SUPPLEMENTARY MATERIAL
Antifungal, phytotoxic and toxic metabolites produced by Penicillium
purpurogenum
He Lia, Jing Weia, Shi-Yin Panb, Jin-Ming Gao a* and Jun-Mian Tiana,*
a
College of Science, Northwest A&F University, Yangling 712100, Shaanxi, PR China;
b
Xi’an No.1 Hospital, Shaanxi Institute of Ophthalmology, Xi’an 710002, Shaanxi PR
China
* Corresponding author. Tel./Fax: +86-29-87092515; Email:
jinminggao@nwsuaf.edu.cn; tianjunmian@hotmail.com
1. Experimental
1.1. General
Column chromatography (CC): silica gel (SiO2: 200–300 mesh, Qingdao Marine
Chemical Group Co.); Sephadex LH-20 (Pharmacia Co.); RP-C18 (Merck).
1
H-,13C-NMR spectra: Bruker Avance-500; ESI-MS: Thermo Fisher LTQ Fleet
instrument spectrometer; UV spectra: Shimadzu UV-2401A spectrophotometer.
1.2. Fungal material and cultivation
The strain Penicillium purpurogenum Stoll (CGMCC 3. 3708) was purchased from
Microbiology Institute of Shaanxi. It was deposited at College of science, Northwest
A&F University.
This fungus was cultivated on sterilized PDA solid medium in Roux flasks (100 g/flask
×200) at 25 °C for 28 days.
1.3. Extraction, isolation and purification
The strain cultures were ultrasonically extracted four times with MeOH. The
solvent was removed under reduced pressure to give a crude extract. The extract was
dissolved in H2O (2 L) and treated four times with petroleum ether to give 1.2 g of
residue A. The remaining layer was partitioned with ethyl acetate. The EtOAc extract
(20 g) was subjected to chromatography on a Si gel column with CHCl3-MeOH (100:1,
v/v), (50:1), (20:1), (10:1) to give fractions B-E (8.8, 3.9, 5.4, 1.28 g), respectively.
The petroleum ether extract A was subjected to Sephadex LH-20 (CHCl3/MeOH,
1:1) to give subfractions A1-A3. Subfraction A1 was subjected to silica gel (petroleum
ether/ EtOAc, 5:1) and Pre-TLC (CHCl3) to give compounds 1 (6 mg), 8 (5 mg).
Subfraction A2 was subjected to silica gel (petroleum ether/ EtOAc, 2:1) to give
compound 2 (5 mg). Subfraction A3 was subjected to silica gel (petroleum ether/
EtOAc, 5:1) and Pre-TLC (CHCl3) to give compounds 3 (2 mg) and 4 (12 mg).
Fraction D (5.4 g) was purified by CC on silica gel (petroleum ether/ acetone,
2:1) to subfractions D1-D3. Subfraction D1 was subjected to Sephadex LH-20
(MeOH), silica gel (CHCl3/MeOH/HCOOH, 200:1:1), RP-C18 (MeOH/H2O, 7:3) to
give 5 (3 mg). Subfraction D2 was chromatographed over Sephadex LH-20
(CHCl3/MeOH, 1:1), silica gel (CHCl3/MeOH, 50:1), Pre-TLC (CHCl3/acetone, 9:1)
to give 9 (20 mg). Subfraction D3 was applied to Sephadex LH-20 (MeOH), silica gel
(CHCl3/acetone, 10:1), Pre-TLC (CHCl3/MeOH, 40:1) to give 6 (7 mg). Fraction E
(1.28 g) was applied to Sephadex LH-20 (MeOH) to give 7 (10 mg).
6,8,1′-tri-O-methyl averantin (1). Red amorphous powder; [α]D20 −15 (c 0.11, CHCl3),
1
H NMR (500 MHz, CDCl3) δ 13.78 (1H, s, 1-OH), 9.45 (1H, s, 3-OH), 7.46 (1H, d, J =
2.4 Hz, H-5), 7.24 (1H, s, H-4), 6.79 (1H, d, J = 2.4 Hz, H-7), 4.97 (1H, dd, J = 7.9, 5.1
Hz, H-1′), 4.03 (3H, s, 8-OCH3), 3.98 (3H, s, 6-OCH3 ), 3.47 (3H, s, 1′-OCH3),
1.94–1.72 (2H, m, H-2′), 1.46 (2H, m, H-3′), 1.30 (4H, overlap, H-4′, 5′ ), 0.88 (3H, m,
H-6′); 13C NMR (125 MHz, CDCl3) δ 186.83 (C-9), 182.55 (C-10), 164.97 (C-6), 162.85
(C-8), 162.25 (C-1), 162.18 (C-3), 137.57 (C-10a), 133.06 (C-4a), 119.01 (C-2), 115.24
(C-8a), 110.26 (C-9a), 108.51 (C-4), 104.89 (C-5), 103.91 (C-7), 79.67 (C-1′), 58.02
(1′-OCH3), 56.63 (8-OCH3), 56.00 (6-OCH3), 34.63 (C-2′), 31.58 (C-4′), 24.95 (C-3′),
22.51 (C-5′), 14.00 (C-6′); ESI-MS (negative) m/z 413.00 [M-H]-.
6,8-di-O-methyl averufnin (2). Red amorphous powder; 1H NMR (500 MHz, CDCl3) δ
13.56 (1H, s, 1-OH), 7.46 (1H, d, J = 2.3Hz, H-5), 7.21 (1 H, s, H-4), 6.78 (1H, d, J =
2.3Hz, H-7), 5.39 (1H, d, J =3.1 Hz, H-1′), 4.02 (3H, s, 8-OCH3), 3.98 (3H, s, 6-OCH3),
2.07–1.60 (6H, m, H-2′, 3′, 4′), 1.58 (3H, s, 6′-CH3);
13
C NMR (125 MHz, CDCl3) δ
186.79 (C-9), 182.60 (C-10), 164.93 (C-6), 162.81 (C-8), 159.55 (C-3, C-1), 137.59
(C-10a), 132.51 (C-4a), 116.81 (C-2), 115.30 (C-8a), 110.01 (C-9a), 107.00 (C-4),
104.85 (C-7), 103.94 (C-5), 100.84 (C-5′), 67.07 (C-1′), 56.62 (8-OCH3), 55.99
(6-OCH3), 35.94 (C-4′), 27.86 (6′-CH3), 27.44 (C-2′), 15.99 (C-3′); ESI-MS (negative)
m/z 395.0 [M-H]-.
6,8-di-O-methyl averufanin (3). Orange amorphous powder; 1H NMR (500 MHz,
CDCl3) δ 13.83 (1H, s, 1-OH), 9.82 (1H, s, 3-OH),7.46 (1H, d, J= 2.3Hz, H-5), 7.24 (1
H, s, H-4), 6.78 (1H, d, J= 2.4Hz, H-7), 5.18 (1H, dd, J =11.4, 2Hz, H-1′), 4.03 (3H, s,
8-OCH3), 3.98 (3H, s, 6-OCH3), 3.74 (1H, m, H-5′), 1.96 (2H, m, H-2′), 1.75 (2H, m,
H-4′), 1.40 (2H, m, H-3′), 1.30 (3H, d, J = 6.1Hz, H-6′); 13C NMR (125 MHz, CDCl3) δ
186.81 (C-9), 182.55 (C-10), 164.94 (C-6), 162.82 (C-8), 162.12 (C-3), 161.18 (C-1),
137.63 (C-10a), 132.76 (C-4a), 120.58 (C-2), 115.30 (C-8a), 110.13 (C-9a), 109.05
(C-4), 104.86 (C-7), 103.91 (C-5), 76.45 (C-1′), 75.80 (C-5′), 56.63 (8-OCH3), 56.00
(6-OCH3), 33.05 (C-4′), 30.06 (C-2′), 23.26 (C-3′), 22.07 (6′-CH3); ESI-MS (negative)
m/z 397.04 [M-H]-.
Aversin (4). Orangeamorphous powder; 1H NMR (500 MHz, CDCl3) δ 13.50 (1 H, s,
1-OH), 7.44 (1H, d, J = 2.3Hz, H-5), 7.21 (1H, s, H-4), 6.78 (1H, d, J = 2.4Hz, H-7),
6.46 (1H, d, J = 5.7Hz, H-4′), 4.15 (2H, m, H-1′, H-3′a), 4.03 (3H, s, 8-OCH3), 3.98 (3H,
s, 6-OCH3), 3.68 (1H, m, H-3′b), 2.37(1H, m, H-2′a), 2.28 (1H, m, H-2′b);
13
C NMR
(125 MHz, CDCl3) δ 187.80 (C-9), 183.16 (C-10), 166.77 (C-3), 165.69 (C-6), 163.56
(C-8), 160.96 (C-1), 138.17 (C-10a), 135.56 (C-4a), 120.80 (C-2), 115.64 (C-8a), 113.63
(C-4′), 113.26 (C-9a), 105.52 (C-7), 104.80 (C-5), 101.83 (C-4), 68.44 (C-3′), 57.32
(8-OCH3), 56.71 (6-OCH3), 45.13 (C-1′), 31.26 (C-2′); ESI-MS (positive) m/z 369.05
[M+H]+.
1,3-dihydroxy 6,8-dimethoxy-9, 10-anthraquinone (5). Red amorphous powder; 1H
NMR (500 MHz, MeOD) δ 7.40 (1H, d, J = 2.0 Hz, H-5), 7.14 (1H, d, J = 2.1 Hz, H-4),
6.88 (1H, d, J = 1.8 Hz, H-7), 6.57 (1H, d, J = 2.1 Hz, H-2), 3.99 (3H, s, 8-OCH3), 3.97
(3 H, s, 6-OCH3); 13C NMR (125 MHz, MeOD) δ 187.48 (C-9), 183.77 (C-10), 166.24
(C-6), 165.86 (C-8), 164.93 (C-3), 164.02 (C-1), 138.21 (C-10a), 135.27 (C-4a), 115.64
(C-8a), 111.42 (C-9a), 109.52 (C-4), 108.51 (C-2), 105.57 (C-5), 105.30 (C-7), 56.93
(8-OCH3), 56.41 (6-OCH3);ESI-MS (positive) m/z 622.62 [2M+Na]+.
6,8-di-O-methylnidurufin (6). Yellow amorphous powder; 1H NMR (500 MHz, CDCl3)
δ 13.58 (1H, s,1-OH), 7.43 (1H, d, J = 2.5 Hz, H-5), 7.20 (1H, s, H-4), 6.77 (1H, d, J =
2.4 Hz, H-7), 5.25 (1H, d, J = 1.6 Hz, H-1′), 4.16 (1H, m, H-2′), 4.02 (3H, s, 6-OCH3),
3.97 (3H, s, 8-OCH3), 2.24-1.73 (4H, m, H-3′, 4′), 1.64 (3H, s, H-6′);
13
C NMR (125
MHz, CDCl3) δ 186.66 (C-9), 182.43 (C-10), 165.01 (C-6), 162.83 (C-8), 159.72 (C-3),
159.18 (C-1), 137.42 (C-10a), 132.89 (C-4a), 115.07 (C-2), 114.72 (C-8a), 109.97
(C-9a), 107.19 (C-4), 104.81 (C-7), 104.03 (C-5), 100.82 (C-5′), 71.31 (C-1′), 65.18
(C-2′), 56.59 (8-OCH3), 55.99 (6-OCH3), 30.73 (C-4′), 27.58 (6′-CH3), 23.15 (C-3′);
ESI-MS (positive) m/z 846.48 [2M+Na]+.
6,8-di-O-methylversiconol (7). Redamorphous powder; 1H NMR (500 MHz, DMSO) δ
14.02 (1H, s, 1-OH), 7.23 (1H, d, J = 2.5 Hz, H-5), 7.12 (1H, s, H-4), 6.97 (1H, d, J =
2.4 Hz, H-7), 3.95 (3H, s, 6-OCH3), 3.93 (3H, s, 8-OCH3), 3.76 (1H, dd, J = 10.1, 7.6
Hz, H-1′a), 3.69 (1H, dd, J = 10.2, 6.5 Hz, H-1′b), 3.48 (1H, m,H-2′), 3.30 – 3.36 (2H,
m, H-4′), 1.94 (2H, m, H-3′);
13
C NMR (125 MHz, DMSO) δ 186.00 (C-9), 181.93
(C-10), 164.59 (C-6), 163.23 (C-1), 162.88 (C-3), 162.28 (C-8), 136.51 (C-10a), 131.09
(C-4a), 123.26 (C-2), 114.01 (C-8a), 109.36 (C-9a), 106.82 (C-4), 104.51 (C-7), 104.41
(C-5), 62.80 (C-1′), 60.07 (C-4′), 56.58 (8-OCH3), 56.05 (6-OCH3), 35.21 (C-2′), 32.64
(C-3′); ESI-MS (negative) m/z 386.92 [M-H]-.
5-methyoxysterigmatocystin (8). Yellow amorphous powder; 1H NMR (500 MHz,
CDCl3) δ 12.62 (1H, s, 8-OH), 7.20 (1H, d, J = 8.9 Hz, H-6), 6.84 (1H, d, J = 7.1 Hz,
H-1′), 6.70 (1H, d, J = 8.9 Hz, H-7), 6.53–6.49 (1H, m, H-4′), 6.45 (1H, s, H-2), 5.52
(1H, t, J = 2.6 Hz, H-3′), 4.93 – 4.78 (1H, m, H-2′), 4.01 (3H, s, 1-OCH3), 3.94 (3H, s,
5-OCH3); 13C NMR (125 MHz, CDCl3) δ 181.27 (C-9), 164.57 (C-3), 163.28 (C-1),
155.26 (C-8), 153.97 (C-10), 145.25 (C-4′), 144.71 (C-13), 139.40 (C-5), 120.42 (C-6),
113.30 (C-1′), 109.57 (C-11), 109.50 (C-7), 106.78 (C-4), 106.07 (C-12), 102.58 (C-3′),
90.61 (C-2), 57.72 (5-OCH3), 56.80 (1-OCH3), 48.14 (C-2′); ESI-MS (positive) m/z
377.03 [M+H]+.
(S)-Ornidazole (9). White amorphous powder; []D20 −10 (c =0.11, CH2C12), lit. []D20
−67.8 (c = 0.99, CH2C12) (Skupin et al. 1997); 1H NMR (500 MHz, CDCl3) δ 2.48 (3H,
s, 2-CH3), 3.67 (1H, dd, J =11.5, 5.0 Hz, H-3′a), 3.74 (1H, dd, J =11.5, 4.1Hz, H-3′b),
4.17-4.24 (2H, m, H-1′a, H-2′), 4.60-4.66 (1H, m, H-1′b), 7.78 (1H, s, H-4);
13
C NMR
(126 MHz, CDCl3) δ 14.48 (2-CH3), 46.91 (C-3′), 49.64 (C-1′), 69.83 (C-2′), 132.46
(C-4), 138.26 (C-5), 151.84 (C-2); ESI-MS (positive) m/z 219.97 [M+H]+.
1.4. Biological tests
Metabolites were tested in vitro for brine shrimp (Artemia salina) bioassay, processed
according to the previously identified method (Li et al. 2012). Brine shrimp eggs were
hatched in artificial seawater prepared from commercial sea salt 25 g/L. After 48 h
incubation at 28 ± 0.5 °C, nauplii were used in subsequent experiments. The tested
compounds 1−9 and a positive control toosendanin were dissolved in DMSO to 2 mM
and 1, 5, 10 μL added to 96-well microplates, then a suspension of nauplii containing
15−20 organisms (199, 195, 190 μL) was added to each well respectively. The final
concentrations of the tested compounds were 10, 50 and 100 μM. Three repetitions were
conducted for each compound. After being incubated at 28 ± 0.5 °C for 24 h, the
microplates were then examined under the microscope to count dead nauplii and total
numbers of brine shrimp in each well. DMSO served as the negative control. The
lethality of each concentration was recorded in Table S1.
Table S1. Toxicity of compounds 1–9 against brine shrimps
concentration (μM)
Compound
a
10 μM
50 μM
100 μM
1
100%
100%
100%
2
10%
15%
23%
3
14%
19%
29%
4
10%
17%
27%
5
9%
12%
27%
6
10%
19%
22%
7
13%
20%
24%
8
100%
100%
100%
9
nta
3%
7%
toosendanin
100%
100%
100%
not tested
The test phytopathogenic fungi used in this study were Botrytis cinerea,
Magnaportheoryzae and Gibberellasaubinettii. All the fungi were isolated from infected
plant organs at the Northwest A&F University. Antifungal activity was assessed by the
microbroth dilution method in 96-well flat-microtiter plates using a potato dextrose (PD)
medium (Li et al. 2012). Compounds were made up to 4 mM in DMSO. Carbendazim
was used as positive controls. The solution of equal concentration of DMSO was used as
a negative control. The fungi were incubated in the PD medium for 18−36 h at 28 ±
0.5 °C at 150rpm and spore concentrations of different microorganism were diluted to
approximately 1 × 106 colony-forming units/mL (CFU/mL) with PD medium. In
microtiter plates, tested compounds, fungal suspension and sterile water were added to
make up final concentrations of the compounds in the range of 3.12−200 μM. Each
measurement consisted of three replicates. Cultures then grew in the dark at 28 ± 0.5 °C
for 48 h. Minimum inhibitory concentrations (MICs) were inspected as the lowest
concentrations in which no fungal growth could be observed. The results were shown in
Table S2.
Table S2. Inhibitory effects of compounds 1-9 on three phytopathogens
phytopathogenic fungi（MIC: μM）
Compound
Botrytis cinerea
Magnaporthe oryzae
Gibberella saubinettii
1
25
50
100
2
50
100
100
3
50
100
>100
4
25
100
25
5
50
100
>100
6
50
100
100
7
25
100
100
8
50
100
100
9
100
100
50
carbendazim
6.25
12.5
6.25
The phytotoxic effects were assessed on radish seedlings according to our reported
method (Li et al. 2014). Briefly, radish seedlings were washed by running water for 120
min and then soaked in 0.3% KMnO4 for 15 min and flushed to colorless. An acetone
solution containing a sample at 100 μM was poured on two sheets of filter paper in
twelve-well microplate. After removing the solvent in vacuo, 200 μL of aseptic water
was added on it. Radish seedlings of uniform shape and size were placed on the filter
paper. In this experiment the seedlings were grown for 4 days at 25℃ under completely
dark conditions. The inhibitory effects were observed after 96 h. Glyphosate was used as
the positive control. Acetone served as a blank control. Each experiment was conducted
three times and presented as mean ± standard deviation of three replicates. The results
were shown in Table S3.
Table S3. Inhibitory effects of compounds 1-9 on the growth of radish seedlings at 100
μM
Inhibition ratio (%)a
Compound
a
root
hypocotyl
1
33.6±3.8
23.2±2.8
2
27.7±4.3
25.7±1.9
3
37.8±4.3
30.6±2.5
4
28.6±6.1
18.4±4.4
5
16.5±2.7
25.9±2.7
6
52.9±2.1
27.6±2.8
7
57.1±1.6
23.2±1.9
8
36.7±2.7
27.3±2.0
9
55.2±2.9
21.0±1.9
glyphosate
75.1±3.9
28.1±2.8
mean±SD.