A new prenylated indole diketopiperazine alkaloid from Eurotium cristatum

Article abstract of DOI:10.3390/molecules191117839

A new prenylated indole diketopiperazine alkaloid, cristatumin F (1), and four known metabolites, echinulin (2), dehydroechinulin (3), neoechinulin A (4) and variecolorin O ( 5), were isolated from the crude extract of the fungus Eurotium cristatum. The structure of 1 was elucidated primarily by NMR and MS methods. The absolute configuration of 1 was assigned using Marfey's method applied to its acid hydrolyzate. Cristatumin F (1) showed modest radical scavenging activity against DPPH radicals, and exhibited marginal attenuation of 3T3L1 pre-adipocytes.

Full text of DOI:10.3390/molecules191117839

Molecules 2014, 19, 17839-17847; doi:10.3390/molecules191117839
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
A New Prenylated Indole Diketopiperazine Alkaloid from
Eurotium cristatum
1
,2
1
1
1
1
1,3
1
Xianwei Zou , Ying Li , Xiaona Zhang , Qian Li , Xuan Liu , Yun Huang , Tao Tang ,
2
2
1,
Saijing Zheng , Weimiao Wang and Jintian Tang *
1
Key Laboratory of Particle & Radiation Imaging, Ministry of Education,
Department of Engineering Physics, Tsinghua University, Beijing 100084, China;
E-Mails: zouxw000@126.com (X.Z.); liying_90126@126.com (Y.L.);
zxn_1228@sina.com (X.Z.); yaoyaoldlq@163.com (Q.L.); liux_54@163.com (X.L.);
sungood591@sina.com (Y.H.); tao_tang1@126.com (T.T.)
2
Shanghai Tobacco Group Corporation, Shanghai 200082, China;
E-Mails: zhengsj@sh.tobacco.com.cn (S.Z.); zdsys@sh.tobacco.com.cn (W.W.)
3
Beijing Longcredit Essence Biotechnology Co. Ltd, Beijing 100085, China
*
Author to whom correspondence should be addressed; E-Mail: tangjt@mail.tsinghua.edu.cn;
Tel.: +86-10-6279-6769; Fax: +86-10-8289-4558.
External Editor: Derek J. McPhee
Received: 11 September 2014; in revised form: 27 October 2014 / Accepted: 29 October 2014 /
Published: 3 November 2014
Abstract: A new prenylated indole diketopiperazine alkaloid, cristatumin F (1), and four
known metabolites, echinulin (2), dehydroechinulin (3), neoechinulin A (4) and
variecolorin O (5), were isolated from the crude extract of the fungus Eurotium cristatum.
The structure of 1 was elucidated primarily by NMR and MS methods. The absolute
configuration of 1 was assigned using Marfey’s method applied to its acid hydrolyzate.
Cristatumin F (1) showed modest radical scavenging activity against DPPH radicals, and
exhibited marginal attenuation of 3T3L1 pre-adipocytes.
Keywords: Eurotium cristatum; diketopiperazine; radical scavenging activity

Molecules 2014, 19
. Introduction
Prenylated indole diketopiperazines are an important class of compounds with diverse chemical
17840
1
structures and biological activities [1]. Prenylated indole alkaloids, generally characterized by a
reverse isoprenic chain in the C-2 position of the indole nucleus, are often found in fungal sources.
This group of alkaloids are an important class of molecules possessing a variety of biological
activities, including antibacterial [2,3], immunosuppressive [4,5], brine shrimp inhibition [3], radical
scavenging [6,7], UV-A protecting [7,8], and cytotoxicity properties [9]. Chinese fuzhuan brick tea is a
unique microbially fermented tea characterized by a period of fungal growth, especially of the fungus
Eurotium cristatum, during its manufacturing process [10]. Biologically active secondary metabolites
have been isolated from the same fungus, including diketopiperazines with antibacterial and
insecticidal activity [3], eurocristatine, a diketopiperazine dimer from the marine sponge-associated
E. cristatum KUFC 7356 [11], and benzaldehyde derivatives with in vitro anti-cancer antivity against
human tumor cell lines [12,13]. During our ongoing search for new bioactive natural products from
unique fungal species, a subculture of an isolate of E. cristatum, a fungus isolated from a sample of
fuzhuan brick tea, was grown in solid-substrate fermentation culture. Fractionation of its organic
solvent extract led to the isolation of a new prenylated indole diketopiperazine alkaloid, named
cristatumin F (1), together with several known metabolites, including echinulin (2) [4],
dehydroechinulin (3) [9], neoechinulin A (4) [8] and variecolorin O (5) [14] (Figure 1). Details of the
isolation, structure elucidation, and biological activities of these compounds are reported herein.
Figure 1. Chemical structures of compounds 1–5.
O
O
O
1
1
NH
2
7
5
22
NH
O
NH
O
1
0
8
1
9
23
12
5
3a
3
14
20
2
HN
HN
HN
5
O
7a
N1
H
N
N
7
17
H
H
19
28
30
32
1
2
3
O
O
NH
O
NH
O
OH
HN
HN
N
N
H
H
5
4
2
. Results and Discussion
Cristatumin F (1) was obtained as a colorless powder with a molecular formula of C31
H
43
N
3
O
2
(
indicating 12 degrees of unsaturation), determined by HRESIMS. The IR spectrum of 1 showed
−1
−1
absorption bands for amine (3459 and 3435 cm ), carbonyls (1676 cm ), and aromatic functionalities
−1
1
13
(
3200 and 1446 cm ) in the molecule. Analysis of the H, C (APT experiment), and HSQC NMR
spectroscopic data of 1 revealed the presence of eight methyl groups, four methylene units, eight
methines, 11 quaternary carbons, 14 aromatic/olefinic carbons (six of which are protonated), and two

Molecules 2014, 19
17841
1
amide/carboxyl carbons. These data, together with three exchangeable protons accounted for all H and
13C-NMR resonances for 1, and are consistent with the molecular formula C
. Interpretation
31
H
43
N
3
O
2
1
1
of the H– H COSY NMR data of 1 identified four isolated proton spin systems corresponding to the
C-4–C-29 (via C-5 and C-7), C-16–C-17, C-8–NH-14 (via C-9), and NH-11–C-22 (via C-12)
fragments. HMBC correlations from CH
C-27, CH -31 and CH -32 to C-29 and C-30, as well as correlations H-29 to C-28, C-31, and C-32
established the two prenyl subunits (C-23–C-27 and C-28–C-32). While those from CH -18 and
CH -19 to C-15 and C-16, H-17 to C-15 and C-16, and from H-16 to C-18 and C-19 completed
3
-26 and CH
3
-27 to C-24 and C-25, H-24 to C-23, C-26, and
3
3
3
3
the C-15–C-19 substructure of 1. The tetrasubstituted indole moiety of 1 was deduced by HMBC
correlations from H-4 to C-3, C-6, C-7a, and C-23; from H-6 to C-4, C-7a, C-23, and C-28; and from
1
1
NH-1 to C-2, C-3, and C-3a, together with the H– H COSY correlation between H-4 and H-6. HMBC
cross-peaks from H -23 to C-4, C-5, and C-6, from H -28 to C-6 and C-7a indicated that C-23 and
C-28 were attached to C-5 and C-7, respectively. HMBC correlations from H-16, CH -18, and CH -19
to C-2 led to the connection of C-2 to C-15. The prenylated indole substructure was established based
2
2
3
3
1
1
on above analysis and those relevant H– H COSY correlations (Figure 2).
Figure 2. Selected 2D NMR correlations for 1.
1
13
Interpretation of the H, C, and 2D-NMR spectra, especially HMBC, revealed the peptidic nature
of 1. A diketopiperazine ring was suggested by the characteristic NMR data of two amide carbonyls
(
C-10, δ
C
168.6; C-13, δ
C
166.5) and two methines (C-9, δ
/δ
H C
4.42/53.6; C-12, δ
H
/δ 3.87/60.8).
C
HMBC correlations from H-9 to C-10, NH-11 to C-9 and C-13, H-12 to -C-13, and NH-14 to C-9,
C-10, and C-12 clearly suggested the existence of the diketopiperazine system in 1. The linkage of the
prenylated indole core with the diketopiperazine part through the methylene bridge was revealed by
1
13
the HMBC correlations from H -8 to C-3 and C-9. Comparison of the H and C-NMR spectroscopic
2
data of 1 with those of 2 revealed their structural similarities. The molecular weight of 1 is 28 mass
units more than that of 2, and this number corresponds to the difference in mass between the valine
(
Val) and the alanine (Ala) residues, suggesting that 1 is a diketopiperazine congener to 2 with an Ala
1
1
unit replaced by a Val unit in the 2,5-diketopeperazine moiety. Analysis of the H– H COSY and
HMBC data of 1 further confirmed the presence of a Val residue. Key HMBC correlations from
NH-11, H-12 and H-20 to C-13, from NH-14 to C-10 and C-12, and from NH-11 to C-9 established the
linkage between C-10, C-12 and C-13 (Figure 2). On the basis of these data, the gross structure of
cristatumin F was established as shown in 1.

Molecules 2014, 19
17842
Compounds 2–5 were identified as echinulin (2) [4], dehydroechinulin (3) [9], neoechinulin A (4) [8]
and variecolorin O (5) [14], respectively, by comparison of the NMR and MS data with reported
spectroscopic data.
Marfey’s method [15,16] was applied to determine the absolute configuration of the Val (C-12)
residue resulting from acid hydrolysis of cristatumin F (1). The 1-fluoro-2,4-dinitrophenyl-5-L-alanine
amide (FDAA) derivatives of the acid hydrolyzate of 1 and the authentic D- and L-valines were
subjected to HPLC-MS analysis. HPLC analysis of FDAA derivatives of the acid hydrolyzates of 1
gave the same retention time as the sample prepared from authentic D-Val. Therefore, C-12 was
assigned the R absolute configuration. The relative configuration of 1 could not be directly assigned by
selective NOE difference experiments (mixing time of 300 ms) due to the lack of enhancement
bewteen H-9 and H-12. However, from biogenetic considerations, C-9 in 1 presumably has the same
absolute configuration as in 2, which was also indirectly supported by the lack of NOE enhancement
2
D
5
between H-9 and H-12. In addition, 1 displayed positive optical rotation ([α]
+11.3), while 2 showed
2
5
20
D
negative value ([α]
D
−23.2 (lit. [α]
−39.1) [3]), both determined in CHCl3. The absolute configuration
of 1 was tentatively assigned as 9 S and 12 R based on the above results. Collectively, these data
permitted assignment of structure 1 to the new natural product, cristatumin F. The new compound 1
displayed modest radical scavenging activity against DPPH, with an IC50 value of 53.6 μM (ascorbic
acid as a positive control, IC50 15 μM), and showed marginal cell prolification inhibition (20.6%
inhibition) against 3T3L1 preadipocytes when tested at 200 μM.
3. Experimental Section
3.1. General
Optical rotations were measured on an Anton Paar MCP 200, and UV data were obtained on a Yoke
UV756CRT spectrophotometer. IR data were recorded using a Nicolet Magna-IR 750 spectrophotometer.
1H and 13C-NMR data were acquired with Bruker Avance-500 spectrometer using solvent signals
(CDCl
3
: δ
H
7.26/δ 77.6) as references. The HSQC and HMBC experiments were optimized for 145.0
C
and 8.0 Hz, respectively. HRESIMS data were obtained using an Agilent Accurate-Mass-Q-TOF
LC/MS 6520 instrument equipped with an electrospray ionization (ESI) source. The fragmentor and
capillary voltages were kept at 125 and 3500 V, respectively. Nitrogen was supplied as the nebulizing
and drying gas. The temperature of the drying gas was set at 300 °C. The flow rate of the drying gas
and the pressure of the nebulizer were 10 L/min and 10 psi, respectively. All MS experiments were
performed in positive ion mode. Full-scan spectra were acquired over a scan range of m/z 100–1000 at
1
.03 spectra/s.
3
.2. Fungal Material
The culture of E. cristatum was isolated from a sample of fuzhuan brick tea collected from Yiyang
city, Hunan Province, People’s Republic of China, in May 2012. Sequence analysis of the ITS region
of the ribosomal DNA suggested highly similarity (98%) with GenBank sequences from the fungus of
E. cristatum EN220 and E. cristatum NRRL 4222. The isolate was identified by one of the authors
(
T.T.) based on morphology and sequence (GenBank Accession No. KM521200) analysis of the ITS

Molecules 2014, 19
17843
region of the rDNA and assigned the accession number JH-01 in J.T.’s culture collection at Tsinghua
Universtiy, Beijing. The fungal strain was cultured on slants of potato dextrose agar (PDA) at 25 °C
3
for 10 days. Agar plugs were cut into small pieces (about 0.5 × 0.5 × 0.5 cm ) under aseptic conditions
and 15 of these pieces were used to inoculate in three Erlenmeyer flasks (250 mL), each containing
50 mL of media (0.4% glucose, 1% malt extract, and 0.4% yeast extract); the final pH of the media
was adjusted to 6.5 and sterilized by autoclave. Three flasks of the inoculated media were incubated at
2
5 °C on a rotary shaker at 170 rpm for five days to prepare the seed culture. Spore inoculum was
6
prepared by suspension in sterile, distilled H
2
O to give a final spore/cell suspension of 1 × 10 /mL.
Fermentation was carried out in forty Fernbach flasks (500 mL), each containing 80 g of rice. Distilled
O (120 mL) was added to each flask, and the contents were soaked overnight before autoclaving at
5 psi for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of the
spore inoculum and incubated at 25 °C for 40 days.
H
2
1
3.3. Extraction and Isolation
The fermented rice substrate was extracted with MeOH (5 × 500 mL), and the organic solvent was
evaporated to dryness under reduced pressure to afford a crude extract (230.0 g), which was then
suspended in water (700 mL) and successively partitioned with petroleum ether, EtOAc and n-BuOH
to give solvent dried fractions PE (4.0 g), EA (12.0 g), NBA (9.0 g), respectively.
A portion of PE (4.0 g) was subjected to silica gel VLC using petroleum ether–EtOAc gradient
elution. The fraction (84.3 mg) eluted with 40% EtOAc was separated by Sephadex LH-20 column
chromatography (CC) eluting with 1:1 CH
2
Cl –MeOH and six fractions 1–6 were collected. Fraction 2
2
was purified by semipreparative RP HPLC (YMC-Pack ODS-A column; 5 μm; 10 × 250 mm;
8
1
5%–98% MeOH in H
2
O for 30 min; 2 mL/min) to afford 1 (3.2 mg, t
R
20.5 min) and 3 (1.5 mg, t
R
8.5 min). A portion of EA (12.0 g) fractionated by silica gel VLC using petroleum ether–EtOAc
gradient elution. The fraction (1.86 g) eluted with 40%–70% EtOAc was chromatographed on a silica
gel (300–400 mesh) column (4.5 × 35 cm) eluting with petroleum ether–Me CO (9:1–1:9). The
fraction (67 mg) eluted with 60% Me CO was separated by Sephadex LH-20 CC eluting with MeOH,
and then the subfractions were purified by RP HPLC (80%–95% MeOH in H O for 30 min; 2 mL/min)
to afford 2 (3.2 mg, t 22.5 min). The fraction (197 mg) eluted with 50% Me CO was separated by
Sephadex LH-20 CC eluting with MeOH, and further purification of the resulting subfractions by RP
HPLC (55%–70% MeCN in H O for 30 min; 2 mL/min) to afford 4 (5.0 mg, t 27.0 min) and 5 (4.5 mg,
2
2
2
R
2
2
R
t
R
25.0 min)
3.4. Spectral Data
2
D
5
Cristatumin F (1): colorless powder; [α]
+11.3 (c 0.3, CHCl ); UV (MeOH) λmax (log ε) 228 (3.57)
3
nm; IR (neat) νmax 3459, 3435, 3200 (br), 3087, 3058, 2965, 2926, 2874, 1676, 1467, 1446, 1377,
−
1
1
1
346, 1102 cm ; H-, 13C-NMR (APT), and HMBC data see Table 1; HRESIMS m/z 490.3434
+
[
M + H] (calcd for C31
H
44
N
3
2
O , 490.3428).

Molecules 2014, 19
Table 1. NMR spectroscopic data of cristatumin F (1) in CDCl
17844
3
(δ in ppm).
a, Mult.
δ
b
(J in Hz)
HMBC (H→C)
No.
δ
C
H
1
-NH
2
8.08, br s
C-2, C-3, C-3a
141.5, qC
103.8, qC
128.9, qC
115.1, CH
133.9, qC
122.9, CH
123.4, qC
132.2, qC
3
3
a
4
7.17, br s
6.83, br s
C-3, C-6, C-7a, C-23
C-4, C-7a, C-23, C28
5
6
7
7
a
8
29.7, CH
2
3.67, dd (14.7, 3.6)
C-2, C-3, C-3a, C-9
C-2, C-3, C-3a, C-9, C-10
C-3, C-8, C-10
3
.20, dd (14.7, 11.7)
9
53.6, CH
168.6, qC
4.42, dd (11.7, 3.6)
1
0
1
1
1-NH
6.01, br s
3.87, br s
C-9, C-13
1
1
2
3
60.8, CH
166.5, qC
C-13, C-21, C-22
4-NH
5.72, br s
C-9, C-10, C-12
1
1
1
5
6
7
39.0, qC
145.8, CH
6.11, dd (17.4, 10.6)
5.19, d (17.4)
C-2, C-15, C-18, C-19
C-15, C-16
112.2, CH
2
5
.17, d (10.6)
1.53, s
C-15, C-16
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
27.8, CH
3
3
C-2, C-15, C-16, C-19
C-2, C-15, C-16, C-18
C-12, C-13, C-21, C-22
C-12, C-20, C-22
C-12, C-20, C-21
C-4, C-5, C-6, C-25
C-23, C-26, C-27
27.7, CH
1.53, s
32.4, CH
2.39, m
18.7, CH
16.1, CH
34.6, CH
3
1.05, d (7.0)
0.94, d (6.8)
3
2
3.41, d (7.2)
124.5, CH
131.6, qC
5.37, t (7.2, 7.2)
17.9, CH
25.8, CH
31.4, CH
3
3
2
1.83, s
1.76, s
C-24, C-25, C-27
C-24, C-25, C-26
C-6, C-7a, C-30
C-28, C-31, C-32
3.55, d (7.0)
5.45, t (7.0,7.0)
122.9, CH
132.9, qC
17.9, CH
3
1.89, s
C-29, C-30, C-32
C-29, C-30, C-31
25.7, CH
3
1.76, s
a: Recorded at 125 MHz; : Recorded at 500 MHz.
b
3
.5. Determination of the Absolute Configuration of Val in 1
A solution of compound 1 (0.5 mg) in 6 N HCl (1 mL) was heated at 105 °C for 24 h. Upon
removal of excess HCl under vacuum, the hydrolyzate was placed in a 1 mL reaction vial and treated
with 1% solution of 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA, 100 μL) in acetone
followed by 1.0 M NaHCO (40 μL). The reaction mixture was heated at 45 °C for 1.5 h and then
3

Molecules 2014, 19
17845
cooled to RT. The mixture was acidified with 2.0 N HCl (20 μL). Likewise, standard D- and L-valine
were derivatized separately. The derivatized hydrolyzate and standard amino acids were subjected to
®
reversed-phase HPLC analysis (DIONEX Acclaim 120 C18 column; 5 μm; 4.6 mm × 250 mm;
1
mL/min) at 35 °C using the following gradient program: solvent A, H
2
O (0.1% CH COOH); solvent
3
B, MeCN; linear gradient, 15%–45% of B in A over 45 min with UV detection at 340 nm. The
retention times for FDAA derivatives of standard L-valine, cristatumin F hydrolysate, and standard
D-valine were 33.49, 41.60, and 41.51 min, respectively.
3
.6. DPPH Radical Scavenging Assay
DPPH assays were carried out according to the method previously described [17], with slight
modifications. In a 96-well microplate, sample at different concentrations in absolute EtOH (50 μL)
was added into wells containing 0.02 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) in EtOH (150 μL)
and mixed well. Reaction mixtures was incubated at 37 °C for 30 min. Absorbance was measured at
5
10 nm by a microplate reader, and percent inhibition was calculated. IC50 values express
the concentration of sample required to scavenge 50% of the DPPH free radicals. Ascorbic acid
was used as a positive control.
3
.7. Cell Viability Assay
The effect of cristatumin F (1) on cellular viability was assessed by using the MTT assay [18,19].
T3L1 cells were seeded at a density of 2000 cells per well in 96-well microtiter plates (Costar,
3
Cambridge, MA, USA) and allowed to adhere. Cells were treated with different concentrations of
nordihydrocapsaicin or compound 1 for 24, 48, or 72 h. At each time point, the wells were washed
three times with warm phosphate buffer solution (PBS) and incubated again for another 4 h with
DMEM containing 5 mg/mL of MTT. After removing the culture medium, 150 μL DMSO was added
to dissolve the precipitates and the resulting solution was measured for absorbance at 490 nm using an
ELISA reader (iMark, Bio Rad 680, Hercules, CA, USA).
4
. Conclusions
Cristatumin F (1) is a new member of prenylated indole diketopiperazine-type of metabolites, and
the presence of a Val unit in 1 is unprecedented. In this work, the discovery of the new indole
diketopiperazine alkaloid from E. cristatum further expanded the structural diversity of the secondary
metabolites produced by this fungal species.
Acknowledgments
We gratefully acknowledge financial support from the National Natural Science Foundation of
China (21402104 and 81102889), the Project funded by China Postdoctoral Science Foundation
(2014T70068 and 2013M530603), and the Opening Fund of Cigarette Smoke Key Laboratory of
China Tobacco (K2013-1-044).

Molecules 2014, 19
17846
Author Contributions
X.-W.Z. and J.-T.T. designed the study; Y.L., X.-N.Z., Q.L., and T.T. performed the experimental
work; X.-W.Z. prepared the manuscript; X.L., Y.H., S.-J.Z., and W.-M.W. contributed helpful
discussion and scientific advice during the preparation of manuscript. All authors discussed and
approved the final version of manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References
1.
2.
3.
Li, S.M. Prenylated indole derivatives from fungi: Structure diversity, biological activities,
biosynthesis and chemoenzymatic synthesis. Nat. Prod. Rep. 2010, 27, 57–78.
Naik, C.G.; Devi, P.; Rodrigues, E. Chrysogenazine Obtained from Fungus Penicillium chrysogenum.
U.S. Patent 20,050,143,392, 30 June 2005.
Du, F.Y.; Li, X.M.; Li, C.S.; Shang, Z.; Wang, B.G. Cristatumins A‒D, new indole alkaloids from
the marine-derived endophytic fungus Eurotium cristatum EN-220. Bioorg. Med. Chem. Lett.
2
012, 22, 4650–4653.
4
.
.
Fujimoto, H.; Fujimaki, T.; Okuyama, E.; Yamazaki, M. Immunomodulatory constituents from
an Ascomycete, Microascus tardifaciens. Chem. Pharm. Bull. 1999, 47, 1426–1432.
Ravikanth, V.; Niranjan Reddy, V.L.; Ramesh, P.; Prabhakar Raoa, T.; Diwan, P.V.; Khar, A.;
Venkateswarlu, Y. An immunosuppressive trytophan-derived alkaloid from Lepidagathis cristata.
Phytochemistry 2001, 58, 1263–1266.
5
6
7
8
.
.
.
.
Wang, W.L.; Lu, Z.Y.; Tao, H.W.; Zhu, T.J.; Fang, Y.C.; Gu, Q.Q.; Zhu, W.M. Isoechinulin-type
alkaloids, variecolorins A‒L, from Halotolerant Aspergillus variecolor. J. Nat. Prod. 2007, 70,
1
558–1564.
Li, Y.; Li, X.F.; Kang, J.S.; Choi, H.D.; Son, B.W. New radical scavenging and ultraviolet-A
protecting prenylated dioxopiperazine alkaloid related to isoechinulin A from a marine isolate of
the fungus Aspergillus. J. Antibiot. 2004, 57, 337–340.
Li, Y.; Li, X.F.; Kim, S.K.; Kang, J.S.; Choi, H.D.; Rho, J.R.; Son, B.W. Golmaenone, a new
diketopiperazine alkaloid from the marine-derived fungus Aspergillus sp. Chem. Pharm. Bull.
2
004, 52, 375–376.
9
1
1
Yan, H.J.; Li, X.M.; Li, C.S.; Wang, B.G. Alkaloid and anthraquinone derivatives produced by
the marine-derived endophytic fungus Eurotium rubrum. Helv. Chim. Acta 2012, 95, 163–168.
0. Xu, A.Q.; Wang, Y.L.; Wen, J.Y.; Liu, P.; Liu, Z.Y.; Li, Z.J. Fungal community associated with
fermentation and storage of fuzhuan brick-tea. Int. J. Food Microbiol. 2011, 146, 14–22.
1. Gomes, N.M.; Dethoup, T.; Singburaudom, N.; Gales, L.; Silva, A.M.S.; Kijjoa, A. Eurocristatine, a
new diketopiperazine dimer from the marine sponge-associated fungus Eurotium cristatum.
Phytochem. Lett. 2012, 5, 717–720.

Molecules 2014, 19
17847
1
1
1
2. Almeida, A.P.; Dethoup, T.; Singburaudom, N.; Lima, R.; Vasconcelos, M.H.; Pinto, M.; Kijjoa, A.
The in vitro anticancer activity of the crude extract of the sponge-associated fungus Eurotium
cristatum and its secondary metabolites. J. Nat. Pharm. 2010, 1, 25–29.
3. Peng, X.Y.; Liang, F.L.; Li, D.L.; Chen, Y.C.; Tao, M.H.; Zhang, W.M.; Zhao, Y.L. Secondary
metabolites of Eurotium cristatum from fu brick tea and their biological activities. Chin. Tradit.
Herb. Drugs 2013, 44, 1881–1886.
4. Zhou, L.N.; Zhu, T.J.; Cai, S.X.; Gu, Q.Q.; Li, D.H. Three new indole-containing
diketopeperazine alkaloids from a deep-ocean sediment derived fungus Penicillium griseofulvum.
Helv. Chim. Acta 2010, 93, 1758–1763.
1
1
1
5. Marfey, P. Determination of D-amino acids. II. Use of a bifunctional reagent, 1,5,-difluoro-2,4-
dinitrobenzene. Carlsberg Res. Comm. 1984, 49, 591–596.
6. Zou, X.W.; Niu, S.B.; Ren, J.W.; Li, E.W.; Liu, X.Z.; Che, Y.S. Verrucamides A–D, antibacterial
cyclopeptides from Myrothecium verrucaria. J. Nat. Prod. 2011, 74, 1111–1116.
7. Van Amsterdam, F.T.M.; Roveri, A.; Maiorino, M.; Ratti, E.; Ursini, F. Lacidipine: A
dihydropyridine calcium antagonist with antioxidant activity. Free Radic. Bio. Med. 1992, 12,
1
83–187.
1
8. Alley, M.C.; Scudiere, D.A.; Monks, A.; Hursey, M.L.; Czerwinski, M.J.; Fine, D.L.; Abbott, B.J.;
Mayo, J.G.; Shoemaker, R.H.; Boyd, M.R. Feasibility of drug screening with panels of human
tumor cell lines using a microculture tetrazolium Assay. Cancer Res. 1988, 48, 589–601.
9. Li, Q.; Zhou, S.Y.; Jing, J.; Yang, T.H.; Duan, S.Y.; Wang, Z.F.; Mei, Q.B.; Liu, L.
Oligosaccharide from apple induces apoptosis and cell cycle arrest in HT29 human colon cancer
cells. Int. J. Biol. Macromol. 2013, 57, 245–254.
1
Sample Availability: Samples of the compounds 1‒5 are available from the authors.
©
2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/4.0/).
(