SUPPLEMENTARY MATERIAL
Triterpenoids from the Fruits and Leaves of the Blackberry
(Rubusallegheniensis) and Their Inhibitory Activities on Foam Cell Formation in
Human Monocyte-Derived Macrophage
MasateruOnoa,*, Shin Yasudaa, HarukiKomatsua, Yukio Fujiwarab, MotohiroTakeyab
and Toshihiro Noharac
*Corresponding author: E-mail: mono@agri.u-tokai.ac.jp
Abstract
From the methanol extract of the fruits of the blackberry (Rubusallegheniensis Port.),
four triterpenoids, pomolic acid (1), tormentic acid (2), euscaphic acid (3), and 1hydroxyeuscaphic acid (4) were isolated, while six triterpenoids, 2, 3, myrianthic acid
(5), ziyu glycoside II (6), sericic acid (7), and 19-hydroxy-2,3-secours-12-ene-2,3,28trioic acid 3-methyl ester (8) were obtained from the methanol extract of the leaves of
this plant.Their structures were determined on the basis of spectral data. Compounds
1–8 were examined the inhibitory effect on foam cell formation in human monocytederived macrophages induced by acetylated low-density lipoproteins at a 50 M
concentration. Among the tested compounds, 1 showed the strongest activity, with the
inhibitory effect being 90%. The inhibitory activities of 2– 8 were evaluated to be 30,
32, 33, 4, 48, 4, and 24%, respectively. Further, the structure-activity relationship of
these compounds was investigated.
Keywords: Rubus allegheniensis; blackberry; triterpenoid; macrophage; foam cell
4. Experimental
4.1.Plant material
Fruits and leaves of R. allegheniensis Port. ‘MertonThornless’ were collected in
August 2000 at the orchard of Tokai University, Kumamoto prefecture, Japan, and
identified by one of the authors (Prof.Haruki Komatsu). A voucher specimen
(RATO2000) has been deposited at the laboratory of Natural Products Chemistry,
School of Agriculture, Tokai University.
4.2 .General experimental procedures
Optical rotations were measured with a JASCO DTP-1000 KUY digital polarimeter
(JASCO, Tokyo, Japan). 1H- and 13C-NMR spectra were recorded using a JEOL alpha
500 spectrometer (JEOL, Tokyo, Japan). Column chromatography was carried out
over silica gel 60 (Merck, Art. 1.09385; Merck, Darmstadt, Germany), Diaion HP20
(Mitsubishi Chemical Industries Co., Ltd., Tokyo, Japan), and Chromatorex ODS
(Fuji Silysia Chemical Ltd., Aichi, Japan). High performance liquid chromatography
(HPLC) separation was run on a Micro pump LC-10AS (Shimadzu, Kyoto, Japan)
with a RIdetector RID-10A (Shimadzu). For HPLC column chromatography, YMCpack SIL-06 (YMC Co., Ltd., Kyoto, Japan, 250 mm×20 mm i. d.), Kusano C.I.G.
Prepacked Si-5 (Kusano Kagakukikai Co., Tokyo, Japan, 100 mm×22 mm i. d.), and
COSMOSIL 5C18 AR-II (Nacalai Tesque, Inc., Kyoto, Japan, 250 mm×20 mm i. d.)
were used. Penicilin G, streptomysin sulfate, and Dulbecco's modified Eagle's medium
(DMEM) were purchased from Invitrogen (Tokyo, Japan). [9,10-3H]oleate was
purchased from GE Healthcare (Tokyo, Japan).
4.3. Extraction and isolation of 1–8
As part of an ongoing study of the constituents of this plant, the current study deals
with the isolation and structural elucidation of four triterpenoids from the fruits and
six triterpenoids from the leaves.
Crushedfruits of R. allegheniensis Port. (1.408 kg) were soaked in MeOH
(1.408 L) at room temperature (this procedure was repeated twice), and the solvent
was removed under reduced pressure to give a syrup (118.8 g). A part (108.8 g) of the
MeOH extract was subjected to Diaion HP20 column chromatography using gradient
mixtures of H2O–MeOH (H2O, 10% MeOH, 30% MeOH, 50% MeOH, 60% MeOH,
70% MeOH, 90% MeOH, MeOH) and acetone as eluentsto give fractions 1–9.
Fraction 7 (2.007 g) was chromatographed over silica gel column using gradient
mixtures of hexane–ethyl acetate (3:1, 2:1, 1:1, 1:2, 0:1) as eluents to give fractions 71–7-5. Fraction 7-4 (528 mg) was subjected to silica gel column chromatography
using gradient mixtures of CHCl3–MeOH (50:1, 45:1, 20:1, 16:1, 10:1, 9:1) as
eluentsto give frs.7-4-1–7-4-13. Fractions 7-4-2 (149 mg) and 7-4-3 (72 mg) were
each subjected to HPLC (YMC-pack SIL-06) with hexane–acetone (1:1) as eluent to
afford 3 (109 mg) from fraction 7-4-2 and 4 (20 mg) from fraction 7-4-3. HPLC
(Kusano C.I.G. Prepacked Si-5) of fraction 7-4-4 (14 mg) with CHCl3–MeOH (20:1)
as eluent gave 2 (5 mg). A part (523 mg) of fraction 8 (1.549 g) was chromatographed
over silica gel column using gradient mixtures of hexane–ethyl acetate (20:1, 10:1,
5:1, 2:1, 1:1, 1:5, 1:10, 1:20, 1:25) as eluentsto give fractions 8-1–8-6. Fraction 8-2
(63 mg) was successively subjected to silica gel column chromatography with CHCl3–
MeOH (200:1) as eluent and HPLC (COSMOSIL 5C18 AR-II) with 80 % MeOH as
eluent to give 1 (6 mg).
The cut fresh leaves (1.032 kg) of the plant were extracted with MeOH (4.200
L) at room temperature, and the solvent was removed under reduced pressure to give a
syrup (124.1 g). The MeOH extract was suspended in H2O (1.200 L) and extracted
with BuOH (1.200 L). The BuOH-soluble fraction (100.1 g) was further partitioned
between 90% MeOH (500 mL) and hexane (1.000 L). The 90% MeOH-soluble
fraction (86.4 g) was chromatographed over Diaion HP20 column using gradient
mixtures of H2O–MeOH (H2O, 50% MeOH, 70% MeOH, MeOH) and acetone as
eluentsto afford fractions 10–14. A part (8.8 g) of fraction 13 (20.9 g) was subjected to
silica gel column chromatography using gradient mixtures of hexane–acetone (10:1,
5:1, 3:1, 0:1:0) as eluentsto furnish fractions 13-1–13-11. Fraction 13-8 (1.304 g) was
subjected to Chromatorex ODS column chromatography using gradient mixtures of
H2O–MeOH (60% MeOH, 70% MeOH, 80% MeOH, 90% MeOH, MeOH) as
eluentsto
afford
fractions
13-8-1–13-8-3.
Fraction
13-8-1
(490
mg)
was
chromatographed over silica gel column using gradient mixtures of hexane–acetone
(5:1, 4:1, 3:1, 2:1, 1:1) and MeOH as eluentsto afford fractions 13-8-1-1–13-8-1-5.
HPLC (COSMOSIL 5C18 AR-II) of fraction 13-8-1-3 (187 mg) with 70% MeOH as
eluent afforded 8 (97 mg) and 3 (20 mg). Fraction 13.10 (1.543 g) was subjected to
silica gel column chromatography using gradient mixtures of CHCl3–MeOH–H2O
(20:1:0, 14:2:0.1, 0:1:0) as eluentsto furnish fractions 13-10-1–13-10-9. Fraction 1310-5 (399 mg) was chromatographed over Chromatorex ODS column using gradient
mixtures of H2O–MeOH (70% MeOH, 90% MeOH, MeOH) as eluentsto give
fractions 13-10-5-1–13-10-5-5. Fractions 13-10-5-1 (227 mg) and 13-10-5-2 (68 mg)
were each subjected to HPLC (COSMOSIL 5C18 AR-II) with 70% MeOH as eluent
to give 5 (26 mg) and 7 (20 mg) from fraction13-10-5-1 and 2 (16 mg), and 6(12 mg)
from fraction 13-10-5-2.
1: amorphous powder, []D17 +25.0° (c 0.5, MeOH). 1H-NMR (in C5D5N, 500 MHz)
: 5.63 (1H, dd, J = 3.5, 3.5 Hz, H-12), 3.46 (1H, dd, J = 5.0, 10.5 Hz, H-3), 3.15 (1H,
ddd, J = 4.5, 13.0, 13.0 Hz, Ha-16), 3.08 (1H, s, H-8), 2.37 (1H, ddd, J = 4.5, 13.0,
13.0 Hz, Ha-15), 1.75 (3H, s, H3-27), 1.47 (3H, s, H3-29), 1.25 (3H, s, H3-23), 1.14
(3H, d, J = 6.5 Hz, H3-30), 1.14 (3H, s, H3-26), 1.05 (3H, s, H3-24), 0.93 (3H, s, H325). 13C-NMR: see Table 1.
2: amorphous powder, []D17 +11.1° (c 0.4, MeOH). 1H-NMR (in C5D5N, 500 MHz)
: 5.59 (1H, dd, J = 3.5, 3.5 Hz, H-12), 4.11 (1H, ddd, J = 4.5, 10.0, 11.0 Hz, H-2),
3.39 (1H, d, J =10.0 Hz, H-3), 3.13 (1H, ddd, J = 4.5, 13.5, 13.5 Hz, Ha-16), 3.06
(1H, s, H-18), 2.35 (1H, ddd, J =5.0, 13.5, 13.5 Hz, Ha-15), 2.25 (1H, dd, J = 4.5,
12.0 Hz, Ha-1), 1.98 (1H, dd, J = 7.5, 10.5 Hz, H-9), 1.72 (3H, s, H3-27), 1.44 (3H, s,
H3-29), 1.28 (3H, s, H3-23), 1.13 (3H, d, J = 7.0 Hz, H3-30), 1.12 (3H, s, H3-26), 1.09
(3H, s, H3-24), 1.02 (3H, s, H3-25). 13C-NMR: see Table 1.
3: amorphous powder, []D17 +18.4° (c 0.2, MeOH). 1H-NMR (in C5D5N, 500 MHz)
: 5.58 (1H, dd, J =3.0, 3.0 Hz, H-12), 4.30 (1H, ddd, J = 2.5, 4.5, 11.5 Hz, H-2), 3.75
(1H, d, J = 2.5 Hz, H-3), 3.10 (1H, ddd, J = 4.5, 13.0, 13.0 Hz, Ha-16), 3.04 (1H, s,
H-18), 2.33 (1H, ddd, J =5.0, 13.0, 13.0 Hz, Ha-15), 1.89 (1H, dd, J = 4.5, 11.5 Hz,
Ha-1), 1.74 (1H, dd, J = 11.5, 11.5 Hz, Hb-1), 1.64 (3H, s, H3-27), 1.42 (3H, s, H329), 1.26 (3H, s, H3-23), 1.11 (3H, d, J = 7.0 Hz, H3-30), 1.11 (3H, s, H3-26), 0.99
(3H, s, H3-24), 0.91 (3H, s, H3-25). 13C-NMR: see Table 1.
4: amorphous powder, []D17 +15.2° (c 0.7, MeOH). 1H-NMR (in C5D5N, 500 MHz)
: 5.70 (1H, dd, J = 3.5,4.0Hz, H-12), 4.16 (1H, dd, J = 2.5, 9.0 Hz, H-2), 4.12 (1H, d,
J = 9.0 Hz, H-1), 3.86 (1H, d, J = 2.5 Hz, H-3), 3.20 (1H, ddd, J = 4.0, 6.0, 19.5 Hz,
Ha-11), 3.11 (1H, ddd, J = 4.5, 13.0, 13.0 Hz, Ha-16), 3.05 (1H, s, H-18), 2.64 (1H,
ddd, J =3.5, 11.0, 19.5 Hz, H-11b), 2.38 (1H, dd, J = 6.0, 11.0 Hz, H-9), 2.35 (1H,
ddd, J = 4.5, 13.0, 13.0 Hz, H-15a), 1.81 (1H, dd, J = 6.0, 8.0 Hz, H-5), 1.68 (3H, s,
H3-27), 1.42 (3H, s, H3-29), 1.28 (3H, s, H3-25), 1.26 (3H, s, H3-23), 1.22 (3H, s, H326), 1.12 (3H, d, J = 6.5 Hz, H3-30), 0.95 (3H, s, H3-24). 13C-NMR: see Table 1.
5: amorphous powder, []D15 +19.1° (c 3.2, MeOH). 1H-NMR (in C5D5N, 500 MHz)
: 5.59 (1H, dd, J = 3.0, 3.0 Hz, H-12), 4.28 (1H, ddd, J =2.5, 4.5, 10.5 Hz, H-2), 4.14
(1H, d, J = 2.5 Hz, H-3), 3.92 (1H, d, J = 11.0 Hz, Ha-23), 3.75 (1H, d, J = 11.0 Hz,
Hb-23), 3.10 (1H, ddd, J = 4.5, 13.5, 13.5 Hz, Ha-16), 3.04 (1H, s, H-18), 2.33 (1H,
ddd, J = 4.5, 13.5, 13.5 Hz, Ha-15), 1.93 (1H, dd, J = 4.5, 12.0 Hz, Ha-1), 1.67 (3H, s,
H3-27), 1.42 (3H, s, H3-29), 1.12 (3H, s, H3-26), 1.12 (3H, d, J = 7.0 Hz, H3-30), 1.03
(3H, s, H3-25), 0.87 (3H, s, H3-24). 13C-NMR: see Table 1.
6: amorphous powder, []D15 +11.1° (c 0.7, MeOH). 1H-NMR (in C5D5N, 500 MHz)
: 5.61 (1H, dd, J = 3.5, 3.5 Hz, H-12 of aglycone (Agl)), 4.78 (1H, d, J = 7.0 Hz, H-1
of Arabinopyranosyl (Ara)), 4.42 (1H, dd, J = 7.0, 8.5 Hz, H-2 of Ara), 4.33 (1H, m,
H-4 of Ara), 4.32 (1H, dd, J = 3.0, 12.0 Hz, Ha-5 of Ara), 4.17 (1H, dd, J = 3.5, 8.5
Hz, H-3 of Ara), 3.84 (1H, dd, J = 2.0, 12.0 Hz, Hb-5 of Ara), 3.35 (1H, dd, J = 4.5,
12.0 Hz, H-3 of Agl), 3.15 (1H, ddd, J = 4.5, 13.5, 13.5 Hz, Ha-16), 3.07 (1H, s, H18), 1.76 (3H, s, H3-27), 1.45 (3H, s, H3-29), 1.29 (3H, s, H3-23), 1.13 (3H, d, J = 7.0
Hz, H3-30), 1.10 (3H, s, H3-26), 0.97 (3H, s, H3-24), 0.89 (3H, s, H3-25). 13C-NMR:
see Table 1.
7: amorphous powder, []D15 +20.4° (c 2.5, MeOH). 1H-NMR (in C5D5N, 500 MHz)
: 5.55 (1H, dd, J = 3.0, 3.0 Hz, H-12), 4.46 (1H, d, J =11.0 Hz, Ha-24), 4.31 (1H,
ddd, J = 4.0, 9.0, 11.0 Hz, H-2), 3.73 (1H, d, J = 11.0 Hz, Hb-24), 3.62 (1H, s, H-8),
3.61 (1H, d, J = 5.0 Hz, H-19), 3.56 (1H, d, J = 9.0 Hz, H-3), 2.83 (1H, ddd, J = 3.0,
13.5, 13.5 Hz, Ha-16), 1.63 (3H, s, H3-27), 1.59 (3H, s, H3-23), 1.20 (3H, s, H3-29),
1.12 (3H, s,H3-30), 1.04 (3H, s, H3-25), 1.02 (3H, s, H3-26). 13C-NMR: see Table 1.
8: amorphous powder, []D15 +16.5° (c 4.2, MeOH). 1H-NMR (in C5D5N, 500 MHz)
: 5.63 (1H, dd, J = 3.5, 3.5 Hz, H-12), 3.69 (3H, s, OCH3), 3.40 (1H, dd, J = 6.0,
10.5 Hz, H-9), 3.06 (1H, s, H-18), 2.99 (1H, dd, J = 3.0, 10.5 Hz, H-5), 2.68 (1H, d, J
= 18.5 Hz, Ha-1), 2.63 (1H, d, J = 18.5 Hz, Hb-1), 1.83 (3H, s, H3-27), 1.40 (3H, s,
H3-23), 1.39 (3H, s, H3-24), 1.34 (3H, s,H3-29), 1.15 (3H, s, H3-26), 1.08 (1H, d, J =
6.0 Hz, H3-30), 1.07 (3H, s, H3-25). 13C-NMR: see Table 1.
4.4. Cell culture and modified LDL preparation
Human monocytes were purified from the blood of healthy volunteers by Ficoll
density gradient centrifugation (Ficoll-Paque from GE Healthcare). Purified
monocytes were suspended in DMEM and seeded onto 24-well plates (4×105/well).
After a 1 hourincubation for adherence, the medium was replaced by RPMI 1640
supplemented with 10% pooled human serum, streptomycin (0.1 mg/mL), and
penicillin G (100 U/mL). The adherent monocytes were incubated for 7 days to
induce their differentiation into macrophages. These HMDM were used for
experiment. Acetyl-LDL was prepared by chemical modification of LDL with acetic
anhydride as previously described (Fujiwara et al., 2011).
4.5. Assay for foam cell formation in HMDM
HMDM monolayers were incubated with 50 g/ml acetyl-LDL for 24 hours in the
presence of 0.1 mM/L [3H]oleate conjugated with BSA, and cellular lipids were
extracted for determination of radioactivity of choresteryl-[3H]oleate as described
previously (Fujiwara et al., 2011). Recent study has reported anti-atherosclerosis
activities of some triterpenoids such as oleanolic acid and ursolic acid (Lee et al.,
2006). In the present study, we have confirmed the IC50 value of oleanolic acid as a
positive control to be 15.3 µM.
References
Fujiwara Y., Hayashida A., Tsurushima K., Nagai R., Yoshitomi M., Daiguji N.,
Sakashita N., Takeya M., Tsukamoto S., Ikeda T. 2011. Triterpenoids
isolated from Zizyphus jujuba inhibit foam cell formation in macrophages. J
Agric Food Chem. 59: 4544–4552.
Lee W. S., Im K.-R., Park Y.-D., Sung N.-D., Jeong T.-S. 2006. Human ACAT-1 and
ACAT-2 inhibitory activities of pentacyclic triterpenes from the leaves of
Lycopus lucidus TURCZ. Biol Pharm Bull. 29: 382–384.
Supplementary Table
Table S1. 13C-NMR data for 1–8 (in C5D5N. 125 MHz)
1
2
3
4
5
6
7
Agl-1 39.1 47.9 42.9 80.9 42.7 38.9 47.5
2 28.2 68.6 66.1 71.5 66.3 26.7 68.7
3 78.3 83.9 79.3 79.7 78.9 88.8 85.8
4 39.4 39.9 38.8 38.5 42.2 39.6 44.0
5 55.9 56.0 48.8 48.6 43.6 56.0 56.6
6 19.0 19.0 18.6 18.9 18.5 18.7 19.4
7 33.7 33.6 33.5 33.9 33.3 33.6 33.6
8 40.4 40.5 40.6 41.2 40.6 40.4 40.1
9 47.8 47.9 47.6 48.7 47.8 47.8 48.5
10 37.4 38.6 38.7 43.8 38.4 37.1 38.5
11 24.1 24.2 24.1 28.1 24.1 24.1 29.1
12 128.1 128.0 128.0 129.6 128.0 128.1 123.3
13 140.0 140.0 140.0 138.9 140.0 140.0 144.9
14 42.2 42.2 42.2 42.1 41.9 42.2 42.2
15 29.4 29.3 29.2 29.4 29.2 29.4 29.3
16 26.5 26.4 26.4 26.5 26.4 26.5 24.5
17 48.3 48.3 48.3 48.4 48.3 48.3 46.1
18 54.7 54.6 54.6 54.6 54.6 54.7 44.8
19 72.8 72.8 72.7 72.7 72.8 72.8 81.3
20 42.4 42.4 42.4 42.4 42.4 42.4 35.7
21 27.0 27.0 27.0 27.0 27.0 27.0 28.4
22 38.5 38.5 38.5 38.5 38.5 38.5 33.7
23 28.8 29.4 29.4 29.4 71.2 28.3 24.1
24 16.8 17.7 22.3 22.4 17.0 17.3 65.6
25 15.6 16.9 16.6 13.0 17.7 15.6 17.2
26 17.3 17.3 17.3 17.7 17.4 16.9 17.4
27 24.7 24.7 24.6 24.8 24.7 24.7 24.8
28 180.7 180.7 180.7 180.8 180.7 180.7 180.9
29 27.2 27.2 27.1 27.1 27.1 27.2 24.9
30 16.5 16.8 16.8 16.8 16.8 16.8 28.8
OCH3
Ara-1
107.4
2
72.9
3
74.6
4
69.4
5
66.6
8
42.4
174.0
179.8
46.7
49.1
21.7
32.9
40.9
39.4
42.0
24.4
128.4
139.6
42.9
29.4
26.9
48.3
54.6
72.7
42.3
26.5
38.4
24.2
27.6
19.3
17.2
24.5
180.7
27.2
16.7
51.8
in ppm from TMS. Agl, Aglycone. Ara, Arabinopyranosy
Table S2. Inhibitory activityof 1–8 on foam cell
formation in HMDM
Sample
Inhibition ()
1
90±4
2
30±5
3
32±3
4
33±5
5
4±2
6
48±4
7
4±2
8
24±3
Data shown represent mean ± S.D from three
experiments.