Nghiên cứu thành phần hóa học và khảo sát hoạt tính gây độc tế bào của các hợp chất phân lập từ vỏ cây dà quánh (ceriops decandra)

Received: 16 April 2020

Revised: 20 August 2020

Accepted: 25 August 2020

DOI: 10.1002/mrc.5091

LETTER - SPECTRAL ASSIGNMENT

Structure elucidation of two new diterpenes from
Vietnamese mangrove Ceriops decandra
Nguyen Van Thanh1
| Kieu Thi Phuong Linh1 | Pham Thanh Binh1
Nguyen Phuong Thao1 | Nguyen The Cuong2 | Tran Thi Bich Ha6 |

|

Nguyen Van Chien3 | Nguyen Quoc Trung4 | Vu Huy Thong5 |
Nguyen Xuan Cuong1 | Nguyen Hoai Nam1 | Chau Van Minh1
1

Department of Bioactive Natural Products, Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, Hanoi, Vietnam

2

Melinh Station for Biodiversity, Institute of Ecology and Biological Resourses, Vietnam Academy of Science and Technology, Hanoi, Vietnam

3

Department of Corrosion and Protection of Metals, Institute for Tropical Technology, Vietnam Academy of Science and Technology, Hanoi, Vietnam

4

Department of Chemistry and Catalytic Materials, Institute of Materials Science, Vietnam Academy of Science and Technology, Hanoi, Vietnam

5

Department of Basic Science, University of Fire Fighting and Prevention, Hanoi, Vietnam

6

Faculty of Quality Standards and Reference Substances, Institute of Drug Quality Control Ho Chi Minh city, Ho Chi Minh, Vietnam

Correspondence
Nguyen Van Thanh, Department of Bioactive Natural Products, Institute of Marine Biochemistry, Vietnam Academy of Science and Technology,
Hanoi, Vietnam.
Email: ,
Funding information
Vietnam Academy of Science and Technology, Grant/Award Number: TĐPCCC.04/18-20

1 | INTRODUCTION
Ceriops decandra (Griff.) W.Theob. (Rhizophoraceae), a
true mangrove plant, occur in Africa, Australia, South
Asia, and many countries of Southest Asia [1]. The bark
of C. decandra is an Indian folk medicine used for the
treatment of diarrhea, amoebiasis, hemorrhage, and
malignant ulcers [2]. The leaf extract has been reported
to exhibite antinociceptive activity [3]. Previous phytochemical investigations on this plant resulted in the isolation of lupane- and ursane-type triterpenoids from the
leaf [4], beyerane-, pimarane-, kaurane-, and abietanetype diterpenoids from the roots [1, 5–7], and abietaneand podocarpane-type diterpenoids from the barks [2,
8]. In an ongoing search for bioactive natural products

from mangroves [9, 10], we report here the isolation,
structure elucidation, and cytotoxicity assay of two new
diterpenes, ceridecandrin A (1) and B (2), from
C. decandra stem barks. Their structures were elucidated
by analysis of HR-QTOF-MS and 1D and 2D NMR spectroscopic data. It should be noted that compound 1 is the
Magn Reson Chem. 2020;1–6.

first example of a 8,15-epoxypimarane-type diterpenoid
possessing a 14,16-ether bridge, and compound 2 is the
second member of a rare class of 10,19-epoxyrosane-type
diterpenoid (Figure 1).

2 | R E S U L T S AN D D I S C U S S I O N
Ceridecandrin A (1) was obtained as white, amorphous
powder. Its molecular formula was determined to be
C20H32O3 on the basis of 13C NMR data (Table 1) and
HR-QTOF-MS ion peaks at m/z 321.2416 [M + H]+
(calcd for C20H33O3+, 321.2424), m/z 343.2244 [M + Na]+
(calcd for C20H32O3Na+, 343.2244), and m/z 338.2687
[M + NH4]+ (calcd for C20H36O3N+, 338.2690), indicating
five index of hydrogen deficiency. The 13C NMR and
HSQC spectra disclosed 20 carbon signals, corresponding
to four methyls, seven sp3 methylenes (including one
oxymethylene at δC 74.2), five sp3 methines (consisting
three bearing oxygen at δC 70.7, 84.1, and 85.1), and four
sp3 quartenary carbons (one oxygenated at δC 89.0). The

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1


2

VAN THANH ET AL.

FIGURE 1

Structures of Compounds

1 and 2

TABLE 1

1

H (500 MHz) and 13C NMR (125 MHz) data for 1 (in CDCl3) and 2 (in CD3OD)
1

2

Position

δC

δ H (J in Hz)

δC

δ H (J in Hz)

1

40.5

β 1.63 (1H, overlapped)
α 0.85 (1H, overlapped)

31.2

β 1.77 (1H, overlapped)
α 1.69 (1H, overlapped)

2

18.5

β 1.48 (1H, overlapped)
α 1.37 (1H, overlapped)

29.8

β 1.95 (1H, dddd, 4.5, 7.0, 14.0, 16.5)
α 1.71 (1H, overlapped)

3

41.8

β 1.41 (1H, overlapped)
α 1.17 (1H, ddd, 3.5, 13.5, 13.5)

75.4

3.56 (1H, br d, 4.0)

4

32.6

-

49.0

-

5

45.2

1.28 (1H, overlapped)

45.6

2.20 (1H, dd, 5.0, 14.0)

6

26.6

1.69 (2H, overlapped)

18.4

β 1.47 (1H, overlapped)
α 1.42 (1H, overlapped)

7

70.7

4.11 (1H, br s)

27.7

α 1.62 (1H, overlapped)
β 1.26 (1H, overlapped)

8

89.0

-

32.9

1.67 (1H, overlapped)

9

49.8

1.25 (1H, overlapped)

40.0

-

10

36.4

-

91.2

-

11

19.3

β 1.75 (1H, dddd, 7.0, 14.0, 14.0, 14.0)
α 1.44 (1H, overlapped)

32.4

α 1.78 (1H, overlapped)

β 1.25 (1H, overlapped)

12

31.2

β 2.00 (1H, dddd, 1.5, 7.0, 14.0)
α 1.59 (1H, overlapped)

33.6

β 1.55 (1H, ddd, 3.5, 13.5, 14.0)
α 1.24 (1H, overlapped)

13

46.3

-

37.1

-

14

85.1

3.61 (1H, br s)

42.0

α 1.27 (1H, overlapped)
β 1.23 (1H, overlapped)

15

84.1

4.01 (1H, br s)

152.3

5.82 (1H, dd, 10.5, 17.5)

16

74.2

α 3.93 (1H, dd, 1.0, 8.0)
β 3.78 (1H, br d, 8.0)

109.3

α 4.93 (1H, dd, 1.0, 17.5)
β 4.87 (1H, overlapped)

17

18.4

1.06 (3H, s)

22.7

1.02 (3H, s)

18

33.5

0.91 (3H, s)

16.1

0.97 (3H, s)

19

22.2

0.86 (3H, s)

77.4

β 3.68 (1H, d, 8.5)
α 3.64 (1H, d, 8.5)

20

15.7

0.90 (3H, s)

15.5

0.96 (3H, s)

presence of five oxygenated carbons and the absence of
any unsaturated carbon, in combination with the molecular formula C20H32O3, suggested that compound 1 was a
pentacyclic diterpenoid with two ether linkages. The 1H

NMR spectrum exhibited four singlet methyl signals at
δH 0.86 (s, H-19), δH 0.90 (s, H-20), δH 0.91 (s, H-18), and
δH 1.06 (s, H-17), three oxygenated methine protons at δH
3.61 (br s, H-14), δH 4.01 (br s, H-15), and δH 4.11 (br s,


VAN THANH ET AL.

3

H-7), and a pair of oxymethylene protons at δH 3.78 (br d,
J = 8.0 Hz, H-16β)/3.93 (dd, J = 1.0, 8.0 Hz, H-16α).
Careful interpretation of correlations observed in the
COSY and HMBC spectra revealed that the planar structure of 1 (Figure 2) was similar to that of 8,15Repoxypimaran-16-ol [7] and ent-8,15R-epoxypimaran-16-ol
[11], except for the presence of an additional hydroxyl
group at C-7, and an ether bridge between C-14 and C-16
in 1. Indeed, the HMBC correlation from H-15 (δH 4.01)
to C-8 (δC 89.0) established the 8,15-epoxy linkage. The

hydroxyl group was located at C-7 due to COSY crosspeaks of H-5/H-6/H-7, as well as the HMBC correlations
from H-5 (δH 1.28) to C-7 (δC 70.7). The connection of
C-14 and C-16 through an oxygen atom was confirmed by
HMBC correlation from H-14 (δH 3.61) to C-16 (δC 74.2).
The relative configuration of 1 was determined by the
analysis of coupling constant and NOESY spectrum. The
small vicinal coupling constant of H-7 (δH 4.11, br s) and
the large vicinal coupling constant of H-3α (δH 1.17,
J = 3.5, 13.5, 13.5 Hz) and H-11β (δH 1.75, J = 7.0, 14.0,
14.0, 14.0 Hz) suggested the equatorial orientation of H-7
and the axial orientations of both H-3α and H-11β. The
NOESY correlations of H-3α/H3-18, H3-18/H-5, H-5/H-

FIGURE 2
(

Key COSY (
) of 1 and 2

) and HMBC correlations

1α, H-1α/H-2α, H-1α/H-9, H-9/H-11α, H-9/H-12α, H-9/
H-14, H-14/H3-17, H3-20/H-1β, H3-20/H-2β, H3-20/H11β, and H-2β/H319 confirmed the structure of pimarane
diterpenoid skeleton of 1 [5] and α-configuration of H-14
(Figure 3). The trans-fusion between the B and C rings of
pimarane scaffold, together with NOESY cross-peaks of
H-16β/H-7 and H-16α/H3-17, indicated that H-15 was
β-orientation. Thus, structure of 1 was determined as
8,15:14,16-diepoxy-7α-hydroxy-pimarane.
Ceridecandrin B (2) was isolated as white, amorphous powder. The molecular formula of 2, C20H32O2,

was deduced from the 13C NMR data, and [M + Cl]−
ion peaks at m/z 339.2071 and 341.2059 with a ratio of
3:1 (calcd for C20H32O2Cl−, 339.2096, and 341.2067) in
the HR-QTOF-MS, corresponding to five index of
hydrogen deficiency. The 13C NMR and HSQC spectra
revealed the presence of 20 carbons, including four
nonprotonated carbons (one bearing oxygen at δC
91.2), nine methylenes (one sp2 carbon at δC 109.3 and
one oxygenated at δC 77.4), four methine (one sp2 carbon at δC 152.3 and one oxymethine at δC 75.4), and
three methyls. The presence of two sp2 carbons and
three oxygenated carbons, along with the HR-MS data
analysis, indicated that 2 was a tetracyclic diterpenoid
with an ether bridge. The 1H NMR spectrum showed
signals for one monosubstituted double bond at δH
5.82 (dd, J = 10.5, 17.5 Hz, H-15), δH 4.87 (H16β)/4.93 (dd, J = 1.0, 17.5 Hz, H-16α), one
oxymethine group at δH 3.56 (br d, J = 4.0 Hz, H-3),
one oxymethylene group at δH 3.64 (d, J = 8.5 Hz, H19α)/3.68 (d, J = 8.5 Hz, H-19β), and three singlet
methyls at δH 0.96 (s, H-20), δH 0.97 (s, H-18), and δH
1.02 (s, H-17). Detailed analysis of COSY and HMBC
correlations (Figure 2) revealed that the planar structure of 2 was closely related to that of euphomianol A
[12], a rosane-type diterpenoid with a 10,19-oxygen
bridge, except for the absence of a hydroxy group at
C-5 in 2. This was confirmed by the COSY cross-peaks
of H-5/H2-6/ H2-7/H-8/H2-14 and the HMBC correlations from H2-19 (δH 3.64/3.68) to C-3 (δC 75.4), C-4
(δC 49.0), C-5 (δC 45.6), C-10 (δC 91.2), and C-18 (δC
16.1).
According to the coupling constant values between
H-3 and H-2β (J = 4.5 Hz), between H-2β and H-1α
(J = 14.0 Hz), and between H-12β and H-11α
(J = 13.5 Hz), H-3 was assigned as equatorial orientation,

and both H-2β and H-12β were in axial orientation. The
NOESY correlations of H-3/H-2β, H-2β/H-19α, H-2β/H1β, H-19β/H-6β, H-6β/H3-20, and H3-20/H-12β revealed
that these protons were cofacial and they were in
β-configuration (Figure 3). By contrast, NOE cross-peaks
of H-1α/H-5, H-5/H-6α, H-5/H-8, H-8/H3-17, and H3-17/
H-12α demonstrated that these protons were α-oriented.


4

VAN THANH ET AL.

FIGURE 3
(

TABLE 2

3 | M A T E R I A L S A ND M E T HO D

Cytotoxicity of Compounds 1 and 2
IC50 (μg/mL)

3.1 | General

Compound

SK-LU-1

HepG2

MCF7

1

58.36 ± 5.83

60.28 ± 2.77

44.17 ± 3.23

22.10 ± 2.65

27.53 ± 1.53

20.02 ± 1.55

0.41 ± 0.05

0.47 ± 0.03

0.35 ± 0.04

2
Ellipticine

a

Key NOESY correlations
) of 1 and 2

a

Positive control substance.

Therefore, the structure of 2 was identified as
10,19-epoxy-3α-hydroxy-rosane.
Compounds 1 and 2 were evaluated for cytotoxicity
against three cancer cell lines: SK-LU-1, HepG2, and
MCF7. Ellipticine was used as a positive control. The
results showed that both compounds exhibited weak
cytotoxicity against three cell lines with IC50 values in
the range of 20.02 to 60.28 μg/mL (Table 2).

Optical rotations were measured using a JASCO P2000 polarimeter (JASCO, Oklahoma, OK, US). The
HR-QTOF-MS were recorded on an Agilent 6530
Accurate-Mass Q-TOF LC/MS system (CA, USA). Column chromatography (CC) was performed on silica gel
(Kieselgel 60, 70–230 mesh and 230–400 mesh, Merck,
Darmstadt, Germany) and YMC*GEL resins (ODS-A,
12 nm S-150 μm, YMC Co., Ltd.). Analytical thin layer
chromatography (TLC) systems were performed on precoated silica gel 60 F254 (1.05554.0001, Merck) and RP18 F254S plates (1.15685.0001, Merck), and the isolated
compounds were visualized by spraying with 10%
H2SO4 in water and then heating for 1.5–2 min. All
procedures were carried out with solvents purchased
from commercial sources that were used without further purification.


VAN THANH ET AL.

3.2 | NMR spectra
NMR spectra were recorded on a Bruker Ascend

500/Avance III HD spectrometer at temperature of
303 K. Compounds 1 and 2 were dissolved in CDCl3 and
CD3OD, respectively, and transferred into 5-mm NMR
tubes. 1H and 13C chemical shifts (δ) were referenced to
tetramethylsilane (TMS) at 0.00 ppm. Coupling constants
(J) were expressed in Hertz (Hz). The 1H NMR experiments were carried out with spectrometer frequency
(SF) = 500.20 MHz, spectral width in Hz
(SWH) = 10,000 Hz, acquisition time (AQ) = 3.2768 s,
number of scans (NS) = 16, relaxation delay (D1) = 1.0 s,
90 pulse width (P1) = 10.00 μs, Fourier transform size
(SI) = 65,536, and line broadening (LB) = 0.3 Hz. The
13
C NMR spectrum was acquired with SF = 125.77 MHz,
SWH = 31,250 Hz, AQ = 1.048 s, NS = 1,536, D1 = 2.0 s,
P1 = 10.00 μs, SI = 32,768, and LB = 1.0 Hz. The 2D
NMR spectra were recorded using Bruker library pulse
sequence condition as follows: for HSQC, NS = 4,
D1 = 2.0 s, SWH = 6009.615 Hz, time domain data points
(TD) = 2048, AQ = 0.1704 s; for HMBC, NS = 16,
SWH = 3012.048 Hz, AQ = 0.3399 s, D1 = 1.3 s,
TD = 2048; for COSY, SWH = 3067.485 Hz, TD = 2048,
NS = 2, AQ = 0.3338 s, D1 = 1.8 s; and for NOESY,
SWH = 2994.012 Hz, TD = 2048, NS = 8, AQ = 0.3420 s,
D1 = 1.8 s.

3.3 | Plant material
The stem barks of C. decandra were collected from Ca
Mau province, Vietnam, in July 2018 and identified by
Dr. Nguyen The Cuong. A voucher specimen (PCCC01-CD) was deposited in the Department of Bioactive
Natural Products, Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST).

3.4 | Extraction and isolation
The air-dried, powdered stem barks of C. decandra
(9 kg) were extracted with EtOAc three times at room
temperature in ultrasonic bath. The crude residue
(100 g) was separated on a silica gel CC and eluted
with n-hexane/EtOAc mixtures of increasing polarity
(100:0 ! 0/100) to obtained 12 fractions, E1–E12.
Fraction E5 was divided into eight fractions, E5A–
E5H, by a silica gel CC (n-hexane/EtOAc, 15/1). Fraction E5H was subjected to a silica gel CC (nhexane/EtOAc, 2.5/1) to give three fractions, E5H1–
E5H3. Fraction E5H1 was further chromatographed on
a silica gel CC (n-hexane/acetone, 2/1) to yield two

5

fractions, E5H1A and E5H1B. Fraction E5H1B was
separated on a silica gel CC (CH2Cl2/EtOAc, 4/1),
followed by a silica gel CC (n-hexane/acetone, 10/1) to
obtained three fractions, E5H1B1–E5H1B3. Fraction
E5H1B1 was purified by a silica gel CC (nhexane/acetone, 10/1) to yield Compound 1 (1.7 mg).
Compound 2 (1.3 mg) was purified by YMC CC
(MeOH/H2O, 6/1) from fraction E5H1B2.

3.5 | Physical and spectroscopic data
Ceridecandrin A (1): white, amorphous powder, α25
D
+52.8 (c 0.8, MeOH); 1H NMR (CDCl3, 500 MHz) and
13
C NMR (CDCl3, 125 MHz) spectral data, see Table 1;
HR-QTOF-MS: m/z 321.2416 [M + H]+ (calcd for

C20H33O3+, 321.2424), m/z 343.2244 [M + Na]+ (calcd for
C20H32O3Na+, 343.2244), and m/z 338.2687 [M + NH4]+
(calcd for C20H36O3N+, 338.2690).
Ceridecandrin B (2): white, amorphous powder, α25
D
+60.7 (c 0.4, MeOH); 1H NMR (CD3OD, 500 MHz) and
13
C NMR (CD3OD, 125 MHz) spectral data, see Table 1;
HR-QTOF-MS: m/z 339.2071, and 341.2059 [M + Cl]−
(calcd for C20H32O2Cl−, 339.2096, and 341.2067).

3.6 | Cytotoxicity assays
The cytotoxicity assays for ceridecandrin A (1) and B (2)
were performed using the SRB method [13] as a reviously
reported protocol [14].
The raw NMR data files of the spectra including the
relevant fid(s) are given in the Supporting Information.
ACKNOWLEDGEMENTS
This research was supported by the Vietnam Academy
of
Science
and
Technology
(project
code:
TĐPCCC.04/18-20). We thank Dr. Dang Vu Luong,
Institute of Chemistry, VAST, for NMR measurement
and Prof. Do Thi Thao, Institute of Biotechnology,
VAST, for cytotoxicity assays.
PE ER RE VI EW

The peer review history for this article is available at
/>ORCID
Nguyen Van Thanh
5999

/>
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SU PP O R TI N G I N F O RMA TI O N
Additional supporting information may be found online
in the Supporting Information section at the end of this
article.
How to cite this article: Van Thanh N,
Linh KTP, Binh PT, et al. Structure elucidation of
two new diterpenes from Vietnamese mangrove
Ceriops decandra. Magn Reson Chem. 2020;1–6.
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