974 0009-3130/20/5605-0974
©
2020 Springer Science+Business Media, LLC
1) College of Pharmaceutical Sciences, Institute of Life Science and Green Development, Hebei University, Baoding,
071002, P. R. China, e-mail: [email protected]; 2) State Key Laboratory of NBC Protection for Civilians, 102205,
Beijing, P. R. China, e-mail: [email protected]. Published in Khimiya Prirodnykh Soedinenii, No. 5, September–October,
2020, pp. 833–835. Original article submitted August 25, 2019.
Chemistry of Natural Compounds, Vol. 56, No. 5, September, 2020
BIOACTIVE METABOLITES FROM Talaromyces purpureogenus,
AN ENDOPHYTIC FUNGUS FROM Panax notoginseng
Li-Xi Feng,
1
Bing-Yang Zhang,
1
Hua-Jie Zhu,
1
Li Pan,
2*
and Fei Cao
1*
Secondary metabolites from endophytic fungi are rich and have many biological activities, including promoting plant
growth, antibacterial, anticancer, and anti-inflammatory properties [1]. Studies on endophytic fungi to discover natural products
with significant activities show great economic value and application prospects [2, 3]. For example, a series of azaphilone
derivatives [4], isocoumarin derivatives [4], diphenylketones [5], xanthones [5], benzofurans [6], sesquiterpene-conjugated
amino acids [7], and diphenyl ether derivatives [8] has been obtained from the endophytic fungus Talaromyces sp. These
compounds exhibit different physiological activities. In order to further discover compounds with novel structure and activities,
the endophytic fungus Talaromyces purpureogenus XL-25 isolated from Panax notoginseng was selected as our research
object for fermentation.
All of the isolated compounds were identified by detailed spectroscopic analysis [9] and by comparison with the data
of those previously reported in the literature. The structures of compounds 1–6 were established as talaroconvolutin A (1) [4],
talaroconvolutin B (2) [4], 3-hydroxy-5-methylphenyl-2,4-dihydroxy-6-methylbenzoate (3) [10], 8-hydroxy-6-methoxy-3-
methylisocoumarin (4) [11], 6,8-dihydroxy-3-methylisocoumarin (5) [12], and purpurester A (6) [13], respectively.
The antibacterial activities of all the isolated compounds (1–6) were evaluated against six pathogenic bacteria strains.
Among them, compound 1 showed pronounced antibacterial activity against Bacillus subtilis with an MIC value of 1.56 μM,
which was close to the positive control ciprofloxacin (MIC = 2.5 μM). Compound 2 had a certain inhibitory activity against
Micrococcus lysodeikticus (MIC = 0.73 μM) and Vibrio parahaemolyticus (MIC = 0.18 μM).
Fungus Material. The fungal strain Talaromyces purpureogenus (XL-25) was isolated from the fresh tissue of Panax
notoginseng, which was collected from medicinal plants from Shijiazhuang, Hebei Province, China, in May, 2016 and identified
by ITS sequence. The strain was deposited at the Key Laboratory of Medicinal Chemistry and Molecular Diagnostics of the
Education Ministry of China, Baoding, P. R. China, with the GenBank accession number KY230505.1.
Culture Conditions. The fungus of Talaromyces purpureogenus was dissected under aseptic conditions and placed
on PDA agar plates (comprising 2% glucose, 2% agar, 20% potato in distilled water). The plates were wrapped in parafilm and
incubated at 28°C for 3–4 days. Then the fungus of Talaromyces purpureogenus XL-25 was set in small scale in rice medium
(100 mL water, 100 g rice, 2.0 g glucose, 20 g peptone) in 1 L Erlenmeyer flasks. Sixty flasks of the fungal strain were
incubated at 28°C for 60 days.
Extraction and Isolation. The fermented solid medium was extracted two or three times with EtOAc until the
organic phase was almost colorless. Then the combined EtOAc layers were evaporated to dryness under reduced pressure to
give a crude extract (69.0 g). The crude extract was then chromatographed on a silica gel column using a stepwise gradient of
petroleum ether (PE)–EtOAñ (100:0 to 0:100) to produce six fractions (Frs. 1–6). Fraction 1 was further purified by Sephadex
LH-20 (CH
2
Cl
2
–MeOH, 1:1), silica gel CC (PE–EtOAc, 2:1), and Sephadex LH-20 (PE–CH
2
Cl
2
–MeOH, 2:1:1), then further
purified on semipreparative HPLC with 70% MeOH–H
2
O to obtained 1 (12 mg), 6 (9 mg), and 5 (8 mg). Fraction 2 was
separated using silica gel CC and then subjected to Sephadex LH-20 CC (MeOH), which was further purified using HPLC
(MeOH–H
2
O, 20:80, 2.0 mL/min) to yield 2 (15 mg), 3 (6 mg), and 4 (5 mg).
DOI 10.1007/s10600-020-03206-9
975
Antibacterial Assay. The antibacterial activity of all the isolated compounds was evaluated by the double dilution
method with 96-well plates [14]. Six pathogenic bacteria strains, including Micrococcus lysodeikticus, Bacillus subtilis,
Micrococcus luteus, Bacillus anthracis, Escherichia coli, and Vibrio parahemolyticus were used, and ciprofloxacin was used
as the positive control. The results were calculated as the minimum inhibitory concentration (MIC).
Talaroconvolutin A (1). C
32
H
41
NO
3
, dark brown oil; [α]
20
D
–106° (c 0.83, CH
2
Cl
2
).
1
H NMR (600 MHz, DMSO-d
6
,
δ, ppm, J/Hz): 10.39 (1H, br.s, 10-OH), 10.08 (1H, br.s, NH-1), 7.76 (1H, s, H-4), 7.54 (2H, d, J = 7.8, H-8, 12), 6.83 (2H, d,
J = 7.7, H-9, 11), 6.44 (1H, s, H-6), 5.39 (1H, s, H-17), 4.64 (1H, d, J = 9.2, H-25), 3.90 (1H, dd, J = 11.9, 7.8, H-14), 3.14 (1H,
d, J = 7.5, H-15), 2.08 (1H, m, H-26), 1.74 (1H, t, J = 11.7, H-22), 1.68 (4H, m, H-20a, 21a), 1.61 (1H, m, H-19), 1.50 (1H, m,
H-18a), 1.46 (3H, s, Me-16), 1.38 (3H, s, Me-24), 1.20, 1.00 (each 1H, m, H-27), 0.94 (1H, m, H-18b), 0.91 (3H, s, 23-Me),
0.84 (3H, d, J = 7.2, Me-19), 0.72 (3H, t, J = 7.0, H-28), 0.64 (3H, d, J = 6.2, Me-26).
13
C NMR (150 MHz, DMSO-d
6
,
δ, ppm): 195.3 (C-13), 169.5 (C-2), 158.9 (C-10), 145.7 (C-4), 135.9 (C-25), 135.6 (C-17), 132.4 (C-24), 132.3 (C-8, 12),
131.2 (C-5), 130.1 (C-3), 129.6 (C-16), 125.2 (C-7), 120.7 (C-6), 116.0 (C-9, 11), 50.2 (C-15), 48.5 (C-14), 48.1 (C-18), 40.0
(C-22), 35.5 (C-20), 34.8 (C-23), 33.4 (C-26), 29.8 (C-27), 26.9 (C-19), 23.8 (C-21), 22.6 (CH
3
-9), 21.8 (CH
3
-16), 20.5 (CH
3
-26),
20.4 (CH
3
-23), 14.0 (CH
3
-24), 11.7 (C-28). ESI-MS m/z 488.45 [M + H]
+
.
Talaroconvolutin B (2). C
32
H
43
NO
4
, yellow oil; [α]
20
D
–106° (c 0.35, CH
2
Cl
2
).
1
H NMR (600 MHz, CDCl
3
,
δ, ppm, J/Hz): 7.25 (1H, s, H-4), 7.03 (2H, d, J = 8.2, H-8, 12), 6.64 (2H, d, J = 8.2, H-9, 11), 6.08 (1H, br.s, 5-OH), 5.33 (1H,
s, H-17), 4.73 (1H, d, J = 9.5, H-25), 3.73 (1H, dd, J = 12.2, 6.7, H-14), 3.05 (1H, d, J = 13.7, H-6a), 2.93 (1H, d, J = 13.7,
H-6b), 2.80 (1H, d, J = 6.7, H-15), 2.16 (1H, m, H-26), 1.70 (1H, m, H-22), 1.68 (2H, m, H-19, 20a), 1.48 (1H, m, H-18a), 1.48
(3H, s, Me-16), 1.43 (1H, m, H-21a), 1.35 (3H, s, Me-24), 1.32, 1.19 (each 1H, m, H-27), 0.90 (3H, s, Me-23), 0.87 (2H, m,
H-20b, 21b), 0.87 (3H, d, J = 7.2, Me-19), 0.86 (3H, t, J = 7.0, H-28), 0.83 (1H, m, H-18b), 0.82 (3H, d, J = 7.0, Me-26).
13
C NMR (150 MHz, CDCl
3
, δ, ppm): 197.4 (C-13), 167.3 (C-2), 155.9 (C-10), 154.5 (C-4), 136.3 (C-3), 135.4 (C-17),
135.1 (C-25), 132.8 (C-24), 130.9 (C-8, 12), 129.7 (C-16), 125.3 (C-7), 114.5 (C-9, 11), 85.9 (C-5), 50.2 (C-15), 48.8 (C-14),
47.9 (C-18), 42.9 (C-6), 39.2 (C-22), 35.4 (C-20), 35.0 (C-23), 33.4 (C-26), 29.7 (C-27), 26.7 (C-19), 23.1 (C-21), 22.5 (CH
3
-19),
21.7 (CH
3
-16), 20.2 (CH
3
-26), 20.1 (CH
3
-23), 13.9 (CH
3
-24), 11.6 (C-28). ESI-MS m/z 506.14 [M + H]
+
.
3-Hydroxy-5-methylphenyl-2,4-dihydroxy-6-methylbenzoate (3). C
15
H
14
O
5
, white powder.
1
H NMR (600 MHz,
DMSO-d
6
, δ, ppm, J/Hz): 9.59 (1H, s, 4-OH), 6.43 (1H, s, H-6′), 6.34 (1H, s, H-2′), 6.32 (1H, s, H-4′), 6.23 (1H, s, H-5), 6.20
(1H, s, H-3), 2.38 (3H, s, CH
3
-5′), 2.21 (3H, s, CH
3
-6′).
13
C NMR (150 MHz, DMSO-d
6
, δ, ppm): 171.9 (C-7), 160.0 (C-4),
158.5 (C-2), 156.0 (C-3′), 148.0 (C-1′), 141.7 (C-5′), 140.4 (C-6), 112.2 (C-6′), 111.4 (C-4′), 110.9 (C-5), 103.9 (C-1), 102.6
(C-2′), 100.5 (C-3), 22.2 (CH
3
-6′), 21.0 (CH
3
-5′). ESI-MS m/z 275 [M + H]
+
.
8-Hydroxy-6-methoxy-3-methylisocoumarin (4). C
11
H
10
O
4
, white powder.
1
H NMR (600 MHz, CD
3
OD, δ, ppm):
6.43 (1H, s, H-7), 6.29 (1H, s, H-5), 6.21 (1H, s, H-4), 3.88 (3H, s, 6-OCH
3
), 2.18 (3H, s, 3-CH
3
).
13
C NMR (150 MHz,
CD
3
OD, δ, ppm): 166.3 (C-1), 163.7 (C-6), 161.1 (C-8), 154.5 (C-3), 142.5 (C-4a), 103.6 (C-4), 102.7 (C-8a), 99.8 (C-5),
98.6 (C-7), 54.9 (6-OCH
3
), 17.8 (CH
3
-3). ESI-MS m/z 207.06 [M + H]
+
.
O
HO
HN
O
HO
O
HN
O
OH
O
OOH
HO
OH
O
R
OOH
O
HO
HO
O
OCH
3
12
3
4, 5
6
2
4
6
9
13
28
26
24
16
14
23
22
20
18
4
6
1
2
71'
2'
5'
1
3
4
5
7
7a
4
3
1
3a
8
9
6
10
HH
4: R = OCH
3
; 5: R = OH
976
6,8-Dihydroxy-3-methylisocoumarin (5). C
10
H
8
O
4
, white powder.
1
H NMR (600 MHz, DMSO-d
6
, δ, ppm, J/Hz):
10.95 (1H, s, 8-OH), 10.84 (1H, s, 6-OH), 6.47 (1H, s, H-4), 6.33 (1H, s, H-5), 6.30 (1H, d, J = 2.1, H-7), 2.20 (3H, s, CH
3
-3).
13
C NMR (150 MHz, DMSO-d
6
, δ, ppm): 165.6 (C-1), 165.4 (C-6), 162.7 (C-8), 154.1 (C-3), 139.7 (C-4a), 104.2 (C-4), 102.3
(C-5), 101.3 (C-7), 97.9 (C-8a), 18.8 (CH
3
-3). ESI-MS m/z 193.05 [M + H]
+
.
Purpurester A (6). C
13
H
16
O
5
, white powder; [α]
20
D
–7.6° (c 0.043, MeOH).
1
H NMR (600 MHz, CD
3
OD, δ, ppm, J/Hz):
6.60 (1H, s, H-4), 3.01 (3H, s, 3-OCH
3
), 2.35 (3H, s, CH
3
-7), 2.13 (2H, m, H-8), 1.28, 1.09 (each 1H, m, H-9), 0.86 (3H, t,
J = 7.4, H-10).
13
C NMR (150 MHz, CD
3
OD, δ, ppm): 169.8 (C-1), 158.9 (C-6), 151.0 (C-5), 127.0 (C-7), 121.7 (C-3a), 115.7
(C-7a), 109.8 (C-3), 107.4 (C-4), 49.7 (3-OCH
3
), 38.6 (C-8), 16.4 (C-9), 12.9 (C-10), 8.0 (CH
3
-7). ESI-MS m/z 253.45 [M + H]
+
.
ACKNOWLEDGMENT
This work was supported by the China Postdoctoral Science Foundation (No. 2019M661045), the Scientific
research Foundation of Hebei educational committee (No. BJ2020048), the National Natural Science Foundation of
China (No. 21877025), and the Natural Science Interdisciplinary Research Program of Hebei University (No. DXK201913).
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