Phytochemistry 71 (2010) 15281533

Contents lists available at ScienceDirect

Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Diterpenoids from Pinus massoniana resin and their cytotoxicity against A431
and A549 cells
Nian-Yun Yang a, Li Liu a, Wei-Wei Tao a, Jin-Ao Duan a,*, Li-Juan Tian b
a
Jiangsu Key Laboratory for TCM Formulae Research, Nanjing University of Traditional Chinese Medicine, Nanjing 210046, China
b
Technique Centre, Jinling Pharmaceutical Co., Ltd., Nanjing 210009, China

a r t i c l e i n f o a b s t r a c t

Article history: Five diterpenoids and 14 known diterpenoids were isolated from the petroleum ether extract of Pinus
Received 2 December 2009 massoniana resin. Their structures were elucidated by spectroscopic data interpretation. The cytotoxic
Received in revised form 13 May 2010 activities of these compounds were evaluated using the MTT method. The results showed that three of
Available online 2 July 2010
the less polar diterpenoids had strong cytotoxicity against A431 and A549 cancer cells, whereas those
of high polarity had none.
Keywords: 2010 Elsevier Ltd. All rights reserved.
Pine resin
Pinus massoniana
Pinaceae
Diterpenoid
Cytotoxicity
A431 cell
A549 cell

1. Introduction including abietane, pimarane and labdane diterpenes and podocar-

Pine resin is a natural resin obtained from coniferous trees and
is commercially produced from Pinus massoniana, Pinus tabulaefor-
mi and other species of Pinus in many countries, and has countless
applications at home and at work. Traditionally, pine resin has
been used to treat inammation, to relieve cough symptoms, and
to alleviate pain. In Chinese medicine, pine resin is used for the
treatment of skin diseases, burn and scald wounds, trachitis, pul-
monary tuberculosis and as a good antiseptic (Hu et al., 1998).
Modern studies have indicated that pine resin mainly consists of
the chemical constituents of abietane and pimarane diterpene
acids, which were reported to have many biological and pharma-
cological functions, including antitumor, anti-inammatory, pesti-
cidal, and antibacterial properties, as well as lowering cholesterol
and inhibiting ATPase activities (Tanaka et al., 2008; Gulacti et al.,
1999; Tanaka and Ezaki, 2006; Fernandez et al., 2001; Hartmann
and Jung, 1981; Saxena et al., 2003; Rubio et al., 2005; Smith
et al., 2005; Degtyarenko et al., 1987; Mellanen and Lehtimaki,
1996; Jeong et al., 2005; Sekido et al., 1990). In our present study,
different solvent extracts of P. massoniana resin were actively
screened, and its petroleum ether extract showed moderate cyto-
toxicities against A431 and A549 cells. So the petroleum ether ex-
tract was chemically investigated, and a series of diterpenoids,
* Corresponding author. Tel./fax: +86 25 8581 1116.
E-mail address: duanja@163.com (J.-A. Duan).
pane norditerpenes, were isolated. In this work, we deal with iso-
lation, structural elucidation and cytotoxicity against human
epithelial carcinoma A431 and human lung cancer A549 cells of
four new diterpenes, one new norditerpene, 12 known diterpenes
and two known norditerpenes from P. massoniana resin.
2. Results and discussion
P. massoniana resin was extracted successively with petro-
leum ether (6090 C) and EtOHH2O (95:5, v/v) under condi-
tions of reux, and the extracts were concentrated in vacuo,
respectively. As mentioned above, the petroleum ether extract
was the important active part. So the petroleum ether extract
was subjected to repeated silica gel liquid chromatography
and reversed-phase preparative HPLC (Sunre C-18) to obtain
four new diterpenes: 8-oxo-8,14-seco-abiet-13(15)-en-15-al-18-
oic acid (1), 7-oxo-13a-ethoxyabiet-8(14)-en-18-oic acid (2),
15,19-dihydroxy-abieta-6,8,11,13-tetraen (3) and 2b-hydroxy-
8(14),15-pimaradien-18-oic acid (4), one new norditerpene: 7b-
hydroxypodocarpen-8(14)-en-13-on-18-oic acid (5), 12 known
diterpenes: 19-acetoxy-8(14),12E,15-labdatrien (6) (Carman
et al., 1969), pimaric acid (7) (Edwards et al., 1960), elliotinol
(8) (Carman and Denni, 1964; Carman and Marty, 1966), abietic
acid (9) (Yadav et al., 2007), dehydroabietic acid (10) (Cheung
et al., 1993), 7a-hydroxy-dehydroabietic acid (11) (Witliam
and Barbara, 1989), 7b-hydroxy-dehydroabietic acid (12)

0031-9422/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2010.06.008
 N.-Y. Yang et al. / Phytochemistry 71 (2010) 15281533 1529

(Witliam and Barbara, 1989), 7-oxo-dehydroabietic acid (13) nic carbons at d 156.5 and 136.9. Detailed analysis of the HMBC
(Tanaka et al., 1997), 15-hydroxy-dehydroabietic acid (14) (Fig. 2) data established correlations between two olenic carbons
(Cheung et al., 1993), 7-oxo-13b-hydroxyabiet-8(14)-en-18-oic [d 156.5 (C-15) and 136.9 (C-13)] and H-14 [d 10.03 (s)], H-17 [d
acid (15) (Ohtsu et al., 2000), 7b-hydroxy-15-en-dehydroabietic 2.13 (s)], H-16 [d 2.00 (s)] and H-12 [d 2.09 (m)], as well as corre-
acid (16) (Daniel and Bernd, 2001), and 3b-hydroxy-8(14),15- lations of C-8 (d 211.8) with H-11 [d 1.56 (m) 1.17 (m)] and H-9
pimaradien-18-ol (17) (Conner and Rowe, 1977), and two [d 2.06 (m)]. Its NOESY spectrum (Fig. 2) demonstrated that H-19
known norditerpenes: 8(14)-podocarpen-13-on-18-oic acid (18) [d 1.12 (s)] and H-20 [d 0.64 (s)] are both in a b-orientation, and
(Cheung et al., 1993), abiesanordine E (19) (Yang et al., 2008) H-5 [d 2.30 (dd J = 10.5, 5.2)] and H-9 [d 2.06 (m)] are both in the
(Fig. 1). Among them, 1 is a rare 8,14-seco-abietane diterpene, a-orientation. The 1H1H COSY and HSQC experiments were also
23 and 916 are abietane diterpenes, 4, 7 and 17 are pimarane carried out to assign all proton and carbon signals for 1. The struc-
diterpenes, 5, 18 and 19 are podocarpane norditerpenes, 6 and 8 ture of 1 was thus assigned as 8-oxo-8,14-seco-abiet-13(15)-en-
are labdane diterpenes, and 6 was isolated as a natural product 15-al-18-oic acid.
for the rst time. Compound 2 was obtained as a colorless oil with the molecular
Compound 1 was obtained as a colorless oil with the molecular formula C22H34O4, as established by a [MH] ion peak at m/z
formula C20H30O4, as established by a [MH] ion peak at m/z 361.2369 in its HR-ESIMS. The IR, 1H, and 13C NMR spectra of 2 (Ta-
333.2059 in its HR-ESIMS. Its IR spectrum showed the presence ble 1) were very similar to those of 15, except that an additional
of carboxyl group (3340, 3218, 1693 cm1) and an aldehyde group ethoxyl signal [dH 3.41 (2H, q, J = 7.0 Hz), 1.09 (3H, t, J = 7.0 Hz);
(1703 cm1), whereas the 1H NMR spectrum (Table 1) displayed dC 58.7, 16.6] was observed in 2. The linkage position for the eth-
partial structural characteristics such as one aldehyde [d 10.03 oxyl moiety was established as C-13 by HMBC correlation for the
(s)] and four methyl groups [d 2.13 (s), 2.00 (s), 1.12 (s) and 0.64 ethoxy methylene proton to C-13 (Fig. 2), and the conguration
(s)]. The 13C NMR spectrum (Table 1) exhibited one ketone (d of the ethoxyl group was determined to be a because the ethoxy
211.8), one aldehyde (d 191.2), one carboxy (d 183.6) and two ole- methylene proton showed NOEs with H-11a [d 1.47 (m)] and

16
17
OCH2CH3 16
16 OH 12
12 17 13 15
15 11
11 12 12
13 15 16
11 13 15 17 11 17 20
13 9 14
20 14CHO 20 20
R2 1
9 9 14 14
1 O 1 1 9
2 10 8
2 10
H
8 2 10 8 2 10 8
H H 3 5 7
3 5 7 7 4 6
3 5 7 3 5
4 6 4 6 4 6 R3 H
H H O H 19 18 R 1
19 18 COOH 19 18 COOH HO 19 18
1 2 3 R1 R2 R3
4 COOH OH H
7 COOH H H
17 CH2 OH H OH

R3
O
12
11 13
20
9 14
1
2 10 8
H
R1
3 5 7
H
4 6 R1 H
R2
H COOH
R2
19 18 COOH H R1 R2 R3
RO R 10 H H H
R1 R2
6 Ac 11 H OH H
5 OH H
12 OH H H
18 H OH 8 H
13 O H
19 H H
14 H H OH

OH

O H OH
H H
COOH COOH COOH
9 15 16

Fig. 1. Structures of compounds 119.

1530 N.-Y. Yang et al. / Phytochemistry 71 (2010) 15281533

Table 1
1 13
H (300 MHz) and C NMR (75 MHz) spectroscopic data of compounds 13 (d ppm).

Position 1 2 3
dH (J, Hz) dC dH (J, Hz) dC dH (J, Hz) dC
1 1.68 m 1.21 m 37.9 1.85 m 1.21 m 39.2 1.43 m 34.5
2 1.66 m 17.6 1.63 m 19.1 1.81 m 18.3
3 1.72 m 1.57 m 37.0 1.72 m 38.4 2.20 m 35.7
4 47.0 47.3 43.8
5 2.30 dd (10.5, 5.2) 48.0 2.38 m 46.0 2.43 m 45.1
6 1.70 m 25.9 2.35 m 40.0 6.00 dd (9.6, 2.8) 129.1
7 2.36 m 42.2 201.8 6.60 dd (9.6, 2.8) 128.5
8 211.8 142.0 132.5
9 2.06 m 63.9 2.07 m 53.1 146.6
10 42.0 36.9 37.3
11 1.56 m 1.17 m 20.8 1.74 m 1.47 m 28.3 7.13 d (8.2) 121.8
12 2.09 m 24.8 1.70 m 1.56 m 19.6 7.29 dd (8.2, 2.2) 123.7
13 136.9 77.0 147.5
14 10.03 s 191.2 6.72 br s 141.0 7.29 d (2.20) 122.6
15 156.5 2.05 m 34.9 72.5
16 2.00 s 23.4 0.90 d (6.7) 17.1 1.56 s 31.7
17 2.13 s 19.3 0.79 d (6.7) 17.9 1.56 s 31.6
18 183.6 182.2 1.01 s 18.2
19 1.12 s 16.3 1.23 s 16.6 3.49 d (11.2) 3.27 d (11.2) 71.5
20 0.64 s 14.8 0.90 s 15.1 1.08 s 20.7
CH3CO
CH3CO
CH3CH2 3.41 q (7.0) 58.7
CH3CH2 1.09 t (7.0) 16.6

H H OCH CH
2 3
H OH
H

CHO
O

H
H

H O
H H
COOH
1 COOH 2 HO
3

HO

H
H H
OH
H
H
COOH H
4 COOH
5

Fig. 2. Key 1H1H NOESY and HMBC correlations of compounds 15.

H-12a [d 1.56 (m)] in the NOESY experiment (Fig. 2). The 1H1H
COSY and HSQC experiments were also run to assign all proton
and carbon signals for 2. Therefore, the structure of 2 was 7-oxo-
13a-ethoxyabiet-8(14)-en-18-oic acid.
Compound 3 was obtained as a colorless oil with the molecular
formula C20H28O2, showing a [M+H]+ ion peak at m/z 301.2158 in
its HR-ESIMS. IR spectrum showed the presence of hydroxyl group
(3303 cm1) and aromatic ring (1611, 1510 cm1). The 1H and 13C
NMR spectral data (Table 1) showed some resemblance to those of
15-hydroxy-6-en-dehydroabietic acid (Lee et al., 2009) except that
two additional hydroxymethyl proton signals at d 3.49 (1H, d,
J = 11.2 Hz) and 3.27 (1H, d, J = 11.2 Hz) appeared in the 1H NMR
spectrum of 3 and an additional hydroxymethyl carbon signal at
d 71.5 appeared in the 13C NMR spectrum of 3, which indicated
the presence of a hydroxymethyl in 3 instead of a carboxyl in 15-
hydroxy-6-en-dehydroabietic acid. The location for the hydroxy-
methyl was established as C-19 by NOE correlation for the
hydroxymethyl proton with Me-20 and HMBC correlation for the
hydroxymethyl proton to C-3 (d 35.7), C-4 (d 43.8) and C-5 (d
45.1) (Fig. 2), and the conguration of the hydroxymethyl was
determined to be b. 1H1H COSY and HSQC experiments were also
run to assign all proton and carbon signals for 3. Therefore, the
 N.-Y. Yang et al. / Phytochemistry 71 (2010) 15281533 1531

structure of 3 was assigned to be 15,19-dihydroxy-abieta- Table 3

6,8,11,13-tetraen. Cytotoxic activities of isolated compounds against A431 and A549 tumor cell lines.

Compound 4 was obtained as a colorless oil with the molecular Compound A431 A549
formula C20H30O3, as established by a [MH] ion peak at m/z IC50 (lM) IC50 (lM)
317.2105 in its HR-ESIMS. The IR, 1H, and 13C NMR spectra of 4 (Ta- 2 56.60 63.17
ble 2) were very similar to those of 7, except that an additional 6 0.38 2.00
oxygenated methine proton [d 4.20 (1H, br s)] appeared in the 1H 7 >100 45.59
8 4.74 19.62
NMR spectrum of 4 and its corresponding carbon (d 68.1) in the 9 65.41 59.49
13
C NMR spectrum of 4. The location for the oxygenated methine 10 68.13 81.25
proton was established as H-2 by HMBC correlation for the oxy- 11 48.55 63.59
genated methine proton to C-4 (d 48.2) and C-10 (d 39.8) 12 >100 61.65
17 1.60 16.44
(Fig. 2).The multiplicity and peak prole (br s) of H-2 signal indi-
Cisplatin 9.10 9.90
cated that H-2 was equatorial and a-oriented. The 1H1H COSY,
1
H1H NOESY (Fig. 2) and HSQC experiments were also run to as-
sign all proton and carbon signals for 4. Therefore, the structure of
4 was determined to be 2b-hydroxy-8(14),15-pimaradien-18-oic in labdane diterpenes signicantly enhances the activity (6 > 8),
acid, or 2b-hydroxy-pimaric acid. and hydroxymethylation of C-18 carboxyl in pimarane diterpenes
Compound 5 was obtained as a colorless oil with the molecular obviously increased the activity (17 > 7). The results also showed
formula C17H24O4 according to its HR-ESIMS, which showed the that all the active compounds are relatively less polar in compari-
[MH] ion peak at m/z 291.1589. The IR, 1H, and 13C NMR spectra son with non-active compounds, which could be responsible for
of 5 (Table 2) were very similar to those of 19, except that the mul- the better penetration of the compound across the phospholipid
tiplicity and coupling constants of the oxygenated methine proton bilayers surrounding the cells. As 6 and 17 are minor compounds,
signal at d 4.17 (1H, dd, J = 9.7, 6.6 Hz) of 5 were different from the observed cytotoxicity of P. massoniana resin extract might be
those of the oxygenated methine proton resonance at d 4.27 (1H, attributable to the major compounds such as 8 and 9. Some abie-
br s) of 19, which suggested that H-7 with the signal at d 4.17 tane, pimarane and labdane diterpenes were reported to show
was axial and a-oriented. The NOEs for H-7 with H-5 [d 2.11 (1H, moderate cytotoxicity against A549 cells (Hebert et al., 2008; Pich-
dd, J = 10.9, 4.8 Hz)] and H-9 [d 2.25 (1H, m)] also supported this ette et al., 2006; Son et al., 2005; Wada and Tanaka, 2006; Zhao
conclusion (Fig. 2). 1H1H COSY, HMBC (Fig. 2) and HSQC experi- et al., 2008), and they are also relatively less polar, while some lab-
ments were also run to assign all proton and carbon signals for 5. dane diterpene glycosides exhibited nonspecic cytotoxicity
So the structure of 5 was established as 7b-hydroxypodocarpen- against A549 (Lee et al., 2005). Few reports can be found on the
8(14)-en-13-on-18-oic acid, or 7-epi-abiesanordine E. cytotoxicity assay of related diterpenes against A431 cells.
The cytotoxic activities of isolated diterpenes were evaluated
using the MTT assay, and IC50 was calculated to quantitatively
compare the cytotoxic potency of the tested compounds. Among 3. Concluding remarks
them, compounds 6, 8 and 17 exhibited strong cytotoxicity against
A431 and A549 cells with lower IC50 (Table 3), compounds 2, 9, 10 The components of plant resin were complicated, and mostly
and 11 showed moderate cytotoxicity against A431 and A549 cells, consisted of terpenoids and their derivatives. This study estab-
and compounds 7 and 12 showed moderate cytotoxicity against lished that P. massoniana resin mainly contained the chemical con-
A549 cells. However, other compounds exhibited weak or no cyto- stituents of many diterpenoids, including abietane, pimarane and
toxicity against A431 and A549 cells. From the observed activity labdane diterpenes and podocarpane norditerpenes. These diterpe-
data, it can be concluded that esterication of C-19 hydroxymethyl noids possessed similar biosynthetic pathway.

Table 2
1 13
H (300 MHz) and C NMR (75 MHz) spectroscopic data of compounds 46 (d ppm).

Position 4 5 6
dH (J, Hz) dC dH (J, Hz) dC dH (J, Hz) dC
1 1.89 m 1.43 m 44.9 1.85 m 1.27 m 40.1 1.8 m 1.14 dd (12.4, 5.4) 39.0
2 4.20 br s 68.1 1.54 m 19.1 1.51 m 18.9
3 2.00 m 1.82 m 42.9 1.63 m 37.1 1.73 m 1.04 m 36.2
4 48.2 47.9 51.9
5 1.99 dd (10.8, 5.0) 49.9 2.11 dd (10.9, 4.8) 46.9 1.30 dd (10.7, 5.0) 56.2
6 1.58 m 1.33 m 25.8 1.63 m 33.9 1.85 m 1.39 m 24.2
7 228 m 2.16 m 36.7 4.17 dd (9.7, 6.6) 72.4 2.40 m 1.98 m 38.3
8 139.4 169.1 147.8
9 1.75 m 53.9 2.25 m 50.8 1.78 m 57.1
10 39.8 39.3 39.4
11 1.55 m 1.45 m 20.2 2.05 m 1.66 m 22.3 2.38 m 2.16 m 23.2
12 1.57 m 36.9 2.40 m 2.31 m 38.0 5.40 br d (6.2) 133.8
13 39.8 202.7 133.5
14 5.16 br s 129.9 6.27 br s 123.1 4.82 s 4.46 s 107.9
15 5.72 dd (17.2, 10.6) 148.3 6.32 dd (17.5, 10.7) 141.6
16 5.00 d (17.2) 4.94 d (10.6) 113.6 5.04 d (17.5) 4.87 d (10.7) 109.9
17 0.97 s 30.1 1.74 s 11.8
18 183.2 182.0 0.97 s 27.6
19 1.38 s 20.0 1.22 s 17.2 4.24 d (11.1) 3.86 d (11.1) 66.8
20 1.00 s 17.8 0.88 s 16.0 0.73 s 15.2
171.3
2.03 s 20.9
1532 N.-Y. Yang et al. / Phytochemistry 71 (2010) 15281533
This work also showed that the cytotoxic constituents from P.
massoniana resin were of low polarity, and that their cytotoxicities
were related to the acetyl and hydroxymethyl groups.
4. Experimental
4.1. General experimental procedures
Optical rotations were measured on a JASCO P-1020 Polarime-
ter. IR spectra were recorded on a Nicolet IR-100 FT-IR spectrome-
ter with KBr disks. NMR spectra were measured on a Bruker AV-
300 MHz (300 MHz for 1H NMR and 75 MHz for 13C NMR). ESIMS
and HR-ESIMS spectra were tested on a Micromass Q/TOF Mass
Spectrometer. Silica gel for column chromatography (CC, 200
300 mesh) and silica gel thin layer chromatography plates were
the products of Qingdao Haiyang Chemical Co., Ltd. (Qingdao, Chi-
na). All solvents used were of analytical grade (Nanjing Chemical
Plant, Nanjing, China). A Waters 25452767UV2489, equipped
with a preparative Sunre C-18 column (30  150 mm), was used
for sample preparation. Cisplatin was purchased from Qilu Phar-
maceutical Co., Ltd. (Jinan, Shangdong, China), whereas fetal bo-
vine serum was from Sijiqing Bioengineering Institute
(Hangzhou, Zhejiang, China), and diphenyltetrazolium bromide
was from Gibco (Grand Island, New York, USA).
4.2. Plant material
P. massoniana resin was collected from Henan Province, China,
in November 2008. A voucher specimen (PR-20081110) is depos-
ited at the Herbarium of Nanjing University of Traditional Chinese
Medicine.
4.3. Extraction and isolation
P. massoniana resin (200 g) was extracted successively with
petroleum ether (6090 C) (2  2 L) and EtOHH2O (95:5, v/v)
(2  2 L) under conditions of reux at 8090 C for 3 h, respec-
tively. The petroleum ether extract (165 g) was subjected to silica
gel CC (2 kg) eluting with petroleum ether (6090 C)acetone in
a stepwise gradient (30:1 ? 3:1), with eight fractions (IVIII) col-
lected. Fraction I was further separated by silica gel CC [petroleum
etherCH2Cl2 (10:1)] to yield 6 (202 mg), whereas fraction II when
applied to silica gel [petroleum etherCH2Cl2 (7:1)] afforded 7
(68.2 g) and 8 (10.1 g). Fraction III was also subjected to silica gel
CC [petroleum etherCH2Cl2 (5:1)] to give 9 (7.5 g) and 10
(650 mg), and fraction IV in the same way [petroleum ether
CH2Cl2 (5:2) as eluant] gave 17 (15 mg) and 2 (37 mg). Fraction V
was separated by HPLC on a Sunre C-18 column using a gradient
of CH3CN in 0.1% AcOH (0.1% AcOH/CH3OH, 30:70) to yield 12
(10 mg), 11 (52 mg), 4 (3 mg) and 13 (5 mg). Fraction VI was fur-
ther subjected to HPLC (Sunre C-18 column) eluted with a gradi-
ent of CH3CN in 0.1% AcOH (0.1% AcOH/CH3OH, 40:60) to yield 16
(3 mg), 1 (65 mg), 3 (2 mg) and 14 (81 mg). Fraction VII on silica gel
CC [petroleum etherCH2Cl2 (1:1)] gave 15 (92 mg) and 18
(175 mg), and fraction VIII on silica gel CC [petroleum ether
CH2Cl2 (1:2)] afforded 19 (107 mg) and 5 (50 mg).
4.3.1. 8-Oxo-8,14-seco-abiet-13(15)-en-15-al-18-oic acid (1)
Colorless oil, a20
D 8.6 (c 0.14, CH3OH); IR (KBr) mmax: 3340,
3218, 2909, 1703, 1693, 1534, 1470 1274, 1061, 1014, 722 cm1;
For 1H and 13C NMR (CD3OD) spectroscopic data, see Table 1; neg-
ative ESIMS m/z: 333 [MH]; HR-ESIMS m/z: 333.2059 [MH]
(calcd. for C20H30O4, 333.2066).
4.3.2. 7-Oxo-13a-ethoxyabiet-8(14)-en-18-oic acid (2)
Colorless oil, a20
D +17.2 (c 0.10, CHCl3); IR (KBr) mmax: 3430
2611, 1710, 1695, 1611, 1464, 1387, 1235, 856 cm1; For 1H and
13
C NMR (CD3OD) spectroscopic data, see Table 1; negative ESIMS
m/z: 361 [MH]; HR-ESIMS m/z: 361.2369 [MH] (calcd. for
C22H34O4, 361.2379).
4.3.3. 15,19-Dihydroxy-abieta-6,8,11,13-tetraen (3)
Colorless oil, a20
D 15.0 (c 0.07, CHCl3); IR (KBr) mmax: 3303,
1611, 1510, 1444, 1360, 1230, 701 cm1; For 1H and 13C NMR
(CD3OD) spectroscopic data, see Table 1; negative ESIMS m/z:
301 [MH]; HR-ESIMS m/z: 301.2158 [MH] (calcd. for
C20H28O2, 301.2168).
4.3.4. 2b-Hydroxy-8(14),15-isopimaradien-18-oic acid (4)
Colorless oil, a20
D +0.5 (c 0.10, CH3OH); IR (KBr) mmax: 3428
2604, 1715, 1651, 1434, 1374, 1245, 991, 911, 858 cm1; For 1H
and 13C NMR (CD3OD) spectroscopic data, see Table 2; negative
ESIMS m/z: 317 [MH]; HR-ESIMS m/z: 317.2105 [MH] (calcd.
for C20H30O3, 317.2117).
4.3.5. 7b-Hydroxypodocarpen-8(14)-en-13-on-18-oic acid (5)
Colorless oil, a20
D 6.5 (c 0.18, CH3OH); IR (KBr) mmax: 3500
2601, 1711, 1685, 1611, 1395, 1265, 1037, 953, 852 cm1; For 1H
and 13C NMR (CD3OD) spectroscopic data, see Table 2; negative
ESIMS m/z: 291 [MH]; HR-ESIMS m/z: 291.1589 [MH] (calcd.
for C17H24O4, 291.1596).
4.3.6. 19-Acetoxy-8(14),12E,15-labdatrien (6)
Colorless oil, a20
D +20.5 (c 0.15, CH3OH); IR (KBr) mmax: 2911,
1733, 1660, 1651, 1544, 1275, 1067, 991, 910, 886 cm1; For 1H
and 13C NMR (CDCl3) spectroscopic data, see Table 2; negative
ESIMS m/z: 331 [MH]; HR-ESIMS m/z: 331.2629 [MH] (calcd.
for C22H34O2, 331.2637).
4.3.7. 7b-Hydroxy-15-en-dehydroabietic acid (16)
Colorless oil, 1H NMR (300 MHz, CD3OD), d: 2.30 (1H, m H-1),
1.37 (1H, m H-1), 1.70 (2H, m H-2), 1.78 (1H, m H-3), 1.66 (1H,
m H-3), 2.19 (1H, br t, J = 7.14 Hz, H-5), 1.82 (2H, m H-6), 4.72
(1H, br t, J = 8.69 Hz, H-7), 7.17 (1H, d, J = 8.34 Hz, H-11), 7.29
(1H, dd, J = 8.34, 2.01 Hz, H-12), 7.59 (1H, d, J = 2.01 Hz, H-14),
5.31 (1H, s, H-16), 4.98 (1H, s, H-16), 2.09 (3H, s, H-17), 1.24 (1H,
s, H-19), 1.25 (1H, s, H-20); 13C NMR (75 MHz, CD3OD), d: 39.5
(C-1), 19.6 (C-2), 38.0 (C-3), 48.3 (C-4), 45.1 (C-5), 33.5 (C-6),
71.4 (C-7), 139.3 (C-8), 150.1 (C-9), 39.1 (C-10), 125.2 (C-11),
125.7 (C-12), 140.0 (C-13), 126.0 (C-14), 144.7 (C-15), 112.0 (C-
16), 22.1 (C-17), 183.0 (C-18), 17.3 (C-19), 25.8 (C-20); negative
ESIMS m/z: 313 [MH]; HR-ESIMS m/z: 313.1794 [MH] (calcd.
for C20H26O3, 313.1804).
4.4. Cytotoxicity bioassays
Compounds were tested against human epithelial carcinoma
(A431) and lung (A549) cell lines using an established colorimetric
diphenyltetrazolium bromide (MTT) assay protocol. Cisplatin was
used as a positive control. Cells were seeded in 96-well culture
plates at densities of 30004000 cells per well. After overnight,
cells were cultured with 10% Fetal Bovine Serum (FBS) in the ab-
sence for 12 h. Then, the tested compounds at different concentra-
tions were added into 96-well plates and cultivated for 72 h, and
20 ll of MTT (5 mg/mL) was added to each well. After 4 h, the cul-
ture medium was removed and the formazan crystal was com-
pletely dissolved with 150 ll dimethylsulfoxide to each well by
vigorously shaking the plate. Finally, formazan absorbance was as-
sessed by a Bio-Tek multi-detection microplate reader at 490 nm.
 N.-Y. Yang et al. / Phytochemistry 71 (2010) 15281533
The IC50 is the concentration of agent that reduced cell growth by
50% under the experimental conditions.
Acknowledgements
This research was supported by the research project of the
Commonweal Foundation of Jiangsu Province. We also thank
Mr. Dong-Jun Chen, Zhenhua Zhu and Da-Wei Qian, Drs. Yu-Ping
Tang and Er-Xin Shang for other helpful assistance.
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