Five new lactone derivatives from the stems of Dendrobium nobile

Article abstract of DOI:10.1016/j.fitote.2016.10.002

Five new lactone derivatives decumbic acids A and B (1 and 2), (?)-decumbic acid (3a), (?)- and (+)-dendrolactone (4a and 4b) together with four known compounds (3b and 5–7) were isolated from the stems of Dendrobium nobile. Their structures were elucidated using comprehensive spectroscopic methods. Compounds 3a and 3b, 4a and 4b were isolated as two pair of enantiomers by chiral HPLC. The absolute configurations of 1, 2, 3a, 4a and 4b were determined by optical rotation and X-ray crystallographic analysis. The inhibitory activities of all compounds against nine phytopathogenic fungi and three cancer cell lines were evaluated.

Full text of DOI:10.1016/j.fitote.2016.10.002

Five new lactone derivatives from the stems of Dendrobium nobile
Xue-Ming Zhou, Cai-Juan Zheng, Jia-Ting Wu, Guang-Ying Chen, Jun
Chen, Chong-Ge Sun
PII:
DOI:
Reference:
S0367-326X(16)30234-9
doi:10.1016/j.fitote.2016.10.002
FITOTE 3507
To appear in:
Fitoterapia
Received date:
Revised date:
Accepted date:
24 August 2016
17 September 2016
5 October 2016
Please cite this article as: Xue-Ming Zhou, Cai-Juan Zheng, Jia-Ting Wu, Guang-Ying
Chen, Jun Chen, Chong-Ge Sun, Five new lactone derivatives from the stems of Dendro-
bium nobile, Fitoterapia (2016), doi:10.1016/j.fitote.2016.10.002
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ACCEPTED MANUSCRIPT
Five New Lactone Derivatives from the Stems of Dendrobium
nobile
Xue-Ming Zhoua, Cai-Juan Zhenga, Jia-Ting Wua, Guang-Ying Chena, Jun Chena, Chong-Ge
Sunb
a
Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, College of Chemistry and
Chemical Engineering, Hainan Normal University, Haikou 571158, China
b Hainan Boying Orchid Industrial Development Co., LTD, Haikou 570105, People’s Republic of China

Corresponding author. Tel.: (86)89865889422; fax: (86)89865889422.
E-mail address: chgying123@163.com

ACCEPTED MANUSCRIPT
Abstract–Five new lactone derivatives decumbic acids A and B (1 and 2),
(-)-decumbic acid (3a), (-)- and (+)-dendrolactone (4a and 4b) together with four
known compounds (3b and 5–7) were isolated from the stems of Dendrobium nobile.
Their structures were elucidated using comprehensive spectroscopic methods.
Compounds 3a and 3b, 4a and 4b were isolated as two pair of enantiomers by chiral
HPLC. The absolute configurations of 1, 2, 3a, 4a and 4b were determined by optical
rotation and X-ray crystallographic analysis. The inhibitory activities of all
compounds against nine phytopathogenic fungi and three cancer cell lines were
evaluated.
Keywords: Dendrobium nobile; butyrolactone; cytotoxic; antifungal
2

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1. Introduction
The genus Dendrobium (Orchidaceae) is represented by about 1100 species of
herbal plant, which are mainly distributed in the southwestern distributed throughout
Asia, Europe and Australia. The stems of several Dendrobium species are used as a
traditional medicine for the treatment of diabetes, fever, chronic atrophic gastritis, and
skin aging diseases [1–6]. Our previous study on Dendrobium nobile, one of the
species in southern China, has led to the isolation of 23 phenanthrene derivatives and
five bibenzyl derivatives, some of the isolated products showed cytotoxic or
antifungal activities [7]. In our ongoing investigation of natural antifungal and
cytotoxic products, five new lactone derivatives decumbic acids A and B (1 and 2),
(-)-decumbic acid (3a), (-)- and (+)-dendrolactone (4a and 4b) together with four
known compounds (Fig. 1), decumbic acid (3b) [8], 4-(3-hydroxyphenyl)-2-butanone
(5) [9], 3-hydroxy-1(3-methoxy-4-hydroxyphenyl)-propan-1-one (6) [10] and
3',4',5',-trimethoxycinnamyl acetate (7) [11] were isolated from the stems of D. nobile.
All compounds were tested for their antifungal activity against nine phytopathogenic
fungi and cytotoxic activity on three cancer cell lines.
2. Experimental
2.1. General experimental procedures
IR spectra were recorded on a Nicolet 6700 spectrophotometer. Optical rotations
were measured on a JASCO P-1020 digital polarimeter. Semi-Preparative HPLC was
performed on an Agilent 1260 LC series with a DAD detector using an Agilent
Eclipse XDB-C18 column (9.4 × 250 mm, 5 µm) and an ULTRON ES-OVM column
(150 × 10 mm, 5 µm). NMR spectra were recorded on a Bruker AV spectrometer (400
MHz for 1H and 100 MHz for 13C). TMS was used as an internal standard. HRESIMS
spectra were measured on a Q-TOF Ultima Global GAA076 LC mass spectrometer.
Silica gel (Qing Dao Hai Yang Chemical Group Co.; 200–300 mesh) were used for
column chromatography (CC). Precoated silica gel plates (Yan Tai Zi Fu Chemical
Group Co.; G60, F-254) were used for thin layer chromatography (TLC). X-ray
diffraction data were collected on an Aglient Technologies Gemini A Ultra system.

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2.2. Plant material
The stems of D. nobile were provided by the Hainan Boying Orchid Industrial
Development Co., LTD in June 2014. A voucher specimen (No. GFM20140612) has
been deposited at the Key Laboratory of Tropical Chemistry of Medicinal Plant of
Ministry of Education, Hainan Normal University (Hainan, China).
2.3. Extraction and isolation
The air-dried and powdered stems (5 kg) of D. nobile were extracted with 70%
EtOH (3 × 20 L, 5 days each) at room temperature. After concentration under reduced
pressure, the water-soluble residue was partitioned successively with petroleum ether
and EtOAc. The EtOAc extract (95 g) was separated using a silica gel column
chromatography (CC) (petroleum ether, EtOAc, MeOH v/v, gradient) to generate
seven fractions (Frs. 1−7). Frs. 2 (17 g) was applied to silica gel CC eluted with
petroleum ether-EtOAc (from 10:1 to 1:1) to afford four subfractions (2a–2d).
Subfraction 2b was further purified by using Semi-Preparative HPLC (CH3CN/H2O,
30 : 70 v/v) to obtain 1 (10 mg), 2 (23 mg) and 3 (50 mg). Compound 3 further
separated by using Semi-Preparative HPLC (CH3CN/H2O, 15 : 85 v/v, ULTRON
ES-OVM column) to obtain 3a (18 mg) and 3b (20 mg). Subfraction 2c were further
separated by Semi-Preparative HPLC (CH3CN/H2O, 35:65 v/v) to obtain 4 (11 mg), 5
(8 mg), 6 (16 mg) and 7 (13 mg). Compound 4 further separated by using
Semi-Preparative HPLC (CH3CN/H2O, 15 : 85 v/v, ULTRON ES-OVM column) to
obtain 4a (3 mg) and 4b (4 mg).
2.4. Spectroscopic data
Decumbic acid A (1): white powder; [α]24 +85 (c 1.2, MeOH); IR (KBr) νmax
D
1
13
1785, 1730 and 1210cm-1; H and C NMR: see Table 1; HRESIMS m/z 185.0808
[M - H]- (calcd for C9H13O4, 185.0814).
Decumbic acid B (2): white powder; [α]24 +21 (c 0.8, MeOH); IR (KBr) νmax
D
1
13
1782, 1722 and 1216cm-1; H and C NMR: see Table 1; HRESIMS m/z 185.0816
[M - H]- (calcd for C9H13O4, 185.0814).
(-)-Decumbic acid (3a): white powder; [α]24 -43 (c 1.5, MeOH); IR (KBr) νmax
D
4

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1
3422, 2962, 2878, 1741, 1665 and 1218cm-1; H and 13C NMR: see Table 1;
HRESIMS m/z 183.0646 [M - H]- (calcd for C9H11O4, 183.0657).
(-)-Dendrolactone (4a): white powder; [α]24D -108 (c 1.0, MeOH); IR (KBr) νmax
1
3425, 1778, 1616 and 1508cm-1; H and 13C NMR: see Table 1; HRESIMS m/z
279.0878 [M - H]- (calcd for C14H15O6, 279.0869).
(+)-Dendrolactone (4b): white powder; [α]24 +110 (c 0.8, MeOH); IR (KBr)
D
1
13
νmax 3424, 1778, 1617 and 1508cm-1; H and C NMR: see Table 1; HRESIMS m/z
279.0872 [M - H]- (calcd for C14H15O6, 279.0869).
Crystal data for decumbic acid (raceme 3): C9H12O4 M = 184.19; monoclinic,
space group P21/c; a = 4.93786 (12) Å, b = 19.0807 (4) Å, c = 10.3251 (2) Å, α = γ =
90.00° β = 101.961 (2)°, V = 951.69 (4) Å3, Z = 4, T = 293 (2) K, Dc = 1.285 g/cm3,
μ(Cu Kα) = 0.854 mm−1, F (000) = 392, 9323 reflections measured, 1517 independent
reflections (Rint = 0.0287). The final R1 values were 0.0365 [I > 2σ(I)]. The final wR2
(F2) values were 0.1028 [I > 2σ(I)]. The final R1 values were 0.0407 (all data). The
final wR2 (F2) values were 0.1075 (all data).
Crystal data for decumbic acid (raceme 4): C14H16O6 M = 280.27; monoclinic,
space group P21/c; a = 10.6264 (4) Å, b = 7.1627 (3) Å, c = 17.1423 (6) Å, α = γ =
90.00° β = 98.792 (3)°, V = 1289.44 (8) Å3, Z = 4, T = 293 (2) K, Dc = 1.444 g/cm3,
μ(Cu Kα) =0.960 mm−1, F (000) = 592, 12642 reflections measured, 1907 independent
reflections (Rint = 0.0333). The final R1 values were 0.0397 [I > 2σ(I)]. The final wR2
(F2) values were 0.1070 [I > 2σ(I)]. The final R1 values were 0.0488 (all data). The
final wR2 (F2) values were 0.1178 (all data).
Crystal X-ray diffraction data was collected on a Bruker APEX DUO
diffractometer with Cu Kα radiation (λ = 1.54184 Å). The structure was solved by
direct methods (SHELXS-97) and refined using full-matrix least-squares difference
Fourier techniques. Carbon and oxygen atoms were refined anisotropically. Hydrogen
atoms were either refined freely with isotropic displacement parameters or positioned
with an idealized geometry and refined riding on their parent C atoms. Crystals
suitable for X-ray diffraction (racemes 3 and 4) were obtained by slow evaporation of
a solution in MeOH/CHCl3 (10:1). Crystallographic data (excluding structure factors)
for racemes 3 and 4 have been deposited with the Cambridge Crystallographic Data

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Centre: CCDC reference number 1499064 and 1499065, respectively. This data can
be obtained, free of charge, from the Cambridge Crystallographic Data Centre via
http://www.ccdc.cam.ac. uk/data_request /cif.
Cells. HeLa, MCF-7 and A549 were provided by College of Pharmacy, Hebei
University and maintained in DMEM medium (Gibco) containing 5% fetal bovine
serum (Gibco) at 37°C in air with 5% CO2.
MTT Assays. Cytotoxic activity was evaluated by the MTT method, as
described previously[12]. In the MTT assay, drug stock solutions were prepared in
DMSO (stored at –70°C). Cells were harvested at the exponential growth phase and
seeded in flat-bottomed 96-well microtiter plates. The cell volume in each well was
180 μL, containing 104 cells per well. The plates were incubated overnight in a 5%
humidified CO2 incubator at 37°C. The compounds were then added to each well at
various concentrations using a constant volume of 20 μL. After 48 h incubation at
37°C in 5% CO2, 50 μL of MTT (1 mg/mL of PBS) was added to each well and again
incubated at 37°C for 4 h. Following incubation, the medium was replaced by DMSO
(150 µL) to dissolve the formazan crystals in each well. The plates were then read at
570 nm wavelength in an enzyme-labeled detector (Elx800, BioTek Instruments, Inc.).
IC50 values of the compounds in different cell lines were determined based on the
dose response curve. Epirubicin was used as a positive control.
Fungal Strains. Alternaria brassicicola, Phytophthora parasitica var.
nicotianae, Colletotrichum capsici, Bipolaris oryzae, Diaporthe medusaea nitschke,
Ceratocystis paradoxa moreau, Exserohilum turcicum, Pestallozzia theae and
Alternaria A. citri were grown on potato dextrose agar.
Antifungal Bioassay. The antifungal bioassays were conducted following the
National Center for Clinical Laboratory Standards (NCCLS) recommendations.
Targeted microbes (three to four colonies) were prepared from broth culture (28°C for
48 h), and the final spore suspensions of fungi were 104 mycelial fragments/mL. Test
compounds (1 mg/mL as the stock solution in DMSO and serial dilutions) were
transferred to a 96-well clear plate in triplicate, and the suspension of the test
6

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organisms was added to each well achieving a final volume of 200 μL. The plates
were stored in controlled-environment cabinets from 4 to 14 days, depending on the
assay, and mycelial growth was then assessed. After incubation, the absorbance at 492
nm was measured with a microplate reader. The MIC was defined as the lowest test
concentration that completely inhibited the growth of the test organisms. Prochloraz
was used as a positive control.
3. Results and discussion
Compound 1 was obtained as white powder, with the molecular formula C9H14O4
1
13
from HRESIMS data combined with H and C NMR spectroscopic data (Table 1).
The 1H NMR data revealed two methyl groups at δH 0.95 (t, J = 7.2 Hz) and 1.32 (d, J
= 7.2 Hz), one oxygenated methine signal at δH 4.71 (td, J = 8.4, 3.2 Hz), two methine
13
signals at δH 3.21 (dd, J = 9.2, 8.4 Hz) and 3.03 (m). The C NMR and 135 DEPT
data showed 9 resonances, including two carboxyl carbons (δC 177.8 and 174.6), one
oxygenated methine carbon (δC 77.4), two sp3 methine carbons (δC 51.8 and 36.7),
two methylene carbons (δC 33.28 and 19.1) and two methyl carbons (δC 14.6 and
13.8). These data closely resembled those of known compound 3 except for the
absence of two olefinic carbons (δC 147.1 and 139.9, C-2 and C-3) and the presence
of two sp3 methine carbons (δC 51.8 and 36.7) in 1. The 2D structure of 1 was thus
1
constructed. It was corroborated by the H-1H COSY and HMBC correlations. The
1H-1H COSY correlations of H-8 to H-2, H-2 to H-3, H-3 to H-4, H-4 to H-5, H-5 to
H-6 and H-6 to H-7 indicated the presence of the C-8/C-2/C-3/C-4/C-5/C-6/C-7
fragment in 1 (Fig. 2). The HMBC correlations from H-2/H-4 to C-9 indicated that
C-9 was linked to C-3. The HMBC correlations from H-8/H-3 to C-1 indicated that
C-2 was linked to C-1. The HMBC correlations from H-4 to C-1 indicated the
presence of the five-membered lactone ring. The relative configuration of 1 was
assigned on the basis of the NOESY experiment. The NOESY correlations of 8-Me
/H-3, 8-Me /H-4 and H-2/H-5 indicated that 8-Me, H-3 and H-4 should be placed on
the same face; H-2 and the propyl group should be placed on the other face (Fig 3).
Compound 1 has the (2R,3R,4R)-form based upon the positive optical rotation of 1
and the reported similar compounds in literatures [13,14]. Thus, the absolute
configuration of 1 was defined as (2R,3R,4R) (Fig. 4). Thus, compound 1 was
identified as a new butyrolactone derivative. And we named compound 1 as decumbic
acid A.

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Compound 2 was also obtained as white powder, with the molecular formula
1
C9H14O4 from HRESIMS data combined with H and 13C NMR spectroscopic data
(Table 1). The 1D NMR data were very similar to those of 1 except for the small
shifting of the chemical shift at C-2, C-3, C-4 and C-5 in 2 (Table 1). Detailed
1
analysis of 2D NMR (HSQC, H-1H COSY and HMBC) spectra confirmed that
compound 2 has the same 2D structure with 1. The relative configuration of 2 was
assigned on the basis of the NOESY experiment. The NOESY correlations of
8-Me/H-3, H-3/H-5 and H-2/H-4 indicated that 8-Me and the propyl group should be
placed on the same face; the carboxyl group, H-2 and H-4 should be placed on the
other face (Fig 3). Compound 2 has the (2S,3S,4R)-form based upon the positive of 2
and the reported similar compounds in literatures [13,14]. Therefore, decumbic acid B
(2) was identified as a new butyrolactone derivative.
Raceme 3 was obtained as colorless crystals. The 1D NMR data were same as
the decumbic acid (known compound 3b) [8]. However, 3 was determined as a
raceme by the crystal space group (Fig. 4). Furthermore, subsequent HPLC separation
on an ULTRON ES-OVM column (150 × 10 mm, 5 µm) was successful in resolving
two stereoisomes (3a and 3b), which were opposite in terms of the specific rotations
(3a, -43 and 3b, +44). 3b was a known compound with the (4R)-form ([α]24D +44). On
the contrary,, the absolute configuration of 3a was defined as (4S) ([α]24D -43) and we
named compound 3a as (-)-decumbic acid.
Raceme 4 was also obtained as colorless crystals. The 1D NMR data were very
similar to those of caruilignan C [15] except for the small shifting of the chemical
shift at C-2−6. Detailed analysis of 2D NMR (HSQC and HMBC) spectra confirmed
that compound 4 has the same 2D structure with caruilignan C. The relative
configuration of 4 was assigned on the basis of the crystal space group (Fig. 5).
Compound 4 as a raceme was also determined by the crystal space group.
(2S,5S,6R)-Form and (2R,5R,6S)-form were found in the crystal space. Furthermore,
subsequent HPLC separation on an ULTRON ES-OVM column (150 × 10 mm, 5 µm)
was also successful in resolving two stereoisomes (4a and 4b), which were opposite
8

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in terms of the specific rotations (4a, -108 and 4b, +110). The (2R,5R,6S)-form for 4b
was based upon the optical rotation. It’s [α]24 (+110) was very close to the reported
D
similar compound (+)-(7S,8R,8'R)-acuminatolide (+100.1) in literatures [16]. On the
contrary, compound 4a has the (2S,5S,6R)-Form. Thus, the absolute configuration of
4a and 4b was defined as (2S,5S,6R) and (2R,5R,6S), respectively. We named
compounds 4a and 4b as (-)- and (+)-dendrolactone, respectively.
The structures of known compounds 3b and 5–7 were identified by comparison
of their spectroscopic data with those in the literature.
Cytotoxic Activities. All compounds were tested for cytotoxic activities against
HeLa, MCF-7 and A549 cells (table 2). Compounds 1–7 showed moderate inhibitory
effects on HeLa, MCF-7 and A549 cells with IC50 values ranging from 15.3 to 30.0
μM. The IC50 values of other compounds higher than 40 μM were regarded as
inactive.
a Epirubicin was used as a positive control.
Antifungal Activities. All compounds were evaluated against the
phytopathogenic fungi and the results are shown in Table 3.
Acknowledgments
This work was supported by The Industry-University-Research Cooperation
Integration Program of Hainan Province (SQ20150CXY0064), the National Natural
Science Foundation of China (31360069), the Hainan province natural science
foundation of innovatie research team project (2016CXTD007), the Innovative
Research Topics of Hainan province Regular IHEs (Hyb2016-15).
Supplementary data
Supplementary matrial 1
Supplementary matrial 2
Supplementary figures

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Fig 1. Structures of compounds 1–7 isolated from D. nobile.
12

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Fig. 2. Key 1H-1H-COSY and HMBC correlations (H→C) of 1 and 2

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Fig. 3. Key NOESY correlations of 1 and 2
14

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Fig. 4. The crystal space group of the raceme 3

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Fig. 5. The crystal space group of the raceme 4
16

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Table 1. NMR Data for compounds 1, 2, 3a, 3b, 4a and 4b
1 (in CDCl3)
2 (in CDCl3)
3a/3b (in CDCl3)
4a/4b (in CDCl3)
position
δH
δC
δH
δC
δH
-
δC
δH
δC
1
2
-
177.8
-
177.7
172.9
139.9
-
178.2
46.2
3.03 (m) 36.7 3.00 (m) 39.9
-
3.46 (m)
4.39 (dd,
9.2, 8.8,
H-3β)
4.20 (dd,
9.2, 2.8,
H-3α)
2.70 (dd,
3.21 (dd,
3
4
51.8
11.2,
10.0)
54.0
-
147.1
70.2
9.2, 8.4)
4.51 (dd,
10.0, 6.8,
H-4β)
4.34 (dd,
10.0, 2.0,
H-4α)
4.71 (td,
8.4, 3.2)
4.51 (td,
8.8, 4.0)
5.13 (dd,
8.0, 2.4)
77.4
79.8
36.9
81.4
34.9
69.9
48.7
1.71/1.82
(m)
1.59/2.11
(m)
5
1.59 (m) 33.2
3.11 (m)
1.43/1.62
19.1
1.45/1.55
(m)
0.97 (t,
7.2)
6
7
18.7 1.44 (m) 18.3 4.59 (d, 6.8) 86.4
0.95 (t,
(m)
0.95 (t,
13.8
13.7
13.8
-
130.0
7.2)
7.2)
1.32 (d,
7.2)
1.33 (d,
7.2)
8 (8a/8b)
14.6
14.5
2.24 (s)
11.2
6.57 (s)
102.9
9 (9a/9b)
10
-
-
174.6
-
-
-
174.7
-
-
-
166.5
-
-
-
147.5
135.0
11
(11a/11b)
-
-
-
-
-
-
3.91 (s)
56.6

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Table 2. The cytotoxic Activities of Isolated Compounds
IC50 (μM)
Compounds
HeLa
25.3
MCF-7
24.3
A549
25.8
1
2
18.2
15.3
19.5
3a
20.2
18.9
20.8
3b
18.8
24.7
21.5
22.0
21.5
22.3
0.5
17.5
21.7
28.2
18.6
21.9
30.0
0.8
19.2
23.4
19.3
20.5
23.6
28.2
1.0
4a
4b
5
6
7
Epirubicin a
18

ACCEPTED MANUSCRIPT
Table 3. The Antifungal Activity of Isolated Compounds
MIC (μg/mL)
P.
C.
Cpds
A.
parasitica
var.
nicotianae
C.
B.
D.medusaea
Nitschke
E.
P.
A.
paradoxa
Moreau
brassicicola
capsici oryzae
turcicum theae citri
>400
50
100
>400
100
>400
>400
50
>400
>400
>400
>400
>400
>400
100
100
100
50
50
100
>400
>400
100
50
>400
50
50
>400
1
2
3a
3b
4a
4b
5
6
>400
>400
>400
>400
>400
>400
50
>400 >400
>400
>400
>400
>400
50
50
>400
12.5
>400
>400
>400
>400
>400
25
>400
>400
50
50
>400
>400 >400
50
25
50
50
>400
>400
50
100
50
>400
>400
100
>400
50
>400
>400
50
25
>400
12.5
100
>400
>400
100
>400
25
50
12.5
>400
25
7
Prochloraza
50
50
a Prochloraz was used as a positive control.

ACCEPTED MANUSCRIPT
Graphical abstract
20