SUPPLEMENTARY MATERIAL
Flavonoids as low-density lipoprotein oxidation inhibitor from Lindera glauca Blume
Gyu-Won Huh1, Ji-Hae Park1, Ji-Hyun Kang2,3, Tae-Sook Jeong3, Hee Cheol Kang4, and
Nam-In Baek1,*
1
The Graduate School of Biotechnology and Institute of Life Sciences & Resources, Kyung
Hee University, Yongin 446-701, Republic of Korea
2
National Research Laboratory of Lipid Metabolism & Atherosclerosis, KRIBB, 52 Eoeundong, Yuseong, Daejeon 305-806, Repuiblic of Korea
3
Department of Food and Nutrition, Chungnam National University, 220 Gung-dong,
Yuseong, Daejeon 305-764, Republic of Korea
4
GFC Co., Ltd., Suwon 443-813, Republic of Korea
1. Experimental
1.1. Plant material
The dried heartwood of grayblue spicebush (L. glauca Bl.) was supplied from GFC
Co., Ltd. (Suwon, Korea) in 2009 and identified by Prof. Dae-Geun Kim, College of
Pharmacy, Woosuk University, Jeonju, Korea. A voucher specimen (KHU-09-1125) was
deposited at the Natural Products Chemistry Laboratory, Kyung Hee University, Yongin,
Korea.
1.2. Reagents
The organic solvents for solid extraction and solvent fractionation were first-grade
reagents supplied by Daejung Chemical Ltd. (Seoul, Korea). Open column chromatography
(c.c.) was performed over with Kieselgel 60 (63~200 μm, Merck, Darmstadt, Germany),
LiChroprep RP-18 (Merck), or Sephadex LH-20 (Amersham Pharmacia Biotech, Uppsala,
Sweden). TLC was performed with Kieselgel 60 F254 and RP-18 F254S (Merck). Deuterated
solvents were purchased from Sigma Aldrich (St. Louis, MO, USA).
1.3. Instrumentation
The TLC spots were monitored using a UV lamp (254 and 366 nm), the Spectroline
Model ENF-240 C/F (Spectronics Corporation, Westbury, NY, USA). 1H-, 13C-NMR, DEPT,
gCOSY, gHSQC, and gHMBC spectra were recorded on a 400 MHz FT-NMR spectrometer
(Varian Inova AS-400, Palo Alto, CA, USA). IR spectra were obtained using a Perkin Elmer
Spectrum One FT-IR spectrometer (Beaconsfield, England). Optical rotations were measured
on a polarimeter P-1020 (JASCO, Tokyo, Japan). EI/MS and FAB/MS data were obtained on
a JMSAX 700 (JEOL, Tokyo, Japan).
1.4. Isolation of flavonoids from the EtOAc fraction of Lindera glauca Blume heartwood
The dried and chipped heartwood of L. glauca (4.9 kg) was extracted repeatedly with
80% aqueous MeOH (24 h, three times) at room temperature. Then, the concentrated extracts
(179 g) were successively partitioned with EtOAc (2 L, three times), n-BuOH (1.8 L, three
times), and H2O (2 L). The partitioned extracts were evaporated in vacuo to obtain residues
of the EtOAc fraction (LGBE, 35 g, yield 0.7%), the n-BuOH fraction (LGBB, 42 g, yield
0.8%), and the H2O fraction (LGBW, 102 g, yield 2.1%). The EtOAc fraction (LGBE, 35 g)
was applied to SiO2 c.c. (Ø 8.0 cm × 19.0 cm) and eluted with n-hexane-EtOAc (1:1, 12.0 L)
and CHCl3-MeOH (10:1⟶5:1, 21.0 L of each). The eluted solutions were monitored by TLC
and produced 13 fractions (LGBE-1 to LGBE-13). The isolation procedure for compounds 13 was reported in our previous study (Huh et al. 2012). Fraction LGBE-8 (3.3 g, Ve/Vt 0.370.55) was subjected to SiO2 c.c. (Ø 5.5 cm × 12.0 cm) and eluted with CHCl3-MeOH-H2O
(15:3:1, lower phase, 6.2 L), yielding 12 fractions (LGBE-8-1 to LGBE-8-12). Fraction
LGBE-8-6 (972 mg, Ve/Vt 0.12-0.24) was applied to SiO2 c.c. (Ø 3.5 cm × 13.0 cm) and
eluted with EtOAc-n-BuOH-H2O (25:2:1, 1.8 L), yielding 12 fractions (LGBE-8-6-1 to
LGEB-8-6-12). Fraction LGBE-8-6-2 (16 mg, Ve/Vt 0.06-0.09) was purified using ODS c.c.
(Ø 2.0 cm × 5.5 cm) and eluted with MeOH-H2O (1:1, 0.78 L) to produce compound 4
(LGBE-8-6-2-6, 2 mg, Ve/Vt 0.07-0.46, ODS TLC Rf 0.38, MeOH-H2O=2:1). Fraction
LGBE-10 (1.5 g, Ve/Vt 0.62-0.68) was subjected to SiO2 c.c. (Ø 4.0 cm × 13.5 cm) and eluted
with CHCl3-MeOH (15:1, 2.9 L), yielding 10 fractions (LGBE-6-1 to LGBE-6-10). Fraction
LGBE-10-16 (81 mg, Ve/Vt 0.39-0.46) was purified using ODS c.c. (Ø 3.5 cm × 6.0 cm) and
eluted with MeOH-H2O (1:2, 0.87 L) to produce compound 5 (LGBE-10-16-11, 11 mg, Ve/Vt
0.17-0.24, SiO2 TLC Rf 0.41, CHCl3-MeOH-H2O=65:35:10). Fraction LGBE-12 (1.5 g,
Ve/Vt 0.85-0.96) was subjected to SiO2 c.c. (Ø 4.0 cm × 15.0 cm) and eluted with CHCl3MeOH-H2O (7:3:1, 12.9 L), yielding 16 fractions (LGBE-12-1 to LGBE-12-16). Fraction
LGBE-12-15 (350 mg, Ve/Vt 0.76-0.98) was applied using ODS c.c. (Ø 3.5 cm × 5.0 cm) and
eluted with acetone-H2O (1:4, 0.34 L), yielding 10 fractions (LGBE-12-15-1 to LGBE-12-1510). LGBE-12-15-3 (87 mg, Ve/Vt 0.04-0.31) was subjected to Sephadex LH-20 c.c. (Ø 2.0
cm × 45.0 cm) and eluted with MeOH-H2O (1:1, 1.7 L) to produce compound 6 (LGBE-1215-3-5, 35 mg, Ve/Vt 0.83-0.86, SiO2 TLC Rf 0.29, CHCl3-MeOH-H2O=8:3:1) and
compound 7 (LGBE-12-15-3-8, 20 mg, Ve/Vt 0.89-0.92, SiO2 TLC Rf 0.24, CHCl3-MeOHH2O=8:3:1). Fraction LGBE-12-15-6 (28 mg, Ve/Vt 0.37-0.43) was applied using Sephadex
LH-20 c.c. (Ø 2.0 cm × 60.0 cm) and eluted with MeOH-H2O (1:1⟶5:1, 0.9 L of each) to
produce compound 8 (LGBE-12-15-6-4, 10 mg, Ve/Vt 0.71-0.82, ODS TLC Rf 0.55, MeOHH2O=1:1).
Chemical characteristics of lindeglaucol (1), lindeglaucone (2), and cilicicone B (3)
were reported in a previous study (Huh et al. 2012).
Tamarixetin 3-O-α-L-rhamnoside (4): Yellowish powder, EI/MS m/z 462.4 [M]+; IR νmax
3300, 1715, 1640 cm-1; 1H-NMR (400 MHz, CD3OD) and
13
C-NMR (100 MHz, CD3OD);
see Table S1.
Procyanidin A2 (5): Brown amorphous powder; positive FAB/MS m/z 577.3 [M+H]+. 1HNMR (400 MHz, CD3OD) and 13C-NMR (100 MHz, CD3OD); see Table S2.
Cinnamtannin B1 (6):
Brown amorphous powder, IR νmax 1610, 1250, 1050 cm-1; positive
FAB/MS m/z 865.4 [M+H]+. 1H-NMR (400 MHz, CD3OD) and
13
C-NMR (100 MHz,
CD3OD); see Table S3.
Cinnamtannin D1 (7): Brown amorphous powder, IR νmax 1612, 1249, 1048 cm-1; positive
FAB/MS m/z 865.4 [M+H]+. 1H-NMR (400 MHz, CD3OD) and
13
C-NMR (100 MHz,
CD3OD); see Table S3.
Procyanidin A1 (8): Brown amorphous powder; positive FAB/MS m/z 577.3 [M+H]+. 1HNMR (400 MHz, CD3OD) and 13C-NMR (100 MHz, CD3OD); see Table S2.
1.5. The evaluation of inhibitory activity on low-density lipoprotein-oxidation
1.5.1. Low-density lipoprotein isolation
LDL was isolated by a standard procedure with a slight modification (Havel et al.
1955). Plasma was obtained from normalipidemic volunteers. EDTA was used as an anti-
coagulant (1.5 mg/mL of blood). After centrifugation at 1,500 × g for 15 min of the whole
blood to obtain plasma, EDTA (0.1%), NaN3 (0.05%), and phenylmethylsulfonylfluoride
(0.015%) were added to prevent lipoprotein modification. LDL was isolated from the plasma
by discontinuous density gradient ultra-centrifugation (Havel et al. 1955). Briefly, the plasma
was centrifuged at 100,000 × g at 4°C for 20 h. After the top layers containing chylomicron
and very low-density lipoprotein (VLDL) were removed, the density of the remaining plasma
fractions increased to 1.064 with NaBr solution and the samples were recentrifuged at
100,000 × g for an additional 24 h. The LDL fraction in the top of the tube was collected and
dialyzed overnight against three changes of phosphate buffer (pH 7.4) containing NaCl (150
mM), in the dark at 4°C to remove NaBr and EDTA. The LDL in PBS was stored at 4°C and
used within 4 weeks. The purity of the fraction was confirmed by agarose gel electrophoresis
and SDS-PAGE as described elsewhere (Markwell et al. 1981). The concentration of LDL
protein was determined using bovine serum albumin (BSA) as a standard (Markwell et al.
1981).
1.5.2. Low-density lipoprotein-oxidation assay
The TBARS assay of Buege and Aust (1978) was used with a slight modification.
The LDL solution (250 μL, 50-100 μg of protein) in 10 mM PBS (pH 7.4) was supplemented
with 10 μM CuSO4. The oxidation was performed in a screw-capped 5-mL glass vial at 37°C
in a shaking water bath. After a 4-hour incubation, the reaction was terminated by the
addition of 1 mL 20% trichloroacetic acid. Following precipitation, 1 mL of 0.67%
thiobarbituric acid in 0.05 N NaOH was added and vortexed, and the final mixture was
heated for 5 min at 95°C, cooled on ice, and centrifuged for 2 min at 1000 × g. The optical
density of the produced MDA was measured at 532 nm. Calibration was completed with an
MDA standard prepared from tetramethoxypropane.
References
Buege JA, Aust SD. 1978. Microsomal lipid peroxidation. Methods Enzymol. 52: 302–310.
Havel RJ, Edger HA, Bradgon JH. 1955. The distribution and chemical composition of
ultracentrifugally separated lipoproteins in human serum. J Clin Invest. 34:1345–1353.
Huh GW, Park JH, Shrestha S, Lee YH, Ahn EM, Kang HC, Kim YB, Baek NI. 2012. New
diarylpropanoids from Lindera glauca Bl. heartwood. Holzforschung 66:585–590.
Markwell MAK, Hass SM, Tolbert NE, Bieber LL. 1981. Protein determination in membrane
and lipoprotein samples: manual and automated procedures. Methods Enzymol. 72:
296–303.
Table S1. 1H-NMR (400 MHz) and
13
C-NMR (100 MHz) data of compound 4 from the
heartwood of Lindera glauca Blume (in CD3OD)
No.
2
3
4
4a
5
6
7
8
8a
1'
2'
3'
4'
5'
6'
4'-OCH3
rhm 1
rhm 2
rhm 3
rhm 4
rhm 5
rhm 6
δC (multiplicity)
159.2 (C)
136.1 (C)
179.6 (C)
105.8 (C)
163.2 (C)
94.8 (CH)
166.3 (C)
99.9 (CH)
158.6 (C)
122.9 (C)
113.4 (CH)
151 (C)
148.9 (C)
116.5 (CH)
124.2 (CH)
56.6 (CH3)
103.4 (CH)
71.9 (CH)
72.1 (CH)
73.1 (CH)
71.9 (CH)
17.7 (CH3)
δH (integration, coupling pattern, J in Hz)
6.70 (1H, br s)
6.71 (1H, br s)
7.44 (1H, d, 2.0)
6.94 (1H, d, 8.4)
7.41 (1H, dd, 2.0, 8.4)
3.86 (3H, s)
6.26 (1H, br s)
5.05 (1H, m)
4.58 (1H, dd, 3.2, 8.8)
4.24 (1H, dd, 8.8, 9.6)
4.17 (1H, dd, 6.0, 9.6)
1.40 (3H, d, 6.0)
Table S2. 1H-NMR (400 MHz) and 13C-NMR (100 MHz) data of compound 5 and 8 from the
heartwood of Lindera glauca Blume (in CD3OD)
compound 5
compound 8
No.
U-2
U-3
U-4
U-4a
U-5
U-6
U-7
U-8
U-8a
U-1'
U-2'
U-3'
U-4'
U-5'
U-6'
L-2
L-3
δC (multiplicity)
100.2 (C)
68.1 (CH)
29.3 (CH)
104.3 (C)
157.0 (C)
98.4 (CH)
158.2 (C)
96.7 (CH)
154.3 (C)
132.5 (C)
115.7 (CH)
146.3 (C)
145.7 (C)
116.1 (CH)
120.4 (CH)
81.8 (CH)
67.0 (CH)
δH (integration, coupling pattern, J in Hz)
4.06 (1H, d, 3.4)
4.40 (1H, d, 3.4)
6.00 (1H, d, 2.0)
6.07 (1H, d, 2.0)
7.15 (1H, d, 1.8)
6.81 (1H, d, 8.4)
7.02 (1H, dd, 8.4, 1.8)
4.92 (1H, br s)
4.24 (1H, m)
δC (multiplicity)
100.1 (C)
68.4 (CH)
29.9 (CH)
103.8 (C)
154.4 (C)
96.5 (CH)
157.8 (C)
98.0 (CH)
156.1 (C)
132.4 (C)
115.8 (CH)
145.7 (C)
146.3 (C)
116.4 (CH)
119.7 (CH)
84.6 (CH)
67.8 (CH)
L-4
29.9 (CH2)
2.94 (1H, dd, 17.4, 4.8) 2.75 (1H, dd, 17.4, 2.2)
30.7 (CH2)
L-4a
L-5
L-6
L-7
L-8
L-8a
L-1'
L-2'
L-3'
L-4'
L-5'
L-6'
102.5 (C)
156.6 (C)
96.7 (CH)
152.3 (C)
107.3 (C)
152.1 (C)
131.2 (C)
115.7 (CH)
146.8 (C)
146.0 (C)
116.0 (CH)
119.8 (CH)
6.09 (1H, s)
7.13 (1H, d, 2.2)
6.81 (1H, d, 8.2)
6.99 (1H, dd, 8.2, 2.2)
103.4 (C)
155.8 (C)
96.5 (CH)
152.7 (C)
107.4 (C)
151.1 (C)
130.8 (C)
116.2 (CH)
146.8 (C)
146.8 (C)
115.8 (CH)
120.8 (CH)
δH (integration, coupling pattern, J in Hz)
4.03 (1H, d, 3.2)
4.31 (1H, d, 3.2)
6.00 (1H, d, 2.4)
6.05 (1H, d, 2.4)
7.41 (1H, d, 2.4)
6.78 (1H, d, 8.4)
7.00 (1H, dd, 2.4, 8.4)
4.66 (1H, d, 8.0)
4.10 (1H, m)
3.00 (1H, dd, 5.6, 16.4)
2.50 (1H, dd, 8.8, 16.4)
6.10 (1H, s)
7.10 (1H, d, 2.0)
6.88 (1H, d, 8.4)
7.00 (1H, dd, 2.0, 8.4)
U represents the upper unit; L indicates the lower unit of compound 5 and 8.
Table S3. 1H-NMR (400 MHz) and 13C-NMR (100 MHz) data of compound 6 and 7 from the
heartwood of Lindera glauca Blume (in CD3OD)
compound 6
No.
U-2
U-3
U-4
U-4a
U-5
U-6
U-7
U-8
U-8a
U-1'
U-2'
U-3'
U-4'
U-5'
U-6'
M-2
M-3
M-4
M-4a
M-5
M-6
M-7
M-8
M-8a
M-1'
M-2'
M-3'
M-4'
M-5'
M-6'
L-2
L-3
L-4
L-4a
L-5
L-6
L-7
L-8
L-8a
L-1'
L-2'
L-3'
L-4'
L-5'
L-6'
δC (multiplicity)
99.9 (C)
67.2 (C)
28.8 (CH)
104.9 (C)
156.7 (C)
98.3 (CH)
157.8 (C)
96.5 (CH)
154.1 (C)
132.4 (C)
115.7 (CH)
145.4 (C)
146.6 (C)
116.1 (CH)
119.9 (CH)
78.9 (CH)
72.6 (CH)
38.3 (CH)
106.7 (C)
156 (C)
96.1 (CH)
151.1 (C)
106.4 (C)
151.8 (C)
131.8 (C)
116.7 (CH)
145.9 (C)
146.3 (C)
115.7 (CH)
121.3 (CH)
80.3 (CH)
67.5 (CH)
29.8 (CH2)
100 (C)
155.5 (C)
96.5 (CH)
155.8 (C)
108.8 (C)
155.8 (C)
133.2 (C)
115.5 (CH)
145.3 (C)
145.7 (C)
116 (CH)
119.4 (CH)
δH (integration, coupling pattern, J in Hz)
3.28 (1H, d, 3.4)
4.15 (1H, d, 3.4)
6.01 (1H, d, 2.4)
5.96 (1H, d, 2.4)
7.03 (1H, d, 1.4)
6.82 (1H, d, 8.0)
6.84 (1H, dd, 8.0, 1.4)
5.70 (1H, br s)
4.12 (1H, d, 1.2)
4.55 (1H, br s)
5.80 (1H, s)
7.31 (1H, d, 2.0)
6.83 (1H, d, 8.4)
7.19 (1H, dd, 8.4, 2.0)
4.38 (1H, s)
3.86 (1H, m)
2.83 (2H, m)
6.10 (1H, s)
6.82 (1H, d, 1.8)
6.75 (1H, d, 8.0)
6.71 (1H, dd, 8.0, 1.8)
compound 7
δC (multiplicity)
100.1 (C)
67.2 (CH)
28.9 (CH)
105.0 (C)
156.7 (C)
98.4 (CH)
157.8 (C)
96.5 (CH)
154.2 (C)
132.4 (C)
115.9 (CH)
145.5 (C)
146.7 (C)
115.7 (CH)
120.0 (CH)
78.7 (CH)
72.5 (CH)
38.3 (CH)
106.6 (C)
155.9 (C)
96.1 (CH)
151.1 (C)
106.3 (C)
151.7 (C)
131.5 (C)
116.5 (CH)
146.0 (C)
146.3 (C)
116.2 (CH)
121.1 (CH)
83.3 (CH)
70.1 (CH)
30.7 (CH2)
100.1 (C)
155.4 (C)
96.5 (CH)
155.6 (C)
108.8 (C)
155.4 (C)
132.7 (C)
115.8 (CH)
145.9 (C)
145.8 (C)
116.2 (CH)
120.0 (CH)
δH (integration, coupling pattern, J in Hz)
3.46 (1H, d, 3.6)
3.99 (1H, d, 3.6)
5.93 (1H, d, 2.4)
6.00 (1H, d, 2.4)
6.74 (1H, d, 2.0)
6.75 (1H, d, 8.0)
6.94 (1H, dd, 2.0, 8.0)
5.51 (1H, br s)
4.05 (1H, d, 1.2)
4.52 (1H, br s)
5.83 (1H, s)
7.22 (1H, d, 2.0)
6.84 (1H, d, 8.4)
7.08 (1H, dd, 2.0, 8.4)
3.95 (1H, d, 9.2)
3.67 (1H, m)
3.04 (1H, dd, 6.4, 16.0) 2.42 (1H, dd, 10.0, 16.0)
6.10 (1H, s)
7.08 (1H, d, 2.4)
6.85 (1H, d, 8.4)
6.66 (1H, d, 8.4)
U represents the upper unit; M designates the middle unit; L indicates the lower unit of
compound 6 and 7.