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Phytomedicine
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Short communication
Ginseng and Velvet Antler Products Quality Supervision & Test Center Certicated by Ministry of Agriculture, Jilin Agricultural University, 130118 Changchun, PR China
College of China Medicinal Material in Jilin Agriculture University, 130118 Changchun, PR China
The Afliated Hospital of Jilin Agricultural University, 130118 Changchun, PR China
a r t i c l e
i n f o
Keywords:
Curcuma wenyujin
Chaetomium globosum L18
Endophytic fungi
Metabolites
Antifungal activity
Cytotoxic activity
a b s t r a c t
An endophytic fungus, strain L18, isolated from the medicinal plant Curcuma wenyujin Y.H. Chen et C.
Ling was identied as Chaetomium globosum Kunze based on morphological characteristics and sequence
data for the internal transcribed spacer (ITS-5.8S-ITS2) of the nuclear ribosomal DNA. A new metabolite
named chaetoglobosin X (1), together with three known compounds erogosterol (2), ergosterol 5,8peroside (3) and 2-methyl-3-hydroxy indole (4), were isolated from C. globosum L18. Their structures
were elucidated by spectroscopic methods including NMR, UV, IR and MS data and comparison with
published data. Chaetoglobosin X (1) is hitherto unknown, whereas 2-methyl-3-hydroxy indole (4) is
reported for the rst time as a fungal metabolite, and erogosterol (2) and ergosterol 5,8-peroside (3)
are known fungal metabolites previously identied in other genera. Chaetoglobosin X (1) exhibited a
broader antifungal spectrum and showed the strongest cytotoxic activity against H22 and MFC cancer
cell lines.
2011 Elsevier GmbH. All rights reserved.
Introduction
Fungal endophytes are microorganisms that colonize living,
internal tissues of plants without causing any immediate, overtly
negative effects (Aly et al. 2008). Endophytic fungi are ubiquitous in plant species and are mutualistic to their host (Li et al.
2007). Some can produce similar or identical biologically active
constituents as the host, such as taxol (Stierle et al. 1993). In
addition, many fungal endophytes produce secondary metabolites
and some of these compounds exhibit antifungal and antibacterial
activity that strongly inhibits the growth of other microorganisms
(Gunatilaka 2006). Other secondary metabolites display antibiotic
activity against pathogens and tumor cells to different degrees (Li
et al. 2000). Recently, endophytic fungi have been recognized as
important sources of a variety of structurally novel active secondary
metabolites with anticancer, antimicrobial and other biological
activities (Yang et al. 2011).
Curcuma is a well-known genus used in traditional Chinese
medicines. Curcuma wenyujin Y.H. Chen et C. Ling, called Erzhu in
Chinese, contains volatile components that are pharmacologically
active and often used for their anticancer activity. The principal
components include -elemene, curzerene, curzerenone, germacrone, curcumol, isocurcumenol and curcumenol (Mau et al. 2003).
Corresponding author. Tel.: +86 0431 8451 7904; fax: +86 0431 8451 0955.
E-mail address: yanhong-w@163.com (X. Wu).
0944-7113/$ see front matter 2011 Elsevier GmbH. All rights reserved.
doi:10.1016/j.phymed.2011.10.011
365
366
Table 1
1
H (600 MHz) and 13 C (150 MHz) NMR spectroscopic data for compound 1 in CDCl3 and DMSO-d6 (J values in Hz).
Position
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
H (, CDCl3 )
3.41(m,1H)
2.28(m,1H)
1.41(m,2H)
8.59 (s,1H)
6.46(d,15.6,1H)
6.81 (s,1H)
6.63 (dd,7.8,15.6, 1 H)
3.55(m,1H)
0.85(t,7.2,3H)
1.04(d,6.6, 3H)
1.62(s,3H)
0.97(s,3H)
0.98(d,1.8,3H)
13
C (, DMSO-d6 )
183.6
51.0
201.3
38.6
28.9
140.6
126.3
151.2
120.7
168.4
105.7
107.8
147.4
87.8
110.5
157.5
160.9
70.0
21.9
25.7
19.5
13.2
12.0
d, J = 6.6), 0.78 (3H, s); 13 C NMR (CDCl3 , 150 MHz) 135.4 (C-6),
135.2 (C-22), 132.3 (C-23), 130.7 (C-7), 82.1 (C-5), 79.4 (C-8), 66.4
(C-3), 56.2 (C-17), 51.7 (C-14), 51.1 (C-9), 44.6 (C-13), 42.8 (C-24),
39.7 (C-20), 39.3 (C-12), 36.9 (C-4), 36.9 (C-10), 34.6 (C-1), 33.0 (C25), 30.1 (C-2), 28.6 (C-16), 23.4 (C-13), 20.9 (C-21), 20.6 (C-15),
19.9 (C-26), 19.6 (C-27), 18.2 (C-19), 17.5 (C-28), 12.9 (C-18); ESIMS: m/z 429.3236 [M] + , 451.3242 [M+Na] + , 427.3275 [MH] ;
(calcd. for C28 H44 O3 , 428.3251).
2-Methyl-3-hydroxyl indole (4)
Light yellow powder, m.p. 133135 C; UV (MeOH) max (log )
257 (4.02), 276 (1.34), 301 (4.04) nm; 1 H NMR (CDCl3 , 600 MHz)
6.34 (1H, d, J = 1.8), 6.26 (1H, d, J = 1.8), 5.99 (1H, s), 5.90 (1H, s), 2.08
(3H, s); 13 C NMR (CDCl3 , 150 MHz) 145.8 (C-2), 138.5 (C-9), 134.3
(C-3), 128.1 (C-6), 116.1 (C-4), 116.0 (C-5), 111.1 (C-7), 104.4 (C3), 9.5 (C-10); ESI-MS: m/z 146.1370 [MH] ; (calcd. for C9 H9 ON,
147.1376).
Antifungal assay
The plant pathogens E. turcicum, F. oxysporium f. sp. cucumeris,
C. lunata, F. graminearum, and F. moniliforme were used in the
antifungal assay. Cultures were obtained from the Plant Pathology
Laboratory of Jilin Agricultural University. The minimum inhibitory
concentrations (MICs) were performed using a modied version
of the 2-fold serial dilution method (Pongcharoen et al. 2008).
DEPT
HMBC
C
CH
C
CH
CH2
C
C
CH
CH
C
CH
C
CH
C
C
C
C
CH
CH3
CH3
CH3
CH3
CH3
C-2
C-1, C-4
C-2, C-4, C-5
C-2, C-5, C-6
C-4, C-19, C-20
C-4, C-5, C-8, C-20, C-21
C-5, C-8, C-9, C-20, C-21
C-9, C-21
C-8, C-11
C-8, C-9, C-11, C-12
C-9, C-12,
C-11, C-22,
C-11, C-12, C-22
C-22
C-2, C-23
C-2, C-23
C-4, C-5
C-5
C-8
C-12
C-2
Cytotoxicity bioassays
MFC (gastric cancer cells in mice) and H22 (hepatic cancer
cells in mice) cell lines were obtained from the Basic Medical
Experimental Animals Laboratory of Jilin University. Cytotoxicity of
compounds 14 were tested against each cell line using the microculture tetrazolium assay as described previously (Ashour et al.
2006). All experiments were carried out in triplicate and repeated
three times, and the target compounds were diluted into different
concentrations and calculate their respective inhibition ratio. Their
inhibition ratio was used as the Y-axis and the concentration as
the X-axis by drawing standard curve for determination of IC50.
As controls, media with 0.1% EGMME/DMSO were included in the
experiments.
367
Fig. 2. Structure of the isolated compound 1 (chaetoglobosin L) and its HMBC correlations and HMQC correlations.
MS data and comparison with published data (Li et al. 2003; Gao
et al. 2000; Xie et al. 2007; Ding and Yang 1999).
Compounds 14 were evaluated against the plant pathogens
Exserohilum turcicum, Fusarium oxysporum f. sp. Cucumerinum,
Curvularia lunata, Fusarium graminearum, and Fusarium moniliforme using the 2-fold serial dilution method. Compounds 2, 3,
and 4 showed no signicant antibiotic activity against the ve fungal strains. Compound 1 exhibited the highest antifungal activity
against E. turcicum, F. oxysporium f. sp. cucumeris and C. lunata with
a MIC value of 3.125 g/ml and showed moderate antifungal activity against F. graminearum and F. moniliforme with a MIC value of
6.25 g/ml.
Biological evaluation of compounds 14 was also carried out
using MFC (Gastric cancer cells in mice) and H22 (hepatic cancer cells in mice) cell lines. The four compounds exhibited clear
differences in cytotoxic activity toward MFC and H22 cells. Compound 1 displayed the strongest cytotoxicity against H22 cells
(IC50 3.125 g/ml) and exhibited moderate cytotoxicity against
MFC cells (IC50 6.25 g/ml), whereas the other compounds were
inactive against H22 and MFC cells.
Conclusions
The isolated endophytic fungus strain L18 was identied as
Chaetomium globosum from its morphological characteristics and
rDNA ITS sequence analysis. A novel compound 1 (chaetoglobosin
X) was isolated from C. globosum L18 together with three known
compounds 2, 3 and 4 (erogosterol, ergosterol 5,8-peroside and
2-methyl-3-hydroxy indole). Compound 4 was isolated from a fungal source for the rst time. Additionally, antimicrobial activity and
cytotoxic activity tests shows compound 1 presents a broader antifungal spectrum and a more effective antifungal activity and shows
an obvious inhibitory effect on MFC and H22 cell lines. Thus C.
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