (Fr. C141 – Fr.
144). Fr. C142 (340 mg) was loaded on a Sephadex LH-20 column (450 g, 2 × 50 cm) and eluted with
80% MeOH and purified by semi-preparative reversed-phase HPLC with a solvent system of 55% MeCN
to yield compound 7 (9 mg). The EtOAc (8.8 g) was separated over an RP-C18 silica gel (230–400 mesh)
column (300 g, 3 × 30 cm) using a solvent system of 30~100% MeOH to give ten fractions (Fr. E1 – Fr.
E10). Fr. E6 (1.0 g) was loaded on a Sephadex LH-20 column (450 g, 2 × 50 cm) and eluted with 80%
MeOH to yield five subfractions (Fr. E61 – Fr. E65). Fr. E65 (220 mg) was purified by semi-preparative
reversed-phase HPLC with a solvent system of 50% MeOH to yield compound 5 (1.5 mg). Fr. E7 (470
mg) was loaded on a Sephadex LH-20 column (450 g, 2 × 50 cm) and eluted with 80% MeOH and
purified by semi-preparative reversed-phase HPLC, as described above, with a solvent system of 55%
MeCN to yield compounds 4 (2 mg) and 6 (5 mg). The BuOH (17.0 g) was chromatographed on a silica
gel (230–400 mesh, 300 g) column eluted with CHCl3-MeOH (5:1 and 1:1) to yield four fraction (Fr. B1
– Fr. B4). Fr. B2 (2.7 g) was separated over an RP-C18 silica gel (230–400 mesh) column (200 g, 1.5 × 30
cm) using a solvent system of 37% MeOH to give nine fractions (Fr. B21 – Fr. B29). Fr. B21 (520 mg)
was loaded on a Sephadex LH-20 column (450 g, 2 × 50 cm) and eluted with 80% MeOH and purified by
semi-preparative reversed-phase HPLC with a solvent system of 10% MeCN to yield compounds 12 (2
mg) and 14 (16 mg). Fr. B22 (500 mg) was loaded on a Sephadex LH-20 column (450 g, 2 × 50 cm) and
eluted with 80% MeOH to yield six subfractions (Fr. B221 – Fr. B226). Fr. B222 (200 mg) was separated
using a Lobar-A Si gel 60 (240 × 10 mm) column (CHCl3/MeOH = 5:1) and purified by semi-preparative
reversed-phase HPLC with a solvent system of 15% MeCN to yield compounds 2 (3 mg), 10 (5 mg), 11
(9 mg) and 13 (4 mg). Fr. B223 (60 mg) was purified by semi-preparative reversed-phase HPLC with a
solvent system of 15% MeCN to yield compound 1 (5 mg). Fr. B225 (40 mg) was purified by
semi-preparative reversed-phase HPLC with a solvent system of 20% MeCN to yield compounds 3 (3
mg) and 15 (3 mg). Fr. B25 (160 mg) was loaded on a Sephadex LH-20 column (450 g, 2 × 50 cm) and
eluted with 80% MeOH and purified by semi-preparative reversed-phase HPLC with a solvent system of
20% MeCN to yield compound 9 (4 mg). Fr. B26 (170 mg) was loaded on a Sephadex LH-20 column

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HETEROCYCLES, Vol. 91, No. 12, 2015
(450 g, 2 × 50 cm) and eluted with 80% MeOH and purified by semi-preparative reversed-phase HPLC
with a solvent system of 20% MeCN to yield compound 8 (6 mg).
Stroside A (1): Brown gum, [α]25 +3.0 (c 0.05, CHCl3); UV (MeOH) λmax (log ε) 272 (0.7), 227 (1.3),
D
207 (1.8) nm; IR (KBr) νmax 3375, 2950, 1653, 1513, 1454, 1419, 1270, 1076, 1018, 713 cm-1; CD
1
(MeOH) λmax (Δε) 208 (+11.1), 222 (–9.3), 238 (+16.1), and 310 (–5.96) nm; H-NMR (CD3OD, 700
MHz) δH 7.69 (1H, dd, J = 8.5, 2.0 Hz, H-6), 7.62 (1H, d, J = 2.0 Hz, H-2), 7.18 (1H, d, J = 8.5 Hz, H-5),
6.90 (1H, d, J = 2.0 Hz, H-2'), 6.76 (1H, dd, J = 8.5, 2.0 Hz, H-6'), 6.74 (1H, d, J = 8.5 Hz, H-5'), 5.01
(1H, d, J = 7.5 Hz, H-1''), 4.78 (1H, dd, J = 8.5, 5.0 Hz, H-8), 4.27 (1H, dd, J = 10.5, 9.0 Hz, H-9a), 3.90
(1H, m, H-6''a), 3.89 (3H, s, 3-OCH3), 3.84 (3H, s, 3'-OCH3), 3.73 (1H, dd, J = 10.5, 5.0 Hz, H-9b), 3.69
(1H, dd, J = 12.5, 6.0 Hz, H-6''b), 3.52 (1H, m, H-2''), 3.48 (1H, m, H-5''), 3.46 (1H, m, H-3''), 3.40 (1H,
m, H-4''); 13C-NMR (CD3OD, 175 MHz) δC 199.3 (C-7), 152.3 (C-4), 150.7 (C-3), 149.5 (C-3'), 147.2
(C-4'), 132.9 (C-1), 129.7 (C-1'), 124.6 (C-6), 122.3 (C-6'), 116.7 (C-5'), 116.3 (C-5), 113.2 (C-2), 112.9
(C-2'), 101.9 (C-1''), 78.4 (C-3''), 78.0 (C-5''), 74.8 (C-2''), 71.4 (C-4''), 65.5 (C-9), 62.6 (C-6''), 56.7 (C-8,
3-OCH3), 56.5 (3'-OCH3); HR-FAB-MS m/z 481.1710 [M+H]+ (calcd for 481.1710).
Stroside B (2): Brown gum, [α]25 +8.0 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 281 (0.5), 230 (1.2),
D
208 (1.9) nm; IR (KBr) νmax 3377, 2945, 1645, 1518, 1456, 1432, 1375, 1275 1158, 1078, 1026, 742 cm-1;
1H-NMR (CD3OD, 700 MHz) δH 6.86 (1H, d, J = 2.0 Hz, H-2), 6.73 (1H, d, J = 7.5 Hz, H-5), 6.69 (1H,
dd, J = 7.5, 2.0 Hz, H-6), 4.28 (1H, d, J = 7.5 Hz, H-1'), 3.98 (1H, m, H-8), 3.93 (1H, dd, J = 10.5, 3.5 Hz,
H-9a), 3.87 (3H, s, 3-OCH3), 3.86 (1H, m, H-6a'), 3.68 (1H, dd, J = 12.0, 5.5 Hz, H-6'b), 3.42 (1H, dd, J
= 10.0, 7.0 Hz, H-9b), 3.37 (1H, m, H-5'), 3.30 (1H, m, H-4'), 3.27 (1H, m, H-3'), 3.24 (1H, m, H-2'), 2.76
(1H, dd, J = 13.0, 6.0 Hz, H-7a), 2.70 (1H, dd, J = 14.5, 7.0 Hz, H-7b); 13C-NMR (CD3OD, 175 MHz) δC
148.9 (C-3), 146.1 (C-4), 131.2 (C-1), 123.0 (C-6), 116.2 (C-5), 114.3 (C-2), 105.0 (C-1'), 78.1 (C-3'),
78.0 (C-5'), 75.3 (C-2'), 74.7 (C-9), 73.2 (C-8), 71.7 (C-4'), 62.7 (C-6'), 56.5 (3-OCH3), 40.5 (C-7);
HR-FAB-MS m/z 361.1499 [M+H]+ (calcd for361.1498).
Stroside C (3): Brown gum, [α]25 +9.3 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 275 (0.5), 228 (0.8),
D
206 (1.5); IR (KBr) νmax 3374, 2929, 2856, 1641, 1519, 1440, 1378, 1261, 1077, 1020, 638cm-1; 1H-NMR
(CD3OD, 700 MHz) δH 7.11 (2H, s, H-2'', 6''), 6.85 (1H, d, J = 1.5 Hz, H-2), 6.71 (1H, d, J = 8.5 Hz, H-5),
6.66 (1H, dd, J = 8.5, 1.5 Hz, H-6), 4.56 (1H, dd, J = 12.0, 2.0 Hz, H-6'a), 4.42 (1H, dd, J = 12.0, 5.0 Hz,
H-6'b), 4.36 (1H, d, J = 7.5 Hz, H-1'), 4.06 (1H, dd, J = 10.0, 6.0 Hz, H-9a), 3.86 (1H, dd, J = 10.0, 7.5
Hz, H-9b), 3.83 (1H, dd, J = 11.0, 6.0 Hz, H-8a), 3.77 (1H, m, H-8b), 3.58 (1H, m, H-5'), 3.42 (1H, m,

HETEROCYCLES, Vol. 91, No. 12, 2015
2363
H-4'), 3.40 (1H, m, H-3'), 3.23 (1H, m, H-2'), 3.03 (1H, m, H-7); 13C-NMR (CD3OD, 175 MHz) δC 168.5
(C-7''), 148.9 (C-3), 146.7 (C-3'', 5''), 146.3 (C-4), 140.0 (C-4''), 133.4 (C-1), 121.9 (C-6), 121.5 (C-1''),
116.2 (C-5), 113.0 (C-2), 110.3 (C-2'', 6''), 105.0 (C-1'), 78.1 (C-3'), 75.7 (C-5'), 75.1 (C-2'), 72.5 (C-9),
71.8 (C-4'), 65.2 (C-8), 64.9 (C-6'), 56.5 (3-OCH3), 49.6 (C-7); HR-FAB-MS m/z 513.1602 [M+H]+
(calcd for 513.1608).
Lagerindiol (4): Yellow gum, [α]25 +7.0 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 335 (0.5), 269 (0.8),
D
206 (1.3); IR (KBr) νmax 3359, 2940, 2831, 1652, 1607, 1498, 1440, 1350, 1252, 1164, 1032, 833, 638
cm-1; 1H-NMR (CD3OD, 700 MHz) δH 7.42 (1H, d, J = 1.5 Hz, H-2'), 7.34 (1H, dd, J = 8.0, 1.5 Hz, H-6'),
7.18 (2H, d, J = 8.0 Hz, H-3'', 5''), 6.88 (1H, d, J = 8.0 Hz, H-5'), 6.74 (2H, d, J = 8.0 Hz, H-2'', 6''), 6.46
(1H, s, H-3), 6.30 (1H, brs, H-8), 6.08 (1H, brs, H-6), 4.84 (1H, d, J = 8.5 Hz, H-7''), 3.96 (1H, brs, H-8''),
3.62 (1H, d, J = 12.0 Hz, H-9''a), 3.39 (1H, td, J = 12.0, 6.0 Hz, H-9''b); 13C-NMR (CD3OD, 175 MHz) δC
182.3 (C-4), 164.9 (C-7), 163.9 (C-2), 161.8 (C-5), 158.0 (C-9, 1''), 147.5 (C-4'), 143.9 (C-3'), 128.7
(C-3'', 5''), 126.8 (C-4''), 123.9 (C-1′), 119.5 (C-6'), 117.4 (C-5'), 115.1 (C-2'', 6''), 114.7 (C-2'), 103.9
(C-10), 103.3 (C-3), 98.9 (C-6), 93.7 (C-8), 78.6 (C-8''), 76.6 (C-7''), 60.5 (C-9''); HR-ESI-MS m/z
435.1076 [M+H-H2O]+ (calcd for 435.1080).
Enzyme hydrolysis and sugar identification Compounds 1 (1.0 mg) and 2 (1.5 mg) in H2O (2 mL) were
o
hydrolyzed with β-glucosidase (30 mg, Emulsin) at 37 C for 2 days. The CHCl3 layers of 1 and 2 were
followed by purification on a silica gel Waters Sep-Pak Vac 6cc (CHCl3-MeOH, 10:1) to afford 1a (0.2
mg) and 2a (0.4 mg). The sugar in the water layer was identified as D-glucose by co-TLC
(CHCl3:MeOH:H2O = 2:1:0.2, Rf value: 0.2 for glucose) and optical rotation {[α]25 +46.6 (c 0.02, H2O)
D
from 1 and [α]25 +43.1 (c 0.10, H2O) from 2}.
D
Evofolin-B (1a): brown gum; FAB-MS: m/z = 319 [M+H]+; 1H-NMR (CD3OD, 700 MHz) δH 7.62 (1H,
dd, J = 8.0, 2.0 Hz, H-6), 7.58 (1H, d, J = 2.0 Hz, H-2), 6.91 (1H, d, J = 2.0 Hz, H-2'), 6.81 (1H, d, J =
8.0 Hz, H-5), 6.77 (1H, dd, J = 8.0, 2.0 Hz, H-6'), 6.74 (1H, d, J = 8.0 Hz, H-5'), 4.77 (1H, dd, J = 8.5, 2.0
Hz, H-8), 4.26 (1H, dd, J = 11.0, 8.0 Hz, H-9a), 3.88 (3H, s, 3-OCH3), 3.84 (3H, s, 3'-OCH3),3.62 (1H, dd,
J = 10.0, 5.0 Hz, H-9b).
3-(4-Hydroxy-3-methoxy)-phenyl-1,2-propandiol (2a): brown gum; FAB-MS: m/z = 199 [M+H]+;
1H-NMR (CD3OD, 700 MHz) δH 6.72 (1H, d, J = 2.0 Hz, H-2), 6.60 (1H, d, J = 2.0 Hz, H-5), 6.55 (1H,
dd, J = 8.0, 2.0 Hz, H-6), 3.73 (3H, s, 3-OCH3), 3.68 (1H, m, H-8), 3.39 (1H, dd, J = 11.0, 4.5 Hz, H-7a),

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3.31 (1H, dd, J = 11.5, 4.0 Hz, H-7b), 2.62 (1H, dd, J = 14.0, 5.5 Hz, H-9a), 2.49 (1H, dd, J = 13.0, 6.5
Hz, H-9b).
Measurement of NO Production and Cell Viability The BV-2 mouse microglial cell line has been
extensively used in published studies as an in vitro culture system for the investigation of primary
microglial function. In this study, BV-2 cells were maintained in DMEM supplemented with 5% FBS and
1% PS. To measure nitric oxide (NO) production, BV-2 cells were plated into a 96 well plate (3™104
cells/well) and treated with 100 ng/mL lipopolysaccharide (LPS) in the presence or absence of isolates
(1-19) for 24 h. Nitrite, a soluble oxidation product of NO, was measured in the culture media using the
Griess reaction. The supernatant (50 μL) was harvested and mixed with an equal volume of Griess
reagent (1% sulfanilamide and 0.1% N-1-naphthylethylenediamine dihydrochloride in 5% phosphoric
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 NO2 concentration. Cell viability was assessed by the MTT assay.
In this study, NG-mono-methyl-L-arginine (L-NMMA, Sigma-Aldrich), a well-known nitric oxide
synthase (NOS) inhibitor, was tested as a positive control.32
Cytotoxicity Assay A sulforhodamine B (SRB) bioassay was used to determine the cytotoxicity of each
compound isolated against four cultured human tumor cell lines.33 The assays were performed at the
Korea Research Institute of Chemical Technology. The cell lines used were A549 (non-small cell lung
carcinoma), SK-OV-3 (ovary malignant ascites), SK-MEL-2 (skin melanoma), and HCT-15 (colon
adenocarcinoma). Doxorubicin was used as a positive control. The cytotoxicities of doxorubicin against
the A549, SK-OV-3, SK-MEL-2, and HCT-15 cell lines were IC50 0.026 ± 0.005, 0.067 ± 0.003, 0.006 ±
0.001, and 0.013 ± 0.017 μM, respectively.
ACKNOWLEDGEMENTS
This research was supported by the Basic Science Research Program through the National Research
Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology
(2013R1A1A2A10005315). We are thankful to the Korea Basic Science Institute (KBSI) for the
measurements of NMR and MS spectra.
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