药学学报 Acta Pharmaceutica Sinica 2015, 50 (2): 203 −206
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203 ·
A novel citrinin derivative from the marine-source
fungus Penicillium citrinum
NI Min1*, LIN Wei-ling1, YANG Peng2, MAO Shui-chun2
(1. Fujian Health College, Fuzhou 350101, China;
2. School of Pharmacy, Nanchang University, Nanchang 330006, China )
Abstract: A novel citrinin derivative, penicitrinol L (1), along with two known analogues, penidicitrinin B
(2) and pennicitrinone A (3) were isolated from the marine-source fungus Penicillium citrinum. The structure of
the new compound was elucidated by spectroscopic methods including one and two-dimensional NMR as well as
high-resolution mass spectrometric analysis. Furthermore, compound 1 showed modest cytotoxic activity
against HL-60 cell line and compound 3 showed weak cytotoxic activity against A375 cell line.
Key words: citrinin derivative; marine-derived fungus; secondary metabolite; cytotoxic activity
CLC number: R284.2
Document code: A
Article ID: 0513-4870 (2015) 02-0203-04
海洋来源真菌 Penicillium citrinum 中的一个新桔霉素衍生物
倪
敏 1*, 林伟铃 1, 杨
(1. 福建卫生职业技术学院, 福建 福州 350101;
鹏 2, 毛水春 2
2. 南昌大学药学院, 江西 南昌 330006)
摘要: 从一株海洋来源真菌 Penicillium citrinum 中分离得到了一个结构新颖的桔霉素衍生物 penicitrinol L
(1) 以及两个已知类似物 penidicitrinin B (2) 和 pennicitrinone A (3)。通过一维和二维核磁以及高分辨质谱等波谱
学方法确定了新化合物的结构。此外, 化合物 1 对人白血病 HL-60 细胞具有中等的抑制活性, 化合物 3 对人黑色
素瘤 A375 细胞具有较弱的抑制活性。
关键词: 桔霉素衍生物; 海洋真菌; 次级代谢物; 细胞毒活性
Malignancies are a kind of the most serious
diseases that damage human health in the modern world.
Although the efficacy of chemotherapy for the majority
of cancer types has improved over the last three decades,
the high toxic effects of chemotherapeutic drugs, causing
a severe reduction in quality of life, are still formidable
problems in clinical medicine [1]. Therefore, many
researchers have begun to investigate natural products,
due to their antitumor activities and low side-effects [2].
Citrinin derivatives are naturally occurring polyReceived 2014-08-21; Accepted 2014-09-24.
Project supported by the Scientific Research Foundation of Fujian Health
Department (2010-2-45) and the Scientific Research
Foundation of Fujian Education Department (JB13323).
*Corresponding author Tel / Fax: 86-591-22869830,
E-mail: [email protected]
ketone compounds from microbes, which have been
reported to possess many kinds of bioactivities, such
as enzyme inhibitory [3], antifouling [3], antioxidant [4],
antitumor [5–8] and antimicrobial [8] properties. Among
these bioactivities, the antitumor property is getting
the most attention. Penicillium citrinum is a rich
source of various citrinin derivatives [4–6]. Our previous
chemical investigation of P. citrinum resulted in the
isolation of a new citrinin derivative, namely, citrinacetal [9].
Our continuing search for bioactive compounds from
this organism has further resulted in the isolation of
another new derivative, namely, penicitrinol L ( 1),
along with two known analogues penidicitrinin B ( 2)[10]
and pennicitrinone A (3)[4] (Figure 1). In this paper,
we report the isolation, structural elucidation and
bioactivity of these metabolites.
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204 ·
Figure 1
药学学报 Acta Pharmaceutica Sinica 2015, 50 (2): 203 −206
Structures of compounds 1−3
Results and discussion
Compound 1, which is trivially named as penicitrinol
L, was obtained as yellow gum and was analyzed to
have the molecular formula C 18H20O4 through negative
high-resolution electrospray ionization mass spectroscopy
(HR-ESI-MS) (m/z: 299.126 4 [M−H]–, Calcd. for
C18H19O4: 299.128 3). Its NMR data (Table 1) revealed
eighteen carbon signals, including three methyls, eight
methines, and seven quaternary carbons.
Table 1 NMR data for 1 (500 MHz for 1H, 125 MHz for
DMSO-d6)
Position
δC
1
71.4
5.54 (1H, s)
3
71.2
3.48 (1H, m)
4, 11
1, 10, 11, 12
4
35.9
2.56 (1H, m)
3, 12
3, 9, 10, 11, 12
5
111.7
6
154.6
7
99.8
8
151.4
9
113.7
10
138.5
δH (J in Hz)
COSY
13
C,
HMBC (H→C)
3, 8, 9, 10, 1', 2', 6'
6.19 (1H, s)
5, 6, 8, 9
11
19.5
1.10 (3H, d, 6.4)
3
3, 4
12
19.4
1.05 (3H, d, 6.8)
4
3, 4, 10
13
10.8
1.97 (3H, s)
1'
133.0
H-11, and H-4 with H-3 and H-12, as well as the HMBC
correlations from H-1 to C-3, C-9 and C-10, H-3 to C-1
and C-10, and H-4 to C-9 and C-10 demonstrated the
existence of a multisubstituted dihydropyrane. The
HMBC correlations of H-13 with C-5, C-6 and C-10,
H-7 with C-5, C-6, C-8 and C-9, 6-OH with C-5, C-6
and C-7, as well as 8-OH with C-7, C-8 and C-9 linked
the second benzene ring to the dihydropyrane via C-9
and C-10. Furthermore, the HMBC correlations of
H-1 with C-1', C-2' and C-6', as well as H-2'/6' with C-1
linked first benzene ring to the dihydropyrane via C-1
and C-1'.
The relative configuration of 1 was assigned on
the basis of the NOESY correlations of H-1 and H-4
with H-11 as well as H-3 with H-12 as shown in Figure
2. The absolute configuration of 1 was determined by
comparison of its specific optical rotation value ([ α]D
+60.5) with those of penicitrinol C ([ α]D +33.2) [11] and
penicitrinol F ([α]D +24.8) [3], which suggested that the
absolute configuration of 1 was also 1R, 3R and 4S.
So, the structure of 1 was elucidated as shown in Figure
1 and named as penicitrinol L.
5, 6, 10
2'/6'
129.3
6.93 (2H, d, 8.5)
3'/5'
1, 4'
3'/5'
114.3
6.63 (2H, d, 8.5)
2'/6'
1', 4'
4'
156.2
4'-OH
9.21 (1H, s)
3', 4', 5'
6-OH
8.93 (1H, s)
5, 6, 7
8-OH
8.74 (1H, s)
7, 8, 9
The plane structure of 1 was revealed through
COSY and HMBC spectrum analyses (Table 1). The
COSY correlations of H-2'/6' with H-3'/5', as well as
the HMBC correlations from H-2'/6' to C-4', H-3'/5'
to C-1' and C-4', and 4'-OH to C-3', C-4' and C-5'
demonstrated the existence of a 1, 4-substituted benzene
ring. The COSY correlations of H-3 with H-4 and
Figure 2
Key NOESY correlations of compound 1
The cytotoxic effects of compounds 1−3 were
evaluated using the MTT method on the HL-60 and
A375 cancer cell lines [12–14]. Compound 1 showed
modest cytotoxic activity against HL-60 cell line with
the IC50 value of 22.7 μg·mL−1, while compound 3
NI Min, et al: A novel citrinin derivative from the marine-source fungus Penicillium citrinum
showed weak cytotoxic activity against A375 cell line
with the IC 50 value of 65.4 μg·mL−1.
Experimental
1
General experimental procedures
Optical rotations were obtained from a Shenguang
SGW-1 digital polarimeter. UV spectra were recorded
on a Shimadzu UV-2450 spectrophotometer. 1H NMR,
13
C NMR, DEPT spectra and 2D NMR were recorded
on a BRUKER BIOSPIN AVANCE III spectrometer
using TMS as the internal standard. ESI-MS were
obtained by an AGILENT 1200/Q-TOF 6510 LC mass
spectrometer. Semipreparative HPLC was performed
using an ODS column (ODS-A, 10 mm × 250 mm, 5 µm)
at 5 mL·min−1.
2 Fungal material
The fungus P. citrinum was isolated from marine
sediments collected from Langqi Island, Fujian, China.
It was identified according to its morphological characteristics and ITS by Beijing Sunbiotech Co., Ltd, and
preserved in our laboratory at −80 ℃. The producing
strain was prepared on Martin medium and stored at
4 ℃.
3 Fermentation and extraction
The fungus was cultured under static conditions at
28 ℃ for 30 days in 1 000 mL conical flasks containing
the liquid medium (400 mL/flask) composed of soytone
(0.1%), soluble starch (1.0%), and seawater (60%).
The fermented whole broth (80 L) was filtered through
cheesecloth to separate the supernatant from the mycelia.
The former was concentrated under reduced pressure
to about a quarter of the original volume and then
extracted three times with EtOAc to give an EtOAc
solution, while the latter was extracted two times with
acetone. The acetone solution was concentrated under
reduced pressure to afford an aqueous solution. The
aqueous solution was extracted two times with EtOAc
to give another EtOAc solution. Both EtOAc solutions
were combined and concentrated under reduced pressure
to give a crude extract (18.9 g).
4 Purification
The crude extract (18.9 g) was separated into
8 fractions on a silica gel column using a step gradient
elution of petroleum ether, CH 2Cl2, and MeOH.
Fraction 3 (3.1 g) eluted with petroleum ether/CH 2Cl2
(1∶3) was further purified on a silica gel column using
a step gradient elution of petroleum ether, CH 2Cl2, and
MeOH. Subfraction 3-2 (0.9 g) eluted with CH 2Cl2 was
further purified on a Sephadex LH-20 (CH 2Cl2/MeOH,
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205 ·
1 ∶ 1), a reversed-phase column (MeOH/H 2O, 3 ∶ 2)
and semipreparative HPLC (45% MeCN), yielding
compounds 1 (5.3 mg), 2 (4.8 mg), and 3 (8.1 mg).
Penicitrinol L (1): yellow gum; [α] 25
+60.5 (c 0.1,
D
MeOH); 1H and 13C NMR see Table 1; HR-ESI-MS m/z
299.126 4 [M−H]− (Calcd for C 18H19O4: 299.128 3); UV
λmax (CH3CN) nm (log ε): 284 (3.44).
5 Biological assay
Cytotoxic activity was evaluated by the MTT
method [11, 12]. The IC 50 values were obtained using the
Bliss method.
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