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CHEMISTRY & BIODIVERSITY – Vol. 5 (2008)
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Cytotoxic Triterpenoids from the Root Bark of Helicteres angustifolia
by Min-Hsiung Pan a ), Chiu-Ming Chen b ), Shwu-Woan Lee c ), and Zong-Tsi Chen* c )
a
) Department of Seafood Science, National Kaohsiung Marine University, No. 142, Hai-Chuan Rd,
Nan-Tzu, Kaohsiung 811, Taiwan, R.O.C.
b
) Department of Chemistry, National Tsing Hua University, Hsinchu 300, Taiwan, R.O.C.
c
) Department of Applied Chemistry, Chia-Nan University of Pharmacy and Science, 60, Erh-Jen Rd,
Sec.1, Jen-Te, Tainan 717, Taiwan, R.O.C. (phone: þ 886-6-2664911, ext. 240; fax: þ 886-6-2667319;
e-mail: [email protected])
Three new triterpenoids, 3b-acetoxy-27-[(E)-cinnamoyloxy]lup-20(29)-en-28-oic acid methyl ester
(1), 3b-acetoxy-27-[(4-hydroxybenzoyl)oxy]lup-20(29)-en-28-oic acid (2), and 3b-acetoxy-27-[(4-hydroxybenzoyl)oxy]olean-12-en-28-oic acid methyl ester (3), together with nine known triterpenoids, 4 –
12, were isolated from the root bark of Helicteres angustifolia. The structures of these compounds were
established on the basis of spectroscopic methods including 2D-NMR experiments. All twelve
compounds were tested for their cytotoxic activities against human colorectal cancer (COLO 205),
human hepatoma (Hep G2), and human gastric cancer (AGS) cell lines in vitro. Among them,
compounds 2, 3, 3b-O-[(E)-coumaroyl]betulinic acid (6), and pyracrenic acid (7) showed significant
cytotoxic activities against human cancer cells COLO 205 and AGS.
Introduction. – Helicteres, a genus of the family Sterculiaceae, comprises ca. 40
species found in tropical Asia and America [1]. Several species of this genus have been
used in folk medicine, for instance, the root of H. isora has been used for the treatment
of empyema, stomach affection, intestinal infection, and diabetes [2], H. ovata and H.
sacarolha have been used for the treatment of syphilis and as a depurant [3], and the
root of H. hirsuta has been used for the treatment of uterus pain [4]. Biological studies
of several species of this genus have demonstrated that the extract of H. isora showed
antinociceptive, antidiabetic, hypolipidemic, and hypoglycaemic activities [2] [5] [6],
the extract of H. hirsuta showed cytotoxic activities [7], and the extract of H.
gardneriana displayed antiprotozoal activity [3]. Previous phytochemical studies of
Helicteres plants have led to the isolation of lignans, neolignans, cucurbitacins,
flavonoids, and rosmarinic acid derivatives [7] [8 – 12].
Helicteres angustifolia L. is a common folk medicine in Taiwan, possessing
antitumor, analgesic, anti-inflammatory, and antibacterial effects [13] 1). The MeOH
extract [14] and cucurbitacin derivatives [15] of the root of this plant were found to
have potent cytotoxic activities. To date, phytochemical studies of this plant have led to
the isolation of sesquiterpenoid quinones [16], flavonoid glycosides [17], triterpenoids
[18] [19], cucurbitacins [20], pregnane, coumarin, and lupane derivatives [15].
1)
H. angustifolia is a common shrubby weed in Taiwan. The root of this plant, known in Chinese as
Hkang-chih-maI, has been commonly used for the treatment of influenzal fever, headache, carbuncle,
hemorrhoid, tonsillitis, pharyngitis, and parotitis in Taiwan.
E 2008 Verlag Helvetica Chimica Acta AG, ZGrich
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CHEMISTRY & BIODIVERSITY – Vol. 5 (2008)
As mentioned above, the diverse phytochemicals and biological activities of
Helicteres plants have been reported, but only a few studies about bioactive compounds
of H. angustifolia have appeared in the literature [15]. These facts motivated us to
further investigate the bioactive constituents from this plant. As a part of our search for
bioactive constituents from natural sources, the CHCl3 extract of the root bark of H.
angustifolia was further investigated to afford three new triterpenoids, namely 3bacetoxy-27-(trans-cinnamoyloxy)lup-20(29)-en-28-oic acid methyl ester (1), 3b-acetoxy-27-[(4-hydroxybenzoyl)oxy]lup-20(29)-en-28-oic acid (2), and 3b-acetoxy-27-[(4hydroxybenzoyl)oxy]olean-12-en-28-oic acid methyl ester (3), together with nine
known triterpenoids, 3b-acetoxy-27-(benzoyloxy)olean-12-en-28-oic acid methyl ester
(4) [18], cylicodiscic acid ( ¼ 3b,27-dihydroxylup-20(29)-en-28-oic acid; 5) [21], 3b-O(trans-coumaroyl)betulinic acid (6) [22], pyracrenic acid ( ¼ 3b-O-(trans-caffeoyl)betulinic acid; 7) [23], 3b-O-(trans-feruloyl)betulinic acid (8) [22], 3b-O-(trans-coumaroyl)betulin (9), 3b-O-(cis-coumaroyl)betulin (10) [24], 3b-O-(trans-caffeoyl)betulin
(11) [23], and 3b-O-(trans-feruloyl)betulin (12) [25]. Here, we report the isolation and
structure elucidation of these compounds. The cytotoxic activities of compounds 1 – 12
CHEMISTRY & BIODIVERSITY – Vol. 5 (2008)
567
against three human cancer cell lines, COLO 205, Hep G2, and AGS were also
evaluated.
Results and Discussion. – 1. Isolation and Structure Elucidation. The CHCl3 extract
of dried powdered root bark of Helicteres angustifolia was further fractionated and
purified by repeated silica-gel column chromatography and preparative TLC to afford
the three new triterpenoids 1 – 3 as well as the nine known ones, 4 – 12. The structures of
these compounds were established by detailed spectral analysis (UV, IR, MS, and NMR
data) and also by comparing with the data reported in the literature. Assignments of the
1
H and 13C signals were performed by extended 1D- and 2D-NMR methods involving
1
H,1H-COSY, NOESY, DEPT, HMQC, and HMBC spectra.
Compound 1 was isolated as colorless amorphous powder. The molecular formula
of 1 was determined as C42H58O6 by high-resolution EI-MS (m/z 658.9220 (M þ ; calc.
658.9228)). The IR spectrum of 1 displayed absorption bands at 1740, 1720 (C¼O),
1680 (conjugated C¼O), 3050, 1650, 890 (terminal C¼C bond), and 1610, 1580, 1505
(Ph group) cm 1. The 13C- and 1H-NMR-spectral data (Tables 1 and 2, resp.) showed
four Me singlets (d(H) 0.81, 0.82, 0.90, 0.99), typical lupene-type triterpene NMR
signals due to an isopropenyl group (d(H) 1.69 (s, Me), 4.62 (br. s, 1 H), 4.75 (br. s,
1 H); d(C) 19.4 (q), 110 (t), 150.2 (s)) and Hb C(19) (d(H) 3.01 (m, 1 H)) [26], a
trans-cinnamoyl moiety (d(H) 6.42 (d, J ¼ 16.0, 1 H), 7.63 (d, J ¼ 16.0, 1 H), 7.39 (m,
3 H), and 7.54 (m, 2 H)), an oxygenated CH2 group (d(H) 4.47 (d, J ¼ 12.5, 1 H), 4.64
(d, J ¼ 12.5, 1 H); d(C) 63.3 (t)), an oxygenated CH group (d(C) 80.8 (d); d(H) 4.45;
overlapped by oxygenated CH2 ), an Ac group (d(H) 2.01 (s, Me); d(C) 21.3 (q), 170.9
(s)), and a MeO group (d(H) 3.68 (s); d(C) 51.4 (q)). The b-equatorial orientation of
the AcO group at C(3) was established by the 13C-NMR data of ring A and the upfieldshift signal due to an axial Me C-atom (C(24), d(C) 16.7 (q)) [27] [28]. These data
revealed that compound 1 was a methyl ester of a betulinic acid derivative bearing a
trans-cinnamoyl moiety and an Ac group. The 13C- and 1H-NMR-spectral data of 1
were very similar to those of 3b-acetoxy-27-(benzoyloxy)lup-20(29)-en-28-oic acid
methyl ester [19] isolated from the same plant, except a trans-cinnamoyl moiety in 1
instead of a benzoyl moiety in the latter. The locations of the AcO group, transcinnamoyloxy moiety, and MeO group were further confirmed by analysis of the
HMBC spectrum (Fig. 1). The location of the AcO group at C(3) was confirmed by
HMBC correlations of HC(3) (d(H) 4.45) with AcO (d(C) 170.9), C(23) (d(C) 27.9),
Fig. 1. Key HMBC correlations observed in 1
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CHEMISTRY & BIODIVERSITY – Vol. 5 (2008)
Table 1.
13
C-NMR Data of 1 – 3. In CDCl3 at 125 MHz; d in ppm.
Position
1
2
3
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
AcO
1’
2’,6’
3’,5’
4’
7’
8’
9’
MeO
38.5 (t)
23.7 (t)
80.8 (d)
37.8 (s)
55.5 (d)
18.2 (t)
35.3 (t)
41.5 (s)
51.8 (d)
37.4 (s)
21.0 (t)
25.2 (t)
39.0 (d)
45.5 (s)
24.1 (t)
32.5 (t)
56.3 (s)
49.7 (d)
46.9 (d)
150.2 (s)
30.4 (t)
36.6 (t)
27.9 (q)
16.7 (q)
16.6 (q)
16.4 (q)
63.3 (t)
176.7 (s)
110.0 (t)
19.4 (q)
170.9 (s), 21.3 (q)
134.4 (s)
128.2 (d)
128.9 (d)
130.3 (d)
144.6 (d)
118.4 (d)
167.0 (s)
51.4 (q)
38.4 (t)
23.6 (t)
81.0 (d)
37.8 (s)
55.4 (d)
18.1 (t)
35.2 (t)
41.4 (s)
51.9 (d)
37.3 (s)
21.0 (t)
25.3 (t)
39.0 (d)
45.6 (s)
24.2 (t)
32.4 (t)
56.5 (s)
49.9 (d)
46.8 (d)
150.1 (s)
30.3 (t)
36.5 (t)
27.8 (q)
16.7 (q)
16.5 (q)
16.3 (q)
63.4 (t)
182.0 (s)
110.0 (t)
19.3 (q)
171.6 (s), 21.2 (q)
122.5 (s)
131.8 (d)
115.4 (d)
160.7 (s)
166.8 (s)
38.0 (t)
23.8 (t)
81.0 (d)
37.6 (s)
55.1 (d)
18.2 (t)
32.9 (t)
39.9 (s)
48.5 (d)
37.0 (s)
23.4 (t)
126.8 (d)
137.4 (s)
45.2 (s)
24.0 (t)
22.7 (t)
46.5 (s)
41.2 (d)
44.5 (t)
30.5 (s)
33.6 (t)
32.3 (t)
28.0 (q)
16.7 (q)
15.6 (q)
18.0 (q)
65.8 (t)
178.3 (s)
32.8 (q)
23.6 (q)
171.3 (s), 21.3 (q)
122.8 (s)
131.7 (d)
115.3 (d)
160.2 (s)
166.3 (s)
51.7 (q)
and C(24) (d(C) 16.7). The location of the trans-cinnamoyloxy moiety at C(27) was
confirmed by HMBC correlations of HC(27) (d(H) 4.47, 4.64) with C(9’) (d(C) 167.0)
and C(8) (d(C) 41.5). The MeO group was located at C(28) based on HMBC
correlation between the MeO H-atom (d(H) 3.68) and C(28) (d(C) 176.7).
Consequently, the structure of 1 was determined as 3b-acetoxy-27-(trans-cinnamoyloxy)lup-20(29)-en-28-oic acid methyl ester.
Compound 2 was isolated as colorless amorphous powder. The molecular formula
of 2 was assigned as C39H54O7 by high-resolution EI-MS (m/z 634.8563 (M þ ; calc.
CHEMISTRY & BIODIVERSITY – Vol. 5 (2008)
569
Table 2. 1H-NMR Data of 1 – 3. In CDCl3 at 500 MHz; d in ppm, J in Hz.
Position 1
2
3
1
2
3
5
6
7
9
11
12
13
15
16
18
19
21
22
23
24
25
26
27
1.62 – 1.65 (m), 1.02 – 1.04 (m)
1.62 – 1.64 (m)
4.45 (dd, J ¼ 10.4, 5.2)
0.85 – 0.87 (m)
1.49 – 1.52 (m), 1.38 – 1.44 (m)
1.37 – 1.44 (m)
1.32 – 1.36 (m)
1.54 – 1.62 (m)
1.76 – 1.82 (m), 1.00 – 1.02 (m)
2.39 – 2.47 (m)
1.93 – 1.97 (m), 1.46 – 1.61 (m)
2.32 – 2.37 (m), 1.30 – 1.35 (m)
1.75 – 1.81 (m)
3.00 – 3.07 (m)
1.96 – 2.00 (m), 1.43 – 1.48 (m)
1.96 – 2.01 (m), 1.44 – 1.49 (m)
0.82 (s)
0.90 (s)
0.80 (s)
1.02 (s)
4.57 (d, J ¼ 12.6),
4.78 (d, J ¼ 12.6)
4.63 (br. s), 4.76 (br. s)
1.70 (s)
2.02 (s)
7.86 (d, J ¼ 8.7)
6.87 (d, J ¼ 8.7)
1.58 – 1.62 (m), 0.89 – 0.92 (m)
1.59 – 1.62 (m)
4.37 (br. t, J ¼ 8.5)
0.79 – 0.81 (m)
1.53 – 1.55 (m), 1.38 – 1.42 (m)
1.53 – 1.57 (m), 1.36 – 1.41 (m)
1.62 – 1.68 (m)
1.74 – 1.86 (m)
5.65 (br. s)
29
30
AcO
2’,6’
3’,5’
4’
7’
8’
MeO
1.63 – 1.65 (m), 1.02 – 1.04 (m)
1.62 – 1.64 (m)
4.43 – 4.46 (m)
0.85 – 0.88 (m)
1.49 – 1.51 (m), 1.39 – 1.43 (m)
1.36 – 1.44 (m)
1.29 – 1.34 (m)
1.56 – 1.65 (m)
1.74 – 1.79 (m), 0.99 – 1.01 (m)
2.30 – 2.38 (m)
1.58 – 1.62 (m), 1.26 – 1.31 (m)
2.20 – 2.24 (m), 1.31 – 1.36 (m)
1.59 – 1.65 (m)
2.97 – 3.05 (m)
1.22 – 1.34 (m)
1.34 – 1.46 (m)
0.82 (s)
0.90 (s)
0.81 (s)
0.99 (s)
4.47 (d, J ¼ 12.5),
4.64 (d, J ¼ 12.5)
4.62 (br. s), 4.75 (br. s)
1.69 (s)
2.01 (s)
7.52 – 7.55 (m)
7.38 – 7.40 (m)
7.38 – 7.40 (m)
7.63 (d, J ¼ 16.0)
6.42 (d, J ¼ 16.0)
3.68 (s)
1.66 – 1.71 (m), 1.16 – 1.21 (m)
1.94 – 1.98 (m), 1.61 – 1.66 (m)
2.94 (dd, J ¼ 2.0, 10.2)
1.37 – 1.40 (m), 1.04 – 1.08 (m)
1.28 – 1.32 (m), 1.13 – 1.17 (m)
1.69 – 1.73 (m), 1.48 – 1.52 (m)
0.82 (s)
0.85 (s)
0.93 (s)
0.76 (s)
4.20 (d, J ¼ 12.8),
4.44 (d, J ¼ 12.8)
0.87 (s)
0.90 (s)
2.02 (s)
7.88 (d, J ¼ 8.8)
6.87 (d, J ¼ 8.8)
3.63 (s)
634.8571)). The IR spectrum of 2 displayed absorption bands at 3450 (OH), 3500 –
2500, 1720 (COOH), 1680 (conjugated C¼O), 1600, 1580, 1510 (phenyl group) cm 1.
The NMR spectra of 2 were similar to those of 1. The 13C- and 1H-NMR-spectral data of
2 (Tables 1 and 2, resp.) also showed typical lupene-type triterpene NMR signals due to
an isopropenyl group and Hb C(19). The NMR spectra of 2 showed a 4-hydroxybenzoyl and an Ac group. The b-equatorial orientation of the AcO group was
established by the signals due to HC(3) (d(H) 4.45 (dd, J ¼ 5.2, 10.4)) [28]. These
data revealed that 2 was a betulinic acid derivative bearing a 4-hydroxybenzoyloxy
moiety and an Ac group.
The NMR-spectral data of 2 were almost identical with those of 3b-acetoxy-27-[(4hydroxylbenzoyl)oxy]lup-20(29)-en-28-oic acid methyl ester [19], except for the signal
due to the MeO group in the latter, which was not observed in the NMR spectra of 2.
The locations of the AcO group and 4-hydroxybenzoyloxy moiety were further
confirmed by analysis of the HMBC spectrum (Fig. 2). The location of the AcO group
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CHEMISTRY & BIODIVERSITY – Vol. 5 (2008)
at C(3) was confirmed by HMBC correlations of HC(3) (d(H) 4.45) with AcO (d(C)
171.6), C(23) (d(C) 27.8), and C(24) (d(C) 16.7). The location of the (4-hydroxybenzoyl)oxy moiety at C(27) was confirmed by HMBC correlations of HC(27) (d(H)
4.57, 4.78) with C(7’) (d(C) 166.8), C(8) (d(C) 41.4), and C(13) (d(C) 39.0). Therefore,
the structure of 2 was determined as 3b-acetoxy-27-[(4-hydroxybenzoyl)oxy]lup20(29)-en-28-oic acid.
Fig. 2. Key HMBC correlations observed in 2
Compound 3 was obtained as colorless amorphous powder. The molecular formula
of 3 was determined as C40H56O7 by high-resolution EI-MS (m/z 648.8833 (M þ ; calc.
648.8840)). The IR spectrum of 3 displayed absorption bands at 3450 (OH), 1740, 1720
(C¼O), 1685 (conjugated C¼O), and 1600, 1585, 1505 (phenyl group) cm 1. The 1Hand 13C-NMR (DEPT), and HMQC spectra indicated the presence of six tertiary Me
groups (d(H) 0.76, 0.82, 0.85, 0.87, 0.90, 0.93 (5s)), a 4-hydroxybenzoyl moiety (d(H)
6.87 (d, J ¼ 8.8, 2 H) and 7.88 (d, J ¼ 8.8, 2 H)), an oxygenated CH2 group (d(H) 4.20
(d, J ¼ 12.8, 1 H), 4.44 (d, J ¼ 12.8, 1 H); d(C) 65.8 (t)), an oxygenated CH group
(d(H) 4.37 (br. t, J ¼ 8.5); d(C) 81.0 (d)), an Ac group (d(H) 2.02 (s, Me); d(C) 21.3
(q), 171.3 (s)), and a MeO group (d(H) 3.63 (s); d(C) 51.7 (q)). Typical oleanene-type
triterpene NMR signals due to an olefinic H-atom HC(12) (d(H) 5.65 (br. s)) and
Hb C(18) (d(H) 2.94 (dd, J ¼ 2.0, 10.2)) [29 – 31] were also observed. The b-equatorial
orientation of the AcO group was established by the signal due to HC(3) (d(H) 4.37
(br. t, J ¼ 8.5)) [30] [31]. These data revealed that 3 was a methyl ester of an oleanolic
acid derivative bearing a (4-hydroxybenzoyl)oxy moiety and an Ac group. The NMRspectral data of 3 were almost the same as those of 3b-acetoxy-27-(benzoyloxy)olean12-en-28-oic acid methyl ester (4) [18], except for the signals due to the 4hydroxybenzoyl moiety in 3 instead of the benzoyl moiety in 4. The locations of the
AcO group, (4-hydroxybenzoyl)oxy moiety, and MeO group were confirmed by
analysis of the HMBC spectrum (Fig. 3). The location of the AcO group at C(3) was
confirmed by HMBC correlations of HC(3) (d(H) 4.37) with AcO (d(C) 171.3),
C(23) (d(C) 28.0), and C(24) (d(C) 16.7). The location of the (4-hydroxybenzoyl)oxy
moiety at C(27) was confirmed by HMBC correlations of HC(27) (d(H) 4.20, 4.44)
with C(7’) (d(C) 166.3), C(8) (d(C) 39.9), C(13) (d(C) 137.4), and C(15) (d(C) 24.0).
The location of the MeO group was confirmed by HMBC correlations between the
MeO H-atom (d(H) 3.63) and C(28) (d(C) 178.3). Therefore, the structure of 3 was
determined as 3b-acetoxy-27-[(4-hydroxybenzoyl)oxy]olean-12-en-28-oic acid methyl
ester.
CHEMISTRY & BIODIVERSITY – Vol. 5 (2008)
571
Fig. 3. Key HMBC correlations observed in 3
2. Biological Studies. The cytotoxic activities of compounds 1 – 12 isolated from the
root bark of H. angustifolia, along with doxorubicin, camptothecin, and paclitaxel
(positive control), against the three human cancer cells COLO 205, Hep G2, and AGS
were evaluated (Table 3). Among these compounds, 1, 4, and four betulin derivatives,
9 – 12, showed no inhibitory activity against these three human cancer cells. The
betulinic acid derivatives 2, 5, 6, 7, and 8, which bear a COOH group, showed moderateto-strong inhibitory activities against the above cancer cells, but compound 5 showed no
inhibitory activity against Hep G2 cell.
Table 3. Cytotoxic Activities of 1 – 12 against Human Cancer Cells
Compound
1
2
3
4
5
6
7
8
9
10
11
12
Doxorubicin
Camptothecin
Paclitaxel
IC50 [mm]
COLO 205
Hep G2
AGS
> 100
22.4 4.7
18.6 3.2
> 100
76.6 6.0
14.6 1.1
16.1 1.2
59.8 7.9
> 100
> 100
> 100
> 100
4.8 0.5
> 100
> 100
> 100
87.6 4.3
77.8 8.3
> 100
> 100
37.3 1.8
19.6 0.8
34.5 0.4
> 100
> 100
> 100
> 100
1.4 0.1
> 100
> 100
> 100
5.4 1.4
16.5 1.9
> 100
92.4 3.2
15.4 0.7
7.4 0.2
26.4 5.8
> 100
> 100
> 100
> 100
1.0 0.1
1.2 0.1
30.1 1.7
On the basis of structure – activity relationships (SAR), the betulinic acid
derivatives 6, 7, and 8, which bear a C(28)OOH group, showed stronger cytotoxic
activities compared to the betulin derivatives 9, 11, and 12, with a CH2(28)OH group.
Triterpenes with a C(28)OOH group were reported to exhibit cytotoxic activities
[32] [33], and this fact was also confirmed in this biological study.
Although the C(28)OOH group is essential for cytotoxicity, it is interesting that
compound 3, which bears a C(28)OOMe group and a (4-hydroxybenzoyl)oxy moiety at
C(27), showed stronger cytotoxic activities compared to the corresponding compound
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CHEMISTRY & BIODIVERSITY – Vol. 5 (2008)
4 bearing a benzoyloxy moiety at C(27). This finding revealed that a (4-hydroxybenzoyl)oxy moiety at C(27) contributes to the cytotoxic activity of 3 [34]. The
structures of compounds 1 and 2 are similar, except for the former bearing a
C(28)OOH group and a (4-hydroxybenzoyl)oxy moiety at C(27) instead of the latter
bearing a CH2(28)OH group and a benzoyloxy moiety at C(27). Compound 2 showed
stronger cytotoxic activities compared to 1. This fact revealed that a C(28)OOH group
and/or a (4-hydroxybenzoyl)oxy moiety at C(27) are the contributors to the cytotoxic
activities of 2. Of compounds 6, 7, and 8, compound 7, bearing a caffeoyl moiety, showed
stronger cytotoxic activities compared to the other two (6 bearing a p-coumaroyl
moiety and 8 bearing a feruloyl moiety) against Hep G2 and AGS cell lines. This fact
revealed that an OH group at C(3’) of the caffeoyl moiety enhanced the cytotoxicity of
7 against these two cell lines.
These results further confirm that the C(28)OOH group is an important contributor
to the cytotoxicity of triterpenoids, but the substituents in the molecules also affect
their cytotoxicities [32] [33] [35].
The authors thank the Chia-Nan University of Pharmacy and Science, Taiwan, R.O.C., for financial
support of this work, and Prof. C. S. Kuoh, Department of Biology, National Cheng Kung University, for
authentification of plant material. The authors also thank Misses J. Z. Wu and L. N. Lai, Department of
Chemistry, National Cheng Kung University, for NMR and MS analysis, resp.
Experimental Part
General. Column chromatography (CC): silica gel 60 (Merck, 70 – 230 mesh). TLC and prep. TLC:
precoated silica-gel plates (Merck, Kieselgel 60 F254, 0.25 mm and 1.00 mm, resp.). Optical rotations:
JASCO DIP-360 digital polarimeter. UV Spectra: Hitachi 200 spectrophotometer; lmax (log e) in nm. IR
Spectra: Perkin-Elmer 781 infrared spectrophotometer; in cm 1. 1H- and 13C-NMR spectra: Bruker AV500 spectrometer at 500 and 125 MHz, resp., in CDCl3 soln.; d in ppm rel. to Me4Si, J in Hz. EI-MS and
HR-EI-MS: JEOL JMS-700 mass spectrometer; in m/z (rel. %).
Plant Material. The root bark of Helicteres angustifolia L. was collected in Puli, Nantou County,
Taiwan. The plant was identified by Prof. C. S. Kuoh, Department of Biology, National Cheng Kung
University. A voucher specimen (CNACNP0512) was deposited with the Natural Product Laboratory of
Department of Applied Chemistry, Chia-Nan University of Pharmacy and Science, Tainan, Taiwan.
Extraction and Isolation. Dried powdered root bark of H. angustifolia L. (3.8 kg) was extracted with
CHCl3 (3 5 l) under reflux. The residue was extracted with MeOH (5 5 l) under reflux. The
concentrated MeOH extract (201 g) was suspended in H2O. The suspension was extracted with AcOEt
and BuOH successively. The CHCl3-soluble fraction (56.4 g) was subjected to silica-gel CC (silica gel
(1.25 kg); hexane/acetone 100 : 1, 50 : 1, 25 : 1, 12 : 1, 6 : 1, 3 : 1, 2 : 1, 1 : 1, 1 : 2, 1 : 4, 1 : 8, 0 : 1) to afford twelve
Fractions (Fr. 1 – 12). Fr. 2 (2.8 g) was further purified by CC (silica gel; hexane/CHCl3 1 : 1) and prep.
TLC (hexane/AcOEt 12 : 1) to afford 1 (15 mg) and 4 (36 mg). Fr. 3 (1.3 g) was further purified by CC
(silica gel; hexane/AcOEt 4 : 1) and prep. TLC (benzene/AcOEt 5 : 1) to afford 3 (12 mg). Fr. 4 (3.6 g)
was further purified by CC (silica gel; hexane/AcOEt 3 : 1) and prep. TLC (benzene/AcOEt 5 : 1) to
afford 9 (53 mg), 10 (6 mg), and 12 (67 mg). Fr. 5 (1.6 g) was further purified by CC (silica gel; hexane/
AcOEt 2 : 1) and prep. TLC (benzene/AcOEt 3 : 1) to afford 2 (6 mg). Fr. 6 (3.1 g) was further purified by
repeated CC (silica gel; hexane/AcOEt/MeOH 4 : 1 : 0.1) to afford 6 (62 mg) and 8 (23 mg). Fr. 7 (4.2 g)
was further purified by repeated CC (silica gel; hexane/AcOEt/MeOH 3 : 1 : 0.1) to afford 7 (780 mg) and
11 (11 mg). Fr. 8 (1.2 g) was further purified by CC (silica gel; hexane/AcOEt/MeOH 3 : 1 : 0.1) and prep.
TLC (benzene/MeOH 12 : 1) to afford 5 (38 mg).
3b-Acetoxy-27-{[(E)-3-phenylprop-2-enoyl]oxy}lup-20(29)-en-28-oic Acid Methyl Ester (1). Colorless amorphous powder. [a]26
D ¼ 14.0 (c ¼ 0.51, CHCl3 ). UV (EtOH): 216 (4.13), 220 (sh), 271 (4.30).
IR (KBr): 3050, 1740, 1720, 1680, 1650, 1610, 1580, 1505, 1460, 990, 890, 760. 1H-NMR (500 MHz,
CHEMISTRY & BIODIVERSITY – Vol. 5 (2008)
573
CDCl3 ): see Table 2. 13C-NMR (125 MHz, CDCl3 ): see Table 1. EI-MS (70 eV): 658 (1.2, M þ ), 598 (8),
510 (99), 497 (47), 450 (27), 437 (14), 391 (10), 276 (13), 260 (17), 255 (10), 247 (20), 201 (25), 189 (42),
131 (100). HR-EI-MS: 658.9220 (M þ , C42H58Oþ6 ; calc. 658.9228).
3b-Acetoxy-27-[(4-hydroxybenzoyl)oxy]lup-20(29)-en-28-oic Acid (2). Colorless amorphous powder. [a]26
D ¼ 10.8 (c ¼ 0.26, CHCl3 ). UV (EtOH): 227 (4.15), 304 (sh), 314 (4.43). IR (KBr): 3450,
3500 – 2500, 1720, 1680, 1640, 1600, 1580, 1510, 1460, 980, 850, 800. 1H-NMR (500 MHz, CDCl3 ): see
Table 2. 13C-NMR (125 MHz, CDCl3 ): see Table 1. EI-MS (70 eV): 634 (2, M þ ), 574 (10), 496 (39), 483
(22), 436 (54), 423 (10), 393 (11), 276 (13), 246 (8), 201 (12), 189 (44), 135 (17), 121 (100). HR-EI-MS:
634.8563 (M þ , C39H54Oþ7 ; calc. 634.8571).
3b-Acetoxy-27-[(4-hydroxybenzoyl)oxy]olean-12-en-28-oic Acid Methyl Ester (3). Colorless amorphous powder. [a]26
D ¼ þ 112.0 (c ¼ 0.43, CHCl3 ). UV (EtOH): 228 (4.16), 304 (sh), 314 (4.45). IR
(KBr): 3450, 3050, 1740, 1720, 1685, 1600, 1585, 1505, 1480, 1460, 1380, 800, 750. 1H-NMR (500 MHz,
CDCl3 ): see Table 2. 13C-NMR (125 MHz, CDCl3 ): see Table 1. EI-MS (70 eV): 648 (1.3, M þ ), 588 (8),
510 (89), 497 (54), 450 (22), 437 (12), 391 (10), 276 (10), 261 (16), 255 (9), 249 (11), 201 (21), 121 (100).
HR-EI-MS: 648.8833 (M þ , C40H56Oþ7 ; calc. 648.8840).
Cytotoxicity Assay. Standard natural-product anticancer agents (doxorubicin, camptothecin, and
paclitaxel) were obtained from commercial sources. The cytotoxicities of compounds 1 – 12 against
human cancer cells COLO 205, Hep G2, and AGS were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay [36].
Briefly, human cancer cells and selected cells were plated at a density of 1 105 cells/ml into 24-well
plates. After overnight growth, cells were pretreated with a series of concentrations of compounds 1 – 12
for 24 h. The final concentration of DMSO in the culture medium was < 0.05%. At the end of treatment,
30 ml of MTT was added, and cells were incubated for an additional 4 h. Cell viability was determined by
scanning with an enzyme-linked immunosorbent assay reader with a 570-nm filter.
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Received June 14, 2007