Two
New
Labdane-Type
Diterpenes
from
the
Wood
of
Cunninghamia konishii
Chi-I Chang a, Yen-Cheng Li b, Yong-Han Hong c, Wei-Yi Cheng c, Che-Yi Chao d, Minoru Tsuzuki e,
Sakae Amagaya e, Ching-Jang Huang f, Yueh-Hsiung Kuo g,h,i*
a
Department of Biological Science and Technology, National Pingtung University of Science and
Technology, Pingtung 912, Taiwan
b
Department of Chemistry, National Taiwan University, Taipei 106, Taiwan
c
Department of Nutrition, I-Shou University, Kaohsiung 824, Taiwan
d
Department of Health and Nutrition Biochnology, Asia University, Taichung 413, Taiwan
e
Department of Biochemistry, Nihon Pharmaceutical University, Saitama 362-0806, Japan
f
Nutritional biochemistry Laboratory, Institute of Microbiology and Biochemistry, National Taiwan
University, Taipei 106, Taiwan
g
Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical
University, Taichung 404, Taiwan
1
h
Tsuzuki Institute for Traditional Medicine, College of Pharmacy, China Medical University, Taichung
404, Taiwan
i
Department of Biotechnology, Asia University, Taichung 413, Taiwan
* Corresponding authors at: Department of Chinese Pharmaceutical Sciences and Chinese Medicine
Resources, China Medical University, No. 91, Hsueh-Shih Rd., Taichung City, 404 Taiwan. Tel.:
+886-4-2205-3366 ext. 5701 (Y.H. Kuo).
E-Mail: kuoyh@mail.cmu.edu.tw (Y.H. Kuo).
2
ABSTRACT
Our preliminary pharmaceutical screening revealed that the methanol extract of wood of C.
konishii possessed significant anti-inflammatory activity. Further phytochemical investigation led to the
isolation of two new labdane-type diterpenoids, (12S,13S)-12,13-epoxy-8(17),14-labdadien-18-ol (1)
and (12S,13S)-12,13-epoxy-8(17),14-labdadien-19-oic acid (2), along with four known labdane-type
diterpenoids,
(12R,13R)-12,13-epoxy-labda-8(17),14-dien-19-oic
acid
(3),
12,13-dihydroxylabda-8(17),14-dien-19-oic acid (4), sclareol (5), and 13-epi-sclareol (6). Their
structures were determined by analysis of spectroscopic data and comparison with the data of known
analogues.
Keywords:
Chinese herb
Taxodiaceae
Cunninghamia konishii
labdane
diterpenoid
3
1. Introduction
Cunninghamia konishii Hayata, an endemic Taiwanese coniferous tree, belongs to Taxodiaceae
family and is distributed in the northern and central part of Taiwan at altitudes of 1,300–2,700 m (Li and
Keng, 1994). The wood of this plant exhibits soft, lightweight, aromatic, and rot-resistant characteristics
and is one of the best materials of buildings and wood products available in Taiwan. Various
phytochemical studies on wood (Chang and Yin, 1991; Chen et al., 2013; Cheng et al., 1979; Cheng et
al., 2011; Cheng et al., 2012; Ikeda and Fujita, 1929a; Ikeda and Fujita, 1929b; Li and Kuo, 1998; Li
and Kuo, 2002), bark (Cheng and Tsai, 1972), leaf (Cheng et al., 2011), and whole plant (He et al., 1997)
of C. konishii have shown that it contains monoterpenes, sesquiterpenes, and diterpenes. Some isolates of
this plant exhibit anti-inflammatory activity (Chen et al., 2013), antifungal activity (Cheng et al., 2011;
Cheng et al., 2012), and cytotoxicity (He et al., 1997). Our preliminary pharmaceutical screening
revealed that the methanol extract of wood of C. konishii possessed significant anti-inflammatory
activity. On the basis of an interest in the discovery of secondary metabolites with anti-inflammatory
activity from this plant, we had reported the isolation and structure elucidation of fifteen diterpenoids
from the MeOH extract of the wood of this plant (Chen et al., 2013; Li and Kuo, 1998; Li and Kuo,
2002). In our continuing investigation on C. konishii, we further identified two new labdane-type
diterpenoids,
(12S,13S)-12,13-epoxy-labda-8(17),14-dien-18-ol
(1)
and
(12S,13S)-12,13-epoxy-labda-8(17),14-dien-19-oic acid (2), as well as four known labdane-type
diterpenoids, (12R,13R)-12,13-epoxy-labda-8(17),14-dien-19-oic acid (3) (Barrero et al., 1991),
4
12,13-dihydroxylabda-8(17),14-dien-19-oic acid (4) (Fang et al., 1993), sclareol (5) (Ulubelen et al.,
1994), and 13-epi-sclareol (6) (Torrenegra et al., 1992), from the wood of C. konishii. (Figure 1). Herein
we reported the extraction, isolation, and structure elucidation of compounds 1 and 2.
2. Results and discussion
The MeOH extract of the wood of C. konishii was suspended in water, and then partitioned with
n-hexane, EtOAc, and n-BuOH by liquid-liquid extraction, successively. The EtOAc fraction containing
nonpolar secondary metabolites was separated by repeated column chromatography and semipreparative
NP-HPLC to afford compounds 1–6.
Compound 1 was obtained as a yellowish oil. Its high resolution electron impact mass spectrum
(HR-EI-MS) showed a molecular ion peak at m/z 304.2412, which corresponded to the molecular
formula, C20H32O2, indicating five degrees of unsaturation. The IR spectrum indicated the presence of
hydroxyl (3420 cm-1), vinyl and terminal double bonds (3084, 1641, 990, 910, and 875 cm-1) groups.
The 1H and 13C NMR spectra of 1 (Table 1) demonstrated three tertiary methyls [δH 0.72, 0.74, 1.39 (3H
each, s)], a hydroxymethylene group [δH 3.08 (1H, d, J = 10.8 Hz), 3.41 (1H, d, J = 10.8 Hz); δC 71.9
(t)], a vinylic ABX system [δH 5.12 (1H, dd, J = 10.5 and 1.1 Hz), 5.26 (1H, d, J = 17.3 and 1.1 Hz), and
5.59 (1H, dd, J = 17.3 and 10.5 Hz); δC 115.7 (t), 141.1 (d)], and a terminal double bond [δH 4.74 (1H,
brs), 4.88 (1H, brs); δC107.8 (t), 147.9 (s)], and a trisubstituted epoxide moiety [δH 2.81 (1H, dd, J = 7.3
and 3.3 Hz); δC 59.6 (s), 64.8 (d)]. 20 carbon signals were found in the
13
C NMR spectrum of 1 and
were assigned by a DEPT experiment as three aliphatic methyl, six aliphatic methylene, two aliphatic
5
methine, two aliphatic quaternary, two olefinic methylene, one olefinic methine, one quaternary
olefinic, one secondary oxygenated, one tertiary oxygenated, and one quaternary oxygenated carbons.
From the above structural characteristics, compound 1 was thus tentatively proposed to be a
labdane-type diterpene (Fang et al., 1993). The 13C NMR resonances of 1 were very similar to the
corresponding signals of methyl (12S,13S)-12,13-epoxy-labda-8(17),14-dien-19-oate in the ring B and
side chain (Barrero et al., 1991), except for the signals of C-1–6 and C-18–20 in the ring A. An
trisubstituted epoxy group [δH 2.81 (1H, dd, J = 7.3, 3.3 Hz); δC 59.6 (s), 64.8 (d)] located at C-12 and
C-13 was confirmed by the HMBC correlations (Figure 1) as follows: H-12 (δH 2.81)/C-13 (δC 59.6);
H-14 (δH 5.59)/C-13; H-15 (δH 5.12)/C-13, and H-16 (δH 1.39)/C-13. The 1H NMR signals of
hydroxymethylene group (δH 3.08, 3.41) showing NOESY correlation with H-5 (δH 1.46) and HMBC
correlations with C-4 (δC 38.0) and C-5 (δC 48.3) assured that the primary alcohol was attached on C-4
and was in equatorial orientation. The α-orientation of the epoxy group at C-12 and C-13 was elucidated
by the NOESY correlation (Figure 1) between H-12 and H-14. Furthermore, the absolute configuration
of C-12 and C-13 in 1 was assigned as (12S,13S) by comparing its NMR signals with those of methyl
(12S,13S)-12,13-epoxylabda-8(17),14-dien-19-oate reported in the literature (Barrero et al., 1991). Thus,
the structure of 1 was determined as (12S,13S)-12,13-epoxy-8(17),14-labdadien-18-ol.
The HR-EI-MS of compound 2 showed an [M]+ ion at m/z 318.2214, which was consistent with the
molecular formula C20H30O3, indicating six degrees of unsaturation. The IR spectrum indicated the
presence of carboxylic group (3300-2500 and 1692 cm-1), vinyl and terminal double bonds (3085, 1647,
6
985, 900, and 880 cm-1). In the 1H and 13C NMR spectra of 2 (Table 1), twenty skeletal carbon
resonances, as well as the signals for a terminal double bond [δH 4.73 (1H, brs), 4.90 (1H, brs); δC107.9
(s), 147.5 (s)] and a vinylic ABX system [δH 5.12 (1H, d, J = 10.8 Hz), 5.25 (1H, d, J = 17.2 Hz), and
5.59 (1H, dd, J = 10.8, 17.2 Hz); δC 115.7 (s), 141.0 (s)], and a trisubstituted epoxide moiety [δH 2.79
(1H, t, J = 6.3 Hz); δC 59.6 (s), 64.8 (d)] were found. From the above evidences, compound 2 was
proposed to be a labdane-type diterpene with a trisubstituted epoxy group (Fang et al., 1993). The 1H
and
13
C NMR resonances of 2 were very similar to the corresponding signals of methyl
(12S,13S)-12,13-epoxy-8(17),14-labdadien-19-oate (Barrero et al., 1991), except for a carboxylic acid
group [δC183.8 (s)] located at C-19 in 2, instead of a methyl ester group in methyl
(12S,13S)-12,13-epoxy-8(17),14-labdadien-19-oate. Thus, the structure of 2 was elucidated as
(12S,13S)-12,13-epoxy-8(17),14-labdadien-19-oic acid.
3. Experimental
3.1. General experimental procedures
Optical rotations were measured using a Jasco-DIP-180 polarimeter. Infrared (IR) spectra were
measured on a Perkin-Elmer-983G FT-IR spectrophotometer. 1H and
13
C NMR and 2D NMR spectra
were obtained using a Varian-Unity-Plus-400 spectrometer with tetramethylsilane (TMS) as the internal
standard. EI-MS and HR-EI-MS were measured with a Jeol-JMS-HX300 mass spectrometer. Silica gel
(230-400 mesh; Merck & Co., Inc.) was used for column chromatography (CC), and pre-coated silica
7
gel (60 F-254; Merck & Co., Inc.) plates were used for TLC. The spots on TLC were detected by
spraying with 5% H2SO4 and then heating at 100 ºC. Semi-preparative HPLC was performed using a
normal phase column (LiChrosorb Si 60, 7 μm, 250 × 10 mm; Merck & Co., Inc.) on a LDC
Analytical-III system.
3.2. Plant Material
The wood of C. konishii was collected at Luantashan, Nantau County, Taiwan, in December 1996.
The plant material was identified by Prof. Shao-Shun Ying, Department of Forestry, National Taiwan
University. A voucher specimen (013492) has been deposited at the Herbarium of the Department of
Botany, National Taiwan University, Taipei, Taiwan.
3.3. Extraction and Isolation
Air dried wood (6.5 kg) of C. konishii was crushed into pieces and extracted with MeOH (60 L) three
times (7 days each time) at room temperature. After removal of the solvent under vacuum, the extract
(60.2 g) was suspended in water (500 mL), and then partitioned sequentially using n-hexane (500 mL ×
3), EtOAc (500 mL×4), and n-BuOH (500 mL×3). The EtOAc fraction (15.6 g) was chromatographed on
silica gel (450 g) using n-hexane–EtOAc and EtOAc–MeOH mixtures as solvent systems to obtain 11
fractions. Fr. 4 from n-hexane–EtOAc (3:7) elution is identified as 1 with some unknown impurities
through 1H NMR analysis. Furture purification by HPLC gave 1 (2.1 mg, tR = 32.2 min) using
n-hexane–CH2Cl2–EtOAc–i-PrOH (8:2:1:0.2). Fr. 5 from n-hexane–EtOAc (2:3) elution was identified
8
as a mixture containing mainly 2, 3 and 4 through 1H NMR analysis. Furture purification by HPLC gave
2 (1.8 mg, tR = 42.2 min), 3 (3.2 mg, tR = 37.6 min) and 4 (4.8 mg, tR = 31.2 min) using
n-hexane–CH2Cl2–EtOAc–i-PrOH
(5:5:1:0.2),
n-hexane–CH2Cl2–EtOAc–i-PrOH
(6:4:1:0.2),
n-hexane–CH2Cl2–EtOAc–i-PrOH (6:5:1:0.2), respectively. Fr. 8 from n-hexane–EtOAc (3:7) elution
was identified as a mixture containing mainly 5 and 6. Furture purification by HPLC gave 5 (1.5 mg, tR =
45.3 min) and 6 (5.2 mg, tR = 39.1 min) using n-hexane–CH2Cl2–EtOAc–i-PrOH (3:2:1:0.2) and
n-hexane–CH2Cl2–EtOAc–i-PrOH (3:1:1:0.2), respectively.
3.3.1. (12S,13S)-12,13-epoxy-labda-8(17),14-dien-18-ol (1)
Yellowish oil; [α] 27D = +18.6 (c 0.27, CHCl3); IR (dry film) νmax 3420, 3084, 1641, 1447, 1386, 1047,
990, 910, and 875 cm-l; 1H and 13C NMR data, see Table 1; EI-MS (%) m/z 304 (54) [M]+, 289 (22), 279
(100), 273 (50), 255 (22), 249 (17), 245 (20), 233 (27). HR-EI-MS [M]+ m/z 304.2412 (calcd for
C20H32O2 304.2404).
3.3.2. (12S,13S)-12,13-epoxy-labda-8(17),14-dien-19-oic acid (2)
Yellowish oil; [α] 27D = +169.0 (c 0.30, CHCl3); IR (dry film) νmax 3300-2500, 3085, 1692, 1647, 1467,
1267, 1183, 985, 900, and 880 cm-l; 1H and 13C NMR data, see Table 1; EI-MS (%) m/z 318 (96) [M]+,
304 (49), 300 (34), 287 (33), 271 (27), 263 (100), 247 (70), 245 (28). HR-EI-MS [M]+ m/z 318.2214
(calcd for C20H30O3 318.2196).
9
Acknowledgements
This work was kindly supported by a grant from the China Medical University (CMU100-S-10), in
part by the Taiwan Department of Heath Clinical Trial and Research Center of Excellence (DOH
102-TD-B-111-004). We thank Ms Shu-Yun Sun for the MS measurements in the Instrumentation Center
of the College of Science, National Taiwan University. We are also grateful to the National Center for
high-performance computing for computer time and facilities.
References
Barrero A, Quintana RM, Altarejos JC. (1991) Biomimetic synthesis of 12-oxy-pimaranes from
12,13-epoxy-labdadienes. Tetrahedron, 47, 4441–4456.
Chang ST, Yin HW. (1991) Identificition of the needle crystal appeared on the wood surface of
Cunninghamia konishii Hyata. Bulletin of Taiwan Forest Research Institute New Series, 6, 57–63.
Chen YC, Li YC, You BJ, Chang WT, Chao LK, Lo LC, Wang SY, Huang GJ, Kuo YH. (2013)
Diterpenoids with anti-inflammatory activity from the wood of Cunninghamia konishii. Molecules,
18, 682–689.
Cheng SS, Chung MJ, Lin CY, Wang YN, Chang ST. (2012) Phytochemicals from Cunninghamia
konishii Hayata act as antifungal agents. Journal of Agricultural and Food Chemistry, 60, 124–128.
10
Cheng YS, Lin CS. (1979) Study of the extractive constituents from the wood of Cunninghamia konishii
Hayata. Journal of the Chinese Chemical Society, 26, 169–172.
Cheng SS, Lin CY, Gu HJ, Chang ST. (2011) Antifungal activities and chemical composition of wood
and leaf essential oils from Cunninghamia konishii. Journal of Wood Chemistry and Technology,
31, 204–217.
Cheng YS, Tsai MD. (1972) Terpenes and sterols of Cunninghamia konishii. Phytochemistry, 11,
2108–2109.
Fang JM, Sou YC, Chiu YH, Cheng YS. (1993) Diterpenes from the bark of Juniperus chinensis.
Phytochemistry, 34, 1581–1584.
He K, Shi G, Zeng L, Ye Q, McLaughlin JL. (1997) Konishiol, a new sesquiterpene, and bioactive
components from Cunninghamia konishii. Planta Medica, 63, 158–160.
Ikeda T, Fujita Y. (1929a) Essential oils of Cunninghamia konishii Hayata. Bulletin of the Chemical
Society of Japan, 50, 32–45.
Ikeda T, Fujita Y. (1929b) The pinene in Cunninghamia konishii Hayata. Bulletin of the Chemical
Society of Japan, 50, 66–70.
Li HL, Keng H. (1994) Flora of Taiwan, 2nd ed. Editorial Committee of the Flora of Taiwan, Taipei,
582–585.
11
Li YC, Kuo YH. (1998) Five New diterpenoids from the wood of Cunninghamia konishii. Journal of
Natural Products, 61, 997–1000.
Li YC, Kuo YH. (2002) Labdane-type diterpenoids from the wood of Cunninghamia konishii. Chemical
& Pharmaceutical Bulletin, 50, 498–500.
Torrenegra R, Pedrozo J, Robles J, Waibel R, Achenbach H. (1992) Diterpenes from Gnaphalium
pellitum and Gnaphalium graveolens. Phytochemistry, 31, 2415–2418.
Ulubelen A, Topcu G, Eris C, Soenmez U, Kartal M, Kurucu S, Bozok-Johansson C. (1994) Terpenoids
from Salvia sclarea. Phytochemistry, 36, 971–974.
12