Drimane-type Sesquiterpenes with Dioxabicyclooctane Skeleton from the
Fruiting Bodies of Nigrofomes melanoporus and Their Cytotoxicity
Hsiu-Hui Chan,1,† Shin-Hun Juang,2,3,† Tran Dinh Thang,4,† Min-Yu Chen,3
Ping-Chung Kuo,5 Mei-Lin Yang,6 Nguyen Thi Ngan,4 Nguyen Ngoc Linh,4 and
Tian-Shung Wu 1,6,7,*
1
Chinese Medicine Research and Development Center, China Medical University
Hospital, Taichung 404, Taiwan.
2
Department of Medical Research, China Medical University Hospital, Taichung 404,
Taiwan
3
Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung
404, Taiwan.
4
Department of Chemistry, Vinh University, Vinh City, Nghean province, Vietnam.
5
Department of Chemistry and Biotechnology, National Formosa University, Yunlin
632, Taiwan.
6
7
Department of Chemistry, National Cheng Kung University, Tainan 701, Taiwan.
Department of Pharmacy, China Medical University, Taichung 404, Taiwan.
* Address for correspondence
Professor Tian-Shung Wu
Department of Chemistry
National Cheng Kung University
1 Ta-Hsueh Road, Tainan 70101, Taiwan.
1
Tel: 886-6-2757575, ext. 65333.
Fax: 886-6-2740552.
E-mail: tswu@mail.ncku.edu.tw.
†
These authors provided equal contributions to this work
2
Abstract
Two new drimane-type compounds, nigrofomin A (1) and B (2) possessing rare
dioxabicyclooctane moiety, were purified from the fruiting bodies of Nigrofomes
melanoporus. Their structures were determined using 1D, 2D-NMR and HR-ESI-MS
spectroscopic analyses. In addition, 1 was established by X-ray crystallographic
studies. Both nigrofomin A (1) and B (2) exhibited cytotoxicity of acute T-cell
leukemia Jurkat, human nasopharyngeal carcinoma (NPC-TW01), and lung cancer
(NCI-H661) cells with IC50 values in the range of 99.44-246.32 μM. Furthermore, the
effects of 1 and 2 on cell-cycle progression of Jurkat cells displayed a
concentration-dependent accumulation in the G0/G1 phase.
Keywords: Nigrofomes melanoporus, Polyporaceae, drimane, sesquiterpenes,
cytotoxicity
Polyporaceae, including Taiwanofungus, Ganoderma, and Poria genus, is a famous
fungus family belonged to the Basidiomycota. The constituents of their fruiting bodies
have been evidenced to be rich sources of biological activities, such as
anti-inflammatory [1, 2], antihypertensive [3, 4], antitumor [5, 6, 7, 8, 9], and
immunomodulatory and adjuvant activities [10]. We had previously reported
cytotoxicity of benzenoids and triterpenoids from Taiwanofungus camphorates [11,
3
12], triterpenoids from Ganoderma lucidum [13], and the chemical constituents from
Cordyceps sinensis [14]. In our continuing program aimed to search for the potential
antitumor agents from natural sources, the fruiting bodies of Nigrofomes melanoporus
(Mont.) Murrill collected in Vietnam were subjected into the chemical investigation
and cytotoxicity studies. In the present paper, two new drimane-type sesquiterpenes
with rarely appeared dioxabicyclooctane skeleton, nigrofomin A (1) and B (2) were
identified with 1D, 2D-NMR and HR-ESI-MS spectroscopic analyses. To the best of
our knowledge, this is the first report of the phytochemical constituents and biological
activities of N. melanoporus.
The air-dried fruiting bodies of the N. melanoporus (1350 g) were extracted with the
solvent mixture of chloroform and methanol at ambient temperature. The
concentrated extract was subjected to silica gel column chromatography and further
resolved with the aid of high performance liquid chromatography to afford
nigrofomin A (1) and B (2) (Fig 2.).
Nigrofomin A (1) was obtained as optically active and colorless prisms, mp 208-210
ºC. The HRESI-MS of 1 displayed a quasimolecular ion peak at m/z 317.1002, which
is consistent with a pseudo-molecular formula of C15H18O6Na (calcd. 317.1001) and
suggested seven degrees of unsaturation. The IR spectrum showed strong absorption
4
peaks for hydroxyl (3360 cm-1), a ketone carbonyl (1771 cm-1), and a lactone carbonyl
(1709 cm-1). The
13
C-NMR, DEPT-135, and HSQC analyses of 1 displayed the
presences of 15 carbon signals, including an aliphatic ketone carbon
210.9), a
lactonic carbon (δ 174.0), two olefinic carbons
s (δ 73.0,
carbons
52.5, 40.8 and 23.6), and two methyls (δ 25.4 and 26.5) carbons. In the 1H-NMR
spectrum of 1, two methyl singlets [δ 1.31 (H-13, s), 1.15 (H-14, s)], an olefinic
proton [δ 6.02 (H-7, br s)], an oxygen-bearing methylene [δ 4.79 (1H, H-12, d, J=10.4
Hz), 4.50 (1H, H-12, d, J=10.4 Hz)], three methylenes [δ 3.53 (H-1α, d, J=13.2 Hz),
2.19 (H-1β, d, J=13.2 Hz), 3.08 (1H, H-3α, d, J=3.8 Hz), 1.91 (1H, H-3β, d, J=3.8
Hz), and 2.60 (2H, H-6, br s)], and an acetal signal [δ 5.62 (H-11, s)] coupled with 13C
signals at δ 25.4, 26.5, 126.3, 40.8, 52.5, 73.0 and 32.6, indicated the occurrence of a
rare dioxabicyclooctane skeleton for a drimane-type sesquiterpene [15, 16]. The
HMBC spectrum of 1 exhibited the crosspeaks of H-3 (δ 3.10, 1.91) and C-2 (δ
210.9)/C-4 (δ 45.7)/C-5 (δ 76.7)/C-13 (δ 25.4)/C-14 (δ 26.5), and H-13 (δ 1.31)/H-14
(δ 1.15) and C-3 (δ 52.5)/C-4 (δ 45.7)/C-5 (δ 76.7). The 2J and 3J HMBC correlations
indicated that the quaternary C-4 was substituted with two methyl groups. The
downfield shifts of C-5 and C-9 were due to the hydroxy substitutions. Furthermore,
the dioxabicyclooctane moiety of 1 connected at C-8, -9, and -10 was established by
5
the HMBC correlations of H-11 (δ 5.62) with C-8 (δ 136.3)/C-12 (δ 73.0)/C-15 (δ
174.0), H-12 (δ 4.79, 4.50) with C-7 (δ 126.3)/C-8 (δ 136.3)/C-9 (δ 82.1), and H-1 (δ
3.53, 2.19) with C-2 (δ 210.9)/C-5 (δ 76.7)/C-9 (δ 82.1)/C-10 (δ 60.1)/C-15 (δ 174.0).
The key NOE correlations in 1 (Fig 2.) were observed between H-14 (δ 1.15) and
H-3α (δ 1.91)/H-3β (δ 3.08)/H-6 (δ 2.60), H-13 (δ 1.31) and H-3α (δ 1.91), and H-6 (δ
2.60) and H-7 (δ 6.02). The relative configuration of 1 was further confirmed by an
X-ray diffraction crystallographic study (Fig. 3). Therefore, the sterochemical
structure of 1 was established as shown, and this compound has been named
nigrofomin A (1).
Nigrofomin B (2) was isolated as an optical active colorless powder and mp 213-215
ºC. The IR absorption bands at 3368, 1771 and 1701 cm-1 indicated the presence of
hydroxyl, ketone and lactone carbonyl groups, respectively. The 1H-NMR spectrum
of 2 was very similar to those of 1 except for the upfield shifts of H-1 (δ 2.27, d,
J=2.0 Hz), H-3δ 2.20, dd, J=12.8, 2.0 Hz and H-13 (δ 1.21, s) and presence of
H-5 signal at δ 3.19 (1H, d, J=10.8 Hz). The HMBC correlations between H-5 (δ 3.19)
and C-3 (δ 56.7), C-9 (δ 80.8), and C-15 (δ 175.0) revealed that a hydroxy group was
absent in C-5 of 2, supported by comparison the molecular formulas of 1 (C15H18O6)
and 2 (C15H18O5). In the NOESY spectrum of 2, the NOE correlations between H-11
(δ 5.67) and H-3α (δ 2.20)/H-12α (δ 4.79), H-5 (δ 3.19) and H-3α, H-3(δ 2.58) and
6
H-14 (δ 1.19), H-3α and H-13 (δ 1.21), and H-3α (δ 2.20)/H-6α (δ 2.49) and H-13,
constructed the relative configuration of 2 which was the same as those of 1 (Fig. 2).
Based on the above observations, the chemical structure of nigrofomin B (2) was
deduced as shown.
To assess the growth inhibitory activity of nigrofomin A (1) (purity > 96%) and B (2)
(purity > 91%) towards tumor cell lines, three different cells from different organs
including acute T-cell leukemia Jurkat, human nasopharyngeal carcinoma
(NPC-TW01), and lung cancer (NCI-H661) were used. Compounds 1 and 2 treatment
showed inhibition of Jurkat cell lines IC
50
of 125 M and 99 M, respectively.
However, NPC-TW01 and NCI-H661 cells are less susceptible to 1 and 2 treatment
with the IC50 values ranged from 159 to 238 M for 1 and 188 to 246 M for 2,
respectively. .Furthermore, our results showed that only acute T-cell leukemia cells
are sensitive to both 1 and 2 treatment with IC50 around 100 M. Our results clearly
demonstrated that significant increase of G0/G1 population with concomitant loss of
the S phases when Jurkat cells treated with both 1 and 2 for 48 hours were noticed.
The accumulation of G0/G1 population increased with treatment dosage increase,
suggested that the cell cycle arrest phenomena by 1 and 2 is in the concentration
dependent manner.
7
This is the first report on the phytochemical study and biological activities of the
Nigrofomes genus. The present study demonstrates that this genus fragment is a
potential source of drimane-type sesquiterpenes possessed rarely occurred
dioxabicyclooctane fragment. Two new compounds, nigrofomin A (1) and B (2)
showed slightlycytotoxicity against the acute T-cell leukemia Jurkat, NPC-TW01 and
NCI-H661 cell lines, with IC50 values ranging from 99.44 to 246.32 μM.
Materials and Methods
The basidiomycete Nigrofomes melanoporus (Mont.) Murrill. was collected at Pumat
National Park of Nghean Province, Vietnam, in November 2009 and identified by Dr.
Ngo Anh, Department of Biology, Hue University. The voucher specimen
(Vin-TSWu 200901) was deposited at the Herbarium of Department of Biology, Vinh
University.
The air-dried fruiting bodies (1350 g) were extracted three times with CHCl3/MeOH
(1 : 1, v/v) at ambient temperature. After removal of the solvent by evaporation in
vacuo, the brown residue (78.0 g) was subjected to silica gel column chromatography
(1kg, 80 × 8 cm), eluting with a hexane–acetone step gradient system (100:0, 50:1,
20:1, 10:1, 2:1, 1:1 , each 1.0 L) to result in 10 fractions. Fraction 7 (5.4 g) was
purified with the aid of silica gel column chromatography (200 g, 40 × 5 cm) eluting
8
with a CHCl3–MeOH step gradient system (100:0, 50:1, 10:1, 2:1, 1:1, each 250 mL)
to afford the sesquiterpene-rich subfraction. This subfraction (74 mg) was further
resolved by RP-HPLC (H2O: MeOH=65:35, v/v) using 5-C18-MS-II Waters column
(20 × 250 mm) in flow rate of 5.0 mL/min and detecting at 197 nm to yield
nigrofomin A (1) (13.2 mg) and B (2) (8.7 mg). The purity of compounds 1 and 2
were analyzed by reversed-phase HPLC (LichroCART 254-4, Purospher STAR
RP-8e, 5 μ m).
Supplementary data
The experimental procedure and the full characterization of compounds 1 and 2 are
available associated with this article can be found. The cytotoxicity assay is also
available as Supporting Information.
Acknowledgement
This study was supported by the National Science Council (Taiwan) and NAFOSTED
(104.01-2010.27) (Vietnam), and in part by Taiwan development of Health Cancer
Research Center of Excellence (DOH100-TD-B-111-004), DMR-96-082 (CMUH)
and DMR-101-105 (CMUH).
Conflict of Interest
The authors have no conflict of interest to report.
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Figure 1. The structures of nigrofomin A (1) and B (2).
H
O
O
11
9
15
O
O
H
12
1
2
O
O
O
O
8
10
OH
3
5
4
6
OH
14
OH
7
H
13
1
2
13
Figure 2. Key NOE correlations of 1 and 2.
11
12
15
11
1
15
1
2
3
3
7
3
13
12
3
5
5
6
7
13
14
6
6
14
1
2
14
Figure 3. Single-crystal X-ray structure of 1.
15
16