Received: 22 June 2020 Revised: 5 November 2020 Accepted: 7 November 2020

DOI: 10.1002/jccs.202000274

ARTICLE

A new alkanol from the endolichenic fungus Daldinia

childiae

Xuan Zhou1 | Cailing Yang1 | Qingfeng Meng2 | Le Liu1 | Shaobin Fu1

1
School of Pharmacy, Zunyi Medical
University, Zunyi, China
Abstract
2
Department of Public Health, Zunyi One new alkanol, 2,4,5-heptanetriol(1), together with four compounds,
Medical University, Zunyi, China 6-heptene-2,4,5-triol (2), rel-(1S, 4S, 5R, 7R, 10R)-10-desmethyl-1-methyl-
11-eudesmene (3), 2,3-dihydro-5-hydroxy-2-methyl-4H-1-benzopyran-4-one
Correspondence
Shaobin Fu, School of Pharmacy, Zunyi (4), (−)-regiolone (5) were isolated from the ethyl acetate extract of rice solid-
Medical University, Zunyi, 563000, China. substrate cultures of endolichenic fungus Daldinia childiae for the first time.
Email: fushb@[Link]
Compounds 1, 2, and 3 were separated firstly from the genus Daldinia. Their
structures were elucidated by spectral data and compound 3 exhibited
α-amylase inhibitory activity.

KEYWORDS
alkanol, Daldinia childiae, endolichenic fungus, secondary metabolites

1 | INTRODUCTION as new sources of rich bioactive secondary metabolites

have become one of the hot spots of natural products.[11]
Lichens are synergetic organisms composed of a mycobiont Genus Daldinia described by Cesati and De Notaris
in relationship with one or more photosynthetic partners belongs to the family Xylariaceae.[12,13] To the best of our
(photobiont) and these organisms grow on various sub- understanding, the fungus Daldinia childiae isolated from
strates even under extreme ecological environments.[1] lichen has rarely been isolated and chemically investigated.
Along with the mycobiont, numerous non-obligate micro- Herein, we reported the isolation and structure elucida-
fungi live in lichen thalli and these microfungi are called tion of compounds 1–5 (Figure 1) from the endolichenic
endolichenic fungi (ELF).[2] Recently, ELF are promising fungus D. childiae inhabiting the lichen Punctelia
bioresources to produce bioactive metabolites that repre- sp. collected from Cloak Mountain Scenic Area in Guizhou
sent unique and diverse structural classes.[3] There were so Province of China. Apart from this, the α-amylase inhibi-
many examples including polyketides from the endo- tory effect of compounds 1, 2, and 3 was evaluated.
lichenic fungus Eupenicillium javanicum,[4] three new
mixed terpenoids produced by an endolichenic fungus
Ulocladium sp.,[5] nodulisporisteroids C-L, new 4-methyl- 2 | RESULTS A ND DISCUSSION
progesteroid derivatives from Nodulisporium sp.,[6] tetramic
acids, and pyridone alkaloids from the endolichenic fungus 2.1 | Metabolites isolation from
Tolypocladium cylindrosporum,[7] quinone derivatives iso- D. childiae
lated from the endolichenic fungus Phialocephala fortinii,[8]
new phomalone derivatives from the endolichenic fungus Compound 1 was obtained as white powder. The IR spec-
Cochliobolus kusanoi,[9] socoumarindole A, a chlorinated trum suggested the presence of hydroxy groups
isocoumarin, and indole alkaloid hybrid metabolite from (3,424 cm−1). Its molecular formula was determined as
an endolichenic fungus Aspergillus sp.[10] Therefore, ELF C7H16O3 on the basis of HR-ESI-MS data at m/z value of

J Chin Chem Soc. 2020;1–4. [Link] © 2020 The Chemical Society Located in Taipei & Wiley-VCH GmbH 1
2 ZHOU ET AL.

FIGURE 1 The structures of

compounds 1–5

TABLE 1 NMR Data of Compound 1 (1H-NMR 400 MHz, 13C-

NMR 100 MHz, CD3OD, δ, ppm, J/Hz)

C atom δH δC HMBC
1 1.18 (3H, d, 6.4) 10.88 C-3
FIGURE 2 Key HMBC correlations of compound 1
2 3.98 (1H, m) 65.63
3 1.45 (1H, m), 1.61 (1H, m) 42.46 C-2
4 3.63 (1H, m) 72.65 (1H, s, H-12b), 1.68 (3H, s, H-13), 1.16 (3H, s, H-14), 0.86
5 3.30 (1H, m) 77.87 (3H, s, H-15). 13C-NMR (100 MHz, CDCl3): 152.42 (C-11),
6 1.33 (1H, m), 1.60 (1H, m) 26.66 C-5
107.72 (C-12), 74.87 (C-1), 55.29 (C-10), 46.06 (C-7), 45.76
(C-5), 38.87 (C-4), 36.05 (C-2), 31.08 (C-3), 29.69 (C-14),
7 0.96 (3H, t, 7.2, 14.4) 24.67 C-6
28.51 (C-8,C-6), 26.12 (C-9), 19.87 (C-13), 16.13 (C-15).
The data were consistent with the literature report.[15]
2,3-Dihydro-5-hydroxy-2-methyl-4H-1-benzopyran-
171.0995 (calc. 171.0992) corresponding to [M + Na]+. 4-one (4). Yellow needles, 1H-NMR (400 MHz, CDCl3):
The 1H-NMR spectrum of 1 showed resonances for two 11.68 (1H, s, Ar-OH), 7.32 (1H, t, J = 8.0 Hz, H-7), 6.46
methyl protons at δH 0.96 (3H, t, J = 7.2, 14.4 Hz, H-7), (1H, d, J = 8.4 Hz, H-6), 6.40 (1H, d, J = 8.0 Hz, H-8),
1.18 (3H, d, J = 6.4 Hz, H-1), four methylene protons at 4.53 (3H, s, H-2), 2.62–2.75 (2H, m, H-3), 1.23 (3H, s,
δH 1.33 (1H, m, H-6a), 1.60 (1H, m, H-6b), 1.45 (1H, m, 2-CH3). 13C-NMR (100 MHz, CDCl3): 198.49 (C-4), 162.02
H-3a), 1.61 (1H, m, H-3b), and three oxygen-bearing (C-9), 161.63 (C-5), 138.14 (C-7), 109.11 (C-6), 107.97 (C-
methine proton at δH 3.30 (1H, m, H-5), 3.63 (1H, m, 10), 107.26 (C-8), 73.77 (C-2), 43.77 (C-3), 20.80 (2-CH3).
H-4), 3.98 (1H, m, H-2). The 13C-NMR spectrum of 1 rev- The data were consistent with the literature report.[16]
ealed the presence of seven carbon signals including (−)-Regiolone (5). Yellow solid, 1H-NMR (400 MHz,
three oxygenated tertiary carbons at δC 77.87 (C-5), 72.65 CDCl3): 12.39 (1H, s, 8-OH), 7.47 (1H, t, J = 8.4, 7.6 Hz,
(C-4), and 65.63 (C-2), two methylene carbons at δC 26.66 H-6), 7.00 (1H, d, J = 7.6 Hz, H-5), 6.90 (1H, d,
(C-6), 42.46 (C-3) and two methyl groups at δC 10.88 (C- J = 8.4 Hz, H-7), 4.89 (1H, dd, J = 3.6 Hz, H-4), 2.98 (1H,
1), 24.67 (C-7). Additionally, the HMBC spectrum indi- ddd, J = 4.8 Hz, H-2α), 2.62 (1H, ddd, J = 4.8 Hz, H-2β).
13
cated H-3b correlated with C-2, H-1 correlated with C-3, C-NMR (100 MHz, CDCl3): 204.20 (C-1), 162.70 (C-8),
H-6a correlated with C-5 and H-7 correlated with C-6 145.80 (C-4a), 136.90 (C-6), 117.70 (C-5), 117.30 (C-7),
(Table 1, Figure 2). Therefore, the structure of compound 115.20 (C-8a), 67.70 (C-4), 34.50 (C-2), 31.20 (C-3). The
1 was identified to be 2,4,5-heptanetriol and first discovered data were consistent with the literature report.[17]
from endolichenic fungus D. childiae.
2,4,5-heptanetriol (1). White powder, ½α25 D : −46.81
(c = 0.30, CH3OH). IR (KBr): 3,424 cm−1. 1H- and 13C- 2.2 | α-Amylase inhibitory assay
NMR data of compound 1 were performed on the Table 1.
6-Heptene-2,4,5-triol (2). Brown oily liquid, 1H-NMR Compound 3 exhibited slight α-amylase inhibitory poten-
(400 MHz, CD3OD): 5.85 (1H, m, H-6), 5.29 (1H, dt, tial at concentration of 1.5 mg/ml with the inhibition rate
J = 10.1, 1.6 Hz, H-7a), 5.11 (1H, dt, J = 16.6, 1.6 Hz, H- up to 15.99% while compounds 1 and 2 showed no
7b), 4.13 (1H, m, H-5), 4.26 (1H, m, H-4), 1.91 (1H, dd, α-amylase inhibitory activity.
J = 6.4, 2.0 Hz, H-3a), 1.64 (1H, m, H-3b), 4.04 (1H, m,
H-2), 1.26 (3H, d, J = 6.4 Hz, H-1). 13C-NMR (100 MHz,
CD3OD): 114.83 (C-7), 137.42 (C-6), 87.55 (C-5), 74.52 3 | EXPERIMENTAL
(C-4), 41.29 (C-3), 76.42 (C-2), 21.09 (C-1). The data were
consistent with the literature report.[14] 3.1 | General experimental procedure
Rel-(1S, 4S, 5R, 7R, 10R)-10-desmethyl-1-methyl-
11-eudesmene (3). Colorless oil, 1H-NMR (400 MHz, Optical rotation was obtained on an I model automatic
CDCl3): 7.24 (1H, s, 10-OH), 4.63 (1H, s, H-12a), 4.56 polarimeter (Rudolph research analytical, USA) using
ZHOU ET AL. 3

MeOH as the solvent at 25 C. IR spectrum was recorded v) to generate five fractions, A-E. Fr. E was repeatedly
on a 650 model advanced FTIR spectrometer (Tianjin purified by silica gel column chromatography, eluting
Gangdong Sci. &Tech. Co. Ltd., Tianjin P. R. China). with PE-EtOAc solvent (3:1, v/v) and PE-EtOAc solvent
Nuclear magnetic resonance (NMR) spectra were acquired (1:3, v/v) to gain compound 1 (70.0 mg). Compound
with an Agilent DD2400-MR spectrometer (Agilent Tech- 2 (84.4 mg) was isolated from the Fr. D on the basis of silica
nologies, Santa Clara, CA) operating at 400 MHz. The gel column chromatography with PE-EtOAc solvent (6:1, v/
High-resolution electrospray ionization mass spectra (HR- v). Fr. B was subjected to silica gel column chromatography
ESI-MS) data were recorded on an Agilent 6210 ESI/TOF eluted with PE-DCM (Methylene chloride) solvent (1:1, v/v)
mass spectrometer (Agilent Technologies, Santa Clara, to give subfractions Fr. B-1 and Fr. B-2. Further purification
CA). Silica gel (100–200 mesh and 200–300 mesh; Qingdao of Fr. B-1 with PE-DCM solvent (1:3, v/v) yielded com-
Marine Chemical Co. Ltd., Qingdao, P. R. China) were pound 3 (84.4 mg). Compound 4 (12.9 mg) was subjected to
used for column chromatography (CC). Solvents including silica gel column chromatography using PE-DCM solvent
petroleum ether, dichloromethane, EtOAc, and MeOH (4:1, v/v). Fr. C was separated by silica gel column chroma-
(Kelong Chemical Industry Co. Ltd., Chengdu, P. R. tography, eluting with PE-EtOAc solvent (15:1, v/v) to
China) used for extraction and chromatographic separation afford compound 5 (7.1 mg).
were of analytical grade and thin-layer chromatography
(TLC) was carried out on silica gel GF254 plates (Qingdao
Marine Chemicals, China). Visualization of the TLC plates 3.4 | α-Amylase inhibitory assay
was performed by UV at 254 nm/365 nm or spraying with
H2SO4/EtOH (1:9, v/v) followed by heating and iodine The inhibition of α-amylase activity was conducted by
staining. The absorbance of compounds were measured by the following method described by Hamdani with slight
a Microplate Reader FC (Thermo Fisher Scientific, Wal- modifications.[18] About 50 μl of 1.5 mg/ml samples was
tham, MA, U.S.A.). Other chemicals and enzymes were incubated with 50 μl α-amylase solution (0.1 U/ml) in
purchased from Yuanye Biotechnology Company, Shang- 100 mmol/L phosphate buffer (pH 6.8) at 37 C for 5 min.
hai, China. After pre-incubation, 50 μl starch solution (1%) was
added to the mixture and re-incubated for 5 min at 37 C.
Finally, the reaction was stopped using 50 μl of
3.2 | Cultivation and fermentation of 3,5-dinitrosalicylic acid (DNS) reagent and heated for
fungal material 5 min in a water bath at 100 C. The reaction mixture
cooled to room temperature was diluted after adding
The strain D. childiae grew on plates of potato dextrose 500 μl phosphate buffer saline (PBS). Then 150 μl mix-
agar (PDA) at 25 C for 4 days. Then, mycelia were inocu- ture was taken and added into a 96-well plate and the
lated into fifteen 250 ml Erlenmeyer flasks, each con- absorbance was measured by a microplate reader at
taining 100 ml potato dextrose broth (PDB), at 25 C on a 540 nm. The sample background group was set using
rotary shaker at 150 rpm to prepare the seed culture. PBS instead of α-amylase, and the blank control group
After 4 days, seed broth (20 ml) was added to 60 Erlen- was prepared using PBS instead of samples, while aca-
meyer flasks (500 ml), each containing 70 g of rice, 3.5 g rbose was used as the positive control. All assays were
glucose and 200 ml distilled water for large-scale fermen- applied in triplicate. The percent inhibition (K%) of amy-
tation before autoclaving at 121 C for 20 min. And these lase activity was calculated using the following formula:
flasks were maintained at room temperature for 30 days



in static conditions. K ð%Þ = 1 − Asample − Abackground =Ablank control  × 100

3.3 | Extraction and isolation of where Ablank control) is the absorbance of the negative con-
metabolites trol, Asample is the absorbance of each sample and
Abackground is the absorbance of the background group.
The fermented rice cultures were cut into small pieces
and extracted exhaustively by EtOAc (10 L × 3). After
concentration and removal of the organic solvent under 4 | CONCLUSIONS
reduced pressure, 20.0 g of the crude extract was
obtained. The crude residue was subjected to silica gel This study investigated chemical constituents of
column chromatography with a gradient of PE (petro- D. childiae from lichen Punctelia sp. Five fermentive
leum ether)-EtOAc (ethyl acetate) from 100:0 to 0:100 (v/ products including one new compound 2,4,5-heptanetriol
4 ZHOU ET AL.

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