Chemico-Biological Interactions 362 (2022) 109998

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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint

Research paper

Structurally diverse steroids from an endophyte of Aspergillus tennesseensis

1022LEF attenuates LPS-induced inflammatory response through the
cholinergic anti-inflammatory pathway
Jun-Cheng Su a, Qianrong Pan b, Xingyuan Xu a, Xia Wei d, Xueping Lei b, **, Peng Zhang c, *
a
State Key Laboratory of Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, China
b
Guangzhou Municipal and Guangdong Provincial Key Laboratory of Molecular Target & Clinical Pharmacology, The NMPA and State Key Laboratory of Respiratory
Disease, School of Pharmaceutical Sciences and the Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou, 511436, China
c
Tobacco Research Institute of Chinese Academy of Agricultural Sciences, Qingdao, 266101, China
d
Pharmaceutical College, Guangxi Medicinal University, Nanning, 530021, China

A R T I C L E I N F O A B S T R A C T

Keywords: The emerging cholinergic anti-inflammatory pathway plays a key role in regulating inflammation. Steroids are
Steroids known to possess remarkable anti-inflammatory activity. However, the links between steroids and the cholin­
Acetylcholinesterase ergic anti-inflammatory pathway remain unidentified. In this study, eight steroids (1–8) featuring five different
Inflammation
structural types were characterized from an endophytic fungus Aspergillus tennesseensis 1022LEF, and were
The cholinergic anti-inflammatory pathway
subsequently evaluated for their potential role in regulating the cholinergic anti-inflammatory pathway. As a
result, compound 8, with the best potency, showed remarkable anti-inflammatory activity at the nanomolar to
low micromolar level. Further pharmacological study indicated that 8 notably increased α7nAchR expression and
inhibited the activation of its down-stream signaling pathways. Collectively, the present study not only high­
lighted the potential correlation between steroids and the cholinergic anti-inflammatory pathway, but also
identified 8 as a dual-functional modulator via directly inhibition to acetylcholinesterase as well as up-regulation
of α7nAchR expression.

1. Introduction
Acetylcholinesterase (ACchE) is a serine hydrolase that hydrolyzes
neurotransmitter acetylcholine into acetic acid and choline [1]. Except
for its “classical” role in synaptic transmission, AchE also possesses
several “non-classical” functions in non-neuronal cells, and is known to
regulate Alzheimer’s disease (AD), tumorigenesis, glaucoma, and
myasthenia gravis [2,3]. Therefore, it is not surprising that AChE has
become an attractive target from the pharmaceutical community, which
resulted in several FDA approved drugs, especially for the treatment of
AD [4,5]. Recently, emerging evidences had demonstrated that AchE
dysfunction could lead to inflammatory disorders, indicating that
acetylcholinesterase inhibitors (AchEIs) could be served as promising
anti-inflammatory agents [6]. This pathway, termed “the cholinergic
anti-inflammatory pathway”, is mediated by the α7 subunit of the
nicotinic acetylcholine (Ach) receptor (α7nAChR) to inhibit the release
of tumor necrosis factor such as TNF-α and other cytokines [7].
Steroids represent a remarkable group of evolutionarily conserved
lipid natural products. While most of the steroids feature a classical
cyclopenta[a]phenanthrene core, in some cases, these molecules may
also act as biogenetic precursors to provide additional skeleton types via
different biosynthetic steps [8]. Meanwhile, steroids are the major
sources of anti-inflammatory drugs, as representative by dexametha­
sone, a glucocorticoid receptor agonist [9]. However, despite of the fact
that the anti-inflammatory effects of steroids are well-documented, few
studies had correlated these phenotypes with the cholinergic
anti-inflammatory pathway. Therefore, it is of great significance to
validate whether this remarkable group of natural products possess an
alternative anti-inflammatory mechanism.
As part of our ongoing efforts to discover bioactive secondary me­
tabolites from endophytic fungi [10–13], eight steroids belonging to five
distinct skeletons were isolated from a tobacco-derived endophytic

* Corresponding author.
** Corresponding author.
E-mail addresses: xuepinglei@gzhmu.edu.cn (X. Lei), zhangpeng@caas.cn (P. Zhang).

https://doi.org/10.1016/j.cbi.2022.109998
Received 13 April 2022; Received in revised form 17 May 2022; Accepted 28 May 2022
Available online 29 May 2022
0009-2797/© 2022 Elsevier B.V. All rights reserved.
J.-C. Su et al. Chemico-Biological Interactions 362 (2022) 109998

fungus Aspergillus tennesseensis 1022LEF (Fig. 1). The structures of the Table 1
1
isolated compounds were established by comprehensive analysis of their H and 13C NMR spectroscopic data of 1 (CDCl3, J in Hz)a.
UV, HRMS, 1D and 2D NMR data, quantum chemical calculation, as well no. δC, type δH
as comparison with those reported in literatures. To uncover the buried
1 35.0, CH2 a 2.17, m
links between steroids, AchE, and the cholinergic anti-inflammatory b 2.00, m
pathway, a series of enzyme-based, cell-based, and in silico experi­ 2 38.1, CH2 a 2.54, m
ments were performed. The most potent compound 8, with a highly b 2.20, m
degraded C21 skeleton, was identified as a dual-functional cholinergic 3 209.5, C
4 43.8, CH2 a 2.44
anti-inflammatory pathway modulator via direct binding and inhibition b 2.04, m
to AchE as well as up-regulation of α7nAchR expression simultaneously. 5 42.7, CH 2.31, m
6 42.2, CH2 a 2.50, m
2. Results and discussion b 2.07, m
7 198.6, C
8 134.9, C
2.1. Structural elucidation 9 162.4, C
10 38.7, C
The molecular formula of tennessoid A (1) was deduced as C28H42O3 11 65.2, CH 4.46, dd (7.1, 6.7)
by its HRESIMS ion peak at 425.3048 [M − H]− (calcd for C28H41O3, 12 49.0, CH2 a 2.25, m
b 1.63, dd (13.7, 4.5)
425.3056), corresponding to 8 degrees of unsaturation (DOUs). The UV 13 46.3, C
spectrum of 1 displayed the absorption maxima at 192, 215, and 246 14 46.4, CH 2.43
nm, suggesting the occurrence of conjugated system in 1. The 1H and 13C 15 28.9, CH2 a 1.85, m
NMR spectra of 1 showed signals corresponding to two keto carbonyl b 1.23, m
16 24.8, CH2 a 2.15
groups (δC 209.5 and 198.6), one tetra-substituted double bond (δC
b 1.47, m
162.4 and 134.9), one terminal double bond [δH 4.71 (1H, s) and 4.65 17 54.8, CH 1.26, m
(1H, s); δC 156.2 and 106.9], one oxygenated methine [δH 4.46 (1H, dd, 18 14.1, CH3 0.53, s
J = 7.1 and 6.7 Hz); δC 65.2], and five methyl groups [δH 1.30 (3H, s), 19 16.6, CH3 1.30, s
1.00 (3H, d, J = 6.8 Hz), 0.99 (3H, d, J = 6.8 Hz), 0.95 (3H, d, J = 6.4 20 35.9, CH 1.35, m
21 18.8, CH3 0.95, d (6.4)
Hz), and 0.53 (3H, s); δC 22.2, 22.1, 18.8, 16.6, and 14.1]. The above-
22 34.4, CH2 a 1.51, m
mentioned functionalities accounted for 4 of 8 DOUs, which required b 1.12, m
1 to possess a tetracyclic skeleton. The full assignments of the 1H and 13C 23 30.9, CH2 a 2.08
NMR spectroscopic data of 1 were approached by 1H–1H COSY, HSQC, b 1.88, m
24 156.2, C
and HMBC experiments (Table 1).
25 33.6, CH 2.20
Five spin systems, namely, H2-1 to H2-2, H2-4 to H2-6, H-11 to H2-12, 26 22.2, CH3 1.00, d (6.8)
H-14 to H3-21/H2-23, and H3-26 to H3-27, could be deduced from the 27 22.1, CH3 0.99, d (6.8)
1
H–1H COSY spectrum of 1 (Fig. 2). In its HMBC spectrum, correlations 28 106.9, CH2 a 4.71, s
from H2-1 to C-3/C-9, from H2-2 to C-4, from H2-4 to C-10, from H-5 to b 4.65, s

C-7, from H2-6 to C-8, from H2-12 to C-9/C-14/C-17, from H-14 to C-7, a
Overlapped signals are reported without designating multiplicity.
from H2-28 to C-23/C-25, as well as from H3-26/H3-27 to C-24 led to the

Fig. 1. Chemical structures of compounds 1–8.

2
J.-C. Su et al.
Fig. 2. Key 1H–1H COSY, HMBC, and NOE correlations of 1.
establishment of a 6/6/6/5 tetra-carbocyclic core, with a C9 side chain
located at C-17. Next, based on the HMBC cross peaks from H3-18 to C-
14/C-17, from H3-19 to C-1/C-5, Me-18 and Me-19 were deduced to be
located at C-13 and C-10, respectively. Moreover, on the basis of the
characteristic down-fielded chemical shift of C-11 (δC 65.2), as well as
the molecular formula information, a hydroxyl group was placed at C-
11. Hence, the planar structure of 1 was established to be a new
ergosterol-type steroid as shown in Fig. 2.
The stereochemistry of 1 was established by combined analysis of its
NOESY spectrum as well as quantum chemical calculations. In detail,
NOE correlations between H3-19 and H-1a/H-6a/H-11, between H-11/
H3-18, and between H3-18 and H-15b/H-20 indicated that these protons
were located at the same side of the 6/6/6/5 tetracyclic skeleton, while
NOE correlations between H-1b and H-5, between H-14 and H-17 sug­
gested that these atoms were located at the opposite orientation. The
above conclusions were further confirmed by comparing the NMR data
of 1 with those reported in literatures, which was in consistance with the
reported ergosteroids [14,15]. Furthermore, the absolute configuration
of 1 was established by TDDFT ECD calculation at PCM/B3LYP/6-31+G
(d) level in methanol. With a good agreement between the calculated
ECD spectrum of 5R,10S,11R,13R,14R,17R,20R-1 and the experimental
curve, the aboslute configuration of 1 was assigned as 5R,10S,11R,13R,
14R,17R,20R (Fig. 3).
Compounds 2–8 were identified as ergosta-4,6,8(14),22-tetraen-3-
one (2) [16,17], 5α,8α-epidioxyergosta-6,22-dien-3β-ol (3) [18], dan­
kasterone B (4) [19], dankasterone A (5) [20,21], herbarulide (6) [22],
7-nor-ergosterolide (7) [15], and demethylincisterol A3 (8) [23] by
comparing of their NMR data with those reported in the literatures.
Among these compounds, 2 and 3 shared a classical cyclopenta[a]
phenanthrene core with an intact C28 skeleton. 4 and 5 are rearranged
steroids with a rare 13(14 → 8)abeo-8-ergostane skeleton. Compound 6
belongs to an unusual class of 5,6-epoxy-5,6-seco-ergosteroid and com­
pound 7 is a C27 7-oxa-ergosteroid. Compound 8 features a highly
degraded steroid scaffold with a C21 skeleton. Except for 2 and 3, all
these known steroids are rarely found in nature, despite of their clear
biogenetic relationship with the classical ergosteroids.
2.2. Compound 8 is an effective AchE inhibitor
Based on the simultaneous discovery of eight steroids featuring five
distinct skeletons, we wondered whether steroids could serve as novel
3
Chemico-Biological Interactions 362 (2022) 109998
Fig. 3. Experimental and calculated ECD spectra of 1.
AchE inhibitors for developing potential anti-inflammatory agents. To
approach this goal, we firstly evaluated the inhibitory activity of these
compounds against AchE at the enzyme level by using the Ellman assay.
As shown in Fig. 4A, at the concentration of 50 μM, compounds 5 and 8,
with a rearranged or a highly degraded ergostane skeleton, showed
significant inhibitory effect against AchE, followed by the commonly
reported ergosteroid 2. Although these compounds belong to steroids of
different structural subtypes, the presence of an α,β-unsaturated ketone
moiety at the southwestward of the molecule seem to be critical for the
inhibitory effect. Among them, compound 8 was identified as the most
potent inhibitor with IC50 value of 11.16 ± 2.79 μM (Fig. 4C), followed
by compound 5 (IC50 = 27.12 ± 1.27 μM).
2.3. Molecular docking
To further understand the possible modes-of-action of these steroid
AchE inhibitors, the binding modes of compounds 8, 5, and 2 with AchE
were obtained by molecular docking. As disclosed from Fig. 5, all these
steroids fitted well with the same peripheral anionic site of AchE, with
the C-17 aliphatic side chains extended into the deep of the pocket to
generate hydrophobic interactions with Tyr72, Tyr124, Val294, Phe295,
and Phe297, whereas the α,β-unsaturated ketone moieties faced the
solvent region. Compound 8, the most potent inhibitor, is anchored at
the pocket entrance through two hydrogen bonds. The imidazole side­
chain of His287 forms a hydrogen bond with the carbonyl group of
compound 8, and the phenol sidechain of Tyr341 is hydrogen-bonded to
the aliphatic alcohol of compound 8 through a water bridge (Fig. 5A). By
contrast, compound 5 showed only one hydrogen-bonding interaction
with Tyr341 (Fig. 5B), whereas compound 2 had no obvious interactions
with either His287 or Tyr341 (Fig. 5C). These observations were in
accordance with the result of the AchE inhibition assay, and may pro­
vide an alternative view to understand why compound 8 possessed the
best inhibitory activity. Thus, compound 8 was selected as representa­
tive compound for further pharmacological research.
2.4. Compound 8 inhibits for nitric oxide (NO) production and
proinflammatory cytokine expression
As described above, compound 8 inhibits AchE via direct binding to
the active site. To validate the anti-inflammatory effect of compound 8
at the cellular level, we measured the LPS-induced NO production and
proinflammatory cytokine expression in RAW264.7 cells with the
treatment of compound 8. As shown in Fig. 6A, compound 8 had a strong
inhibitory effect against LPS-induced NO production, a critical signaling
molecule during inflammation process. The suppressive effect of
J.-C. Su et al. Chemico-Biological Interactions 362 (2022) 109998

Fig. 4. The effect of compounds on AchE activity. (A) The effect of compounds 1–8 on AchE activity at 50 μM. Tacrine (TA) was set as positive control with final
concentration of 0.333 μM. (B) The AchE inhibition curves of compound 5 and (C) compound 8. The data are presented as mean ± SD.

Fig. 5. In silico analysis of the interactions of compounds 8 (A), 5 (B), and 2 (C) with AchE protein (PDB: 6F25).

compound 8 was in a dose dependent manner, with inhibitory ratio of
40%, 60%, and 91% at 0.625 μM, 1.25 μM and 2.5 μM, respectively.
Notably, no obvious cytotoxicity was observed at these concentrations.
The secretions of IL-6 and TNF-α, two crucial proinflammatory media­
tors, were also measured in after co-incubation with 8 (Fig. 6B). LPS
stimulation dramatically (P < 0.001) increased TNF-α and IL-6 secretion
in comparison with the control group, whereas compound 8 treatment
significantly reduced TNF-α and IL-6 production in a dose dependent
manner (Fig. 6B). Similarly, RT-qPCR assay also confirmed that com­
pound 8 treatments obviously reduced the mRNA of TNF-α and IL-6 in a
dose dependent manner (Fig. 6C). All these results showed that com­
pound 8 was a potential anti-inflammatory agent.

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J.-C. Su et al. Chemico-Biological Interactions 362 (2022) 109998

Fig. 6. Compound 8 suppressed LPS-induced NO production and inflammatory factors expression. (A) Compound 8 reduced LPS-induced NO production in RAW
246.7 cells. The RAW 246.7 cells were stimulated with 20 ng/mL and different concentration of compound 8 for 24 h. The condition medium was collected and
subjected for NO production. (B&C) Compound 8 downregulated LPS-induced inflammatory cytokines in RAW 246.7 cells. The cells were treated with different
concentration of compound 8, the condition medium was collected for Elisa assay (B). The cells were collected and subjected for RT-qPCR assay (C). The data are
presented as mean ± SD, ***P < 0.001 compared with the control group, #P < 0.05 and ###P < 0.001 compared with LPS group.

2.5. Compound 8 suppresses LPS-induced activation NF-κB and Stat3
signaling pathway in RAW 246.7 cells
Since NF-κB transcription factors and the corresponding signaling
pathways are central regulator for inflammation response [24,25], we
evaluated the effect of compound 8 on the critical members of NF-κB
signaling pathway using Western blotting assay, including IKKα, IKKβ,
p-IKKα/βSer176/180, IκBα, p-IκBαSer32, NF-κB p65, p–NF–κB p65Ser536. We
found that LPS stimulation significantly increased the phosphorylation
of IKKa/β, IκBα, NF-κB p65, which indicates the activation of NF-κB
signaling pathway [26]. Whereas, compound 8 treatments obviously
reduced LPS-induced up-regulation of p-IKKα/βSer176/180, p-IκBαSer32,
p–NF–κB p65Ser536 without affecting the total IKKa/β, IκBα, and NF-κB
p65 expression (Fig. 7). We also found that compound 8 treatments
significantly suppressed LPS-induced phosphorylation of Stat3, another
regulator of inflammation [27]. These results suggested that compound
8 suppressed LPS-induced activation NF-κB and Stat3 signaling
pathway.
2.6. Compound 8 increases alpha7 nicotinic acetylcholine receptor
(α7nAchR) expression
Considering the fact that compound 8 exhibited much greater ac­
tivity in cells than at the enzyme level, we speculated that compound 8
may possess an alternative anti-inflammatory mechanism apart from
direct binding and inhibition of AchE. It had been demonstrated that the
activation of α7nAChR, a major receptor of Ach, had obvious inhibitory
effects on LPS-mediated NF-κB and Stat3 signaling pathways [28,29].
Thus, we detected the effect of compound 8 on α7nAchR expression
using Western blotting assay. Surprisingly, the results showed that LPS
stimulation obviously reduced α7nAchR expression, but compound 8
significantly increased α7nAchR expression in a dose dependent manner
(Fig. 8A). Moreover, we found that methyllycaconitine pretreatment, an
inhibitor of α7nAchR [30], obviously attenuated compound 8 mediated
inhibitory effect on the secretion of TNF-α and IL-6 (Fig. 8B). Taken
these together, our result indicated that compound 8 might suppress
LPS-induced inflammation by increasing α7nAchR expression.
3. Conclusions
Although steroids are known to possess remarkable anti-
inflammatory activity, either as endogenous molecules or exogenous
drugs, the targets and pharmacological mechanisms of these small
molecules are not fully understood. In this study, eight steroids featuring
five different structural types were isolated from a tabacco-derived
fungus A. tennesseensis. To seek whether steroids possess an alternative
anti-inflammatory mechanism beyond glucocorticoid receptor activa­
tion, compound 8 was selected as a chemical probe for further mecha­
nism investigation. As a result, compound 8 strongly suppressed LPS-
induced NO production and inflammatory factors expression,
including TNF-α and IL-6. In addition, treatment with compound 8
notably increased α7nAchR expression and inhibited the phosphoryla­
tion of its down-stream NF-κB and Stat3 signaling pathway. Moreover,
in the competitive experiments, the inhibitory effect on TNF-α and IL-6
secretion mediated by compound 8 was obviously attenuated by the
α7nAchR antagonist. Taken together, we speculate that the anti-
inflammatory effect of compound 8 may due to its AchE suppressive
effect by direct binding to the enzyme and up-regulation of α7nAchR
expression.

5
J.-C. Su et al.
4. Experimental section
4.1. General experimental procedures
Optical rotations were acquired using a Jasco P-1020 digital polar­
imeter (Jasco, Tokyo, Japan) with MeOH as solvent. The UV measure­
ments were performed on a Shimadzu UV-2700 spectrophotometer
(Shimadzu, Kyoto, Japan). The ECD spectrum was obtained by a
Chirascan-plus Circular Dichroism Spectrometers (Applied Photo­
physics, Leatherhead, UK). 1D (1H and 13C) and 2D (HSQC, HMBC,
COSY, NOESY) NMR spectra were recorded on an Agilent DD2 NMR
spectrometer (Agilent Technologies, Waldbronn, Germany) operating at
500 MHz for the 1H channel and 125 MHz for the 13C channel. Chemical
shifts were referenced to the residual solvent values of 7.26/77.16 ppm
(1H/13C) for CDCl3 and 2.50/39.52 ppm (1H/13C) for DMSO‑d6,
respectively. The HRESIMS data were obtained with a Waters ACQUITY
UPLC I-Class-Vion IMS Q-TOF mass spectrometer (Waters Corp., Mas­
sachusetts, America). Silica gel (200–300 mesh, Haiyang Chemical
Factory, Qingdao, China), Lobar LiChroprep RP-18 (40–60 μm, Merck,
Darmstadt, Germany), and Sephadex LH-20 (Merck) were applied for
column chromatography. Preparative thin-layer chromatography was
performed with precoated TLC plates (GF254, Haiyang Chemical Fac­
tory). All solvents used for extraction and purification were of either
analytical or HPLC purity.
6
Chemico-Biological Interactions 362 (2022) 109998
Fig. 7. Compound 8 inhibited LPS-induced activation
of NF-kB and Stat3 signaling pathway. The RAW
246.7 cells were exposed with 20 ng/mL LPS for 24 h,
and then treated with or without various concentra­
tions of compound 8 for 24 h. And then, the cells were
collected and subjected for Western blotting assay to
detect several critical regulators of NF-κB and Stat3
signaling pathway. β-actin was set as loading control.
The representative blots were presented in (A), the
quantitative data of blots was measured using Image J
and showed in (B). The data are presented as mean ±
SD, ***P < 0.001 compared with the control group,
#
P < 0.05, ##P < 0.01 and ###P < 0.001 compared
with LPS group.
4.2. Fungal material
The candidate fungal strain Aspergillus tennesseensis 1022LEF was
previously isolated from healthy leaves of cultivated tobacco, Nicotiana
tabacum L., which was collected from Hubei, China
(108◦ 23′ 12′′ –110◦ 38′ 08′′ E, 29◦ 07′ 10′′ –31◦ 24′ 13′′ N). This fungal strain
was taxonomically identified based on the molecular protocol by
sequencing of the ITS region of the rDNA. A BLAST search result
demonstrated that the submitted sequence was most similar (99%) to
that of A. tennesseensis. This fungus was then assigned the GenBank
number of MW898422 and was deposited in the China General Micro­
biological Culture Collection Center (CGMCC) with a deposition number
of 21469.
4.3. Fermentation, extraction, and purification
Large-scale fermentation of A. tennesseensis 1022LEF was statically
performed at 28 ◦ C in a total of 150 of 1 L Erlenmeyer flasks, with 300
mL of commercially available potato dextrose broth medium (Solarbio
Life Sciences CO., LTD., Beijing, China) in each flask. Since the myce­
lium was grown slowly in medium, the cultivation was extended to 50
days. Subsequently, the mycelium and the broth were filtered and
extracted exhaustively with EtOAc to afford 20.2 g of crude extracts. The
obtained extracts were partitioned between petroleum ether (PE) and
MeOH. The MeOH fractionation was subjected to silica gel vacuum
liquid chromatography with mixtures of PE–EtOAc (from 10:1 to 1:1, v/
v) and CH2Cl2–MeOH (from 20:1 to 5:1) gradient systems. As a result,
J.-C. Su et al.
three major fractions, Fr.1–Fr.3, were obtained and combined by TLC
and HPLC analysis. Fr.1 (4.8 g), eluted with PE–EtOAc 2:1 and 1:1, was
refractionated by reversed-phase column chromatography (CC) eluting
with a stepped MeOH–H2O gradient system (from 1:9 to 10:0) to afford
six subfractions, Fr.1.1–Fr.1.6. Fr.1.2 was chromatographed on silica gel
CC (CH2Cl2–MeOH as the mobile phase, from 30:1 to 20:1) to afford 4
(5.8 mg) and 8 (13.6 mg). The new compound 1 (7.0 mg) was isolated
from Fr.1.3 through prep.-TLC (plate: 20 × 20 cm; developing solvents:
PE–acetone, 5:1), while 7 (8.8 mg) was obtained from Fr.1.6 by
Sephadex LH-20 with CH2Cl2–MeOH 1:1 as the mobile phase. Fr.2 (2.4
g) eluting with CH2Cl2–MeOH 20:1 was subjected to silica gel CC
(CH2Cl2–MeOH, from 50:1 to 20:1), and then followed by prep.-TLC
(CH2Cl2-acetone, 20:1) and Sephadex LH-20 to yield compounds 5
(9.0 mg), 6 (3.9 mg), and 2 (7.7 mg), respectively. Finally, Fr.3 (1.8 g,
eluted with CH2Cl2–MeOH 10:1) was purified by silica gel CC
(PE–EtOAc, from 10:1 to 2:1) to yield 3 (26.8 mg).
Tennessoid A (1): Colorless oil; [α] +25.8 (c = 0.10, MeOH); UV
(MeOH) λmax (log ε) 192 (3.22), 215 (3.69), 246 (3.98) nm; CD (MeOH):
λmax (Δε) = 200 (13.75), 237 (− 19.48), 282 (2.72); (+)-HRESIMS m/z
427.3209 [M + H]+ (calcd for C28H43O3, 427.3212), (− )-HRESIMS m/z
425.3048 [M − H]− (calcd for C28H41O3, 425.3056); For 1H and 13C
NMR data, see Table 1.
4.4. Quantum chemical calculations
The random conformational search of 1 was performed under
MMFF94s molecular force field using SYBYL X 2.1.1, with an energy
cutoff of 10 kcal mol− 1 to the global minima. The obtained conformers
were optimized using Gaussion09 at B3LYP/6-31+G(d) level in gas
phase followed by ECD calculations at B3LYP/6-31+G(d) level in
methanol. The overall ECD spectrum was weighted according to Boltz­
mann distribution, and the predicted curve was subsequently compared
with the experimental ones, respectively [31,32].
7
Chemico-Biological Interactions 362 (2022) 109998
Fig. 8. Compound 8 mediated inhibitory effects on
LPS-induced inflammation may partly dependent on
α7nAchR activation. (A) Compound 8 increased the
expression of α7nAchR. (B) Methyllycaconitine
treatment (100 nM) significantly weaken the inhibi­
tory effect of compound 8 (1.25 μM) on LPS-induced
inflammation. After indicated treatment, the culture
supernatant was collected and applied for Elisa assay
to detect TNF-α and IL-6 secretion. The data are
presented as mean ± SD, ***P < 0.001 compared
with the control group, #P < 0.05, ##P < 0.01 and
###
P < 0.001 compared with LPS group, &&P < 0.01
compared with compound 8 group.
4.5. Molecular docking
The ligand structures used for molecular docking were generated
with ChemDraw and prepared by LigPrep module of Glide in
Schrödinger. The receptor structure (PDB ID: 6F25) was downloaded
from the PDB Bank (http://www.rcsb.org) and prepared by hydroge­
nation and dewatered through Protein Preparation Wizard module. The
binding site used for docking was generated by Receptor Grid Genera­
tion module and the site size was controlled at 15 Å × 15 Å × 15 Å. The
dockings were carried out with Ligand Docking module. The in­
teractions between protein and ligands were analyzed by Pymol (version
2.2.2).
4.6. Anti-inflammatory activity
4.6.1. Reagents
The Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St
Louis, MO, USA). Radioimmunoprecipitation assay (RIPA) buffer and
Griess reagent for NO assay kit were supplied by Yeasen Biotech
(Shanghai, China). Quantikine Elisa kit of TNF-a and IL-6 were obtained
from NeoBioscience (Shenzhen, China). Antibodies against IKKα, IKKβ,
p-IKKa/βSer176/180, IκBα, p-IκBαSer32, NF-κB p65, p–NF–κB p65Ser536,
Stat3, p-Stat3Tyr705 and HRP-conjugated anti-rabbit IgG antibody were
provided by Cell Signaling Technology (Boston, MA, United States), and
the antibody of a7nAchR was purchased from Abcam (Cambridge, Mass,
UK). S-Acetylthiocholine iodide, S-butyrylthiocholine iodide, 5, 5′ -
dithio-bis-(2-nitrobenzoic) acid (DTNB, Ellman’s reagent) and AchE
derived from human erythrocytes were purchased from Sigma-Aldrich
(St. Louis, MO, USA). Methyllycaconitine was purchased from Target­
Mol, USA.
4.6.2. Cell culture and treatment
Murine macrophage RAW246.7 cells were obtained from American
Type Culture Collection (ATCC, Manassas, VA, USA). The cells were
maintained with RIPM-1640 medium supplemented with 10% fetal
J.-C. Su et al.
bovine serum and 1% penicillin/streptomycin solution in humidified
atmosphere at 37 ◦ C with 5% CO2. For inducing inflammation, the cells
were treated with 20 ng/mL LPS for 24 h following with or without
various concentration of compound 8 treatment for indicated times.
4.6.3. AchE activity assay
The enzymatic activity was detected based on Ellman assay as pre­
vious described [33]. In brief, compounds were dissolved in DMSO. The
reaction mixture (totally 200 μL) containing phosphate buffer (pH 8.0),
test compound, and acetyl cholinesterase (0.02 U/mL), was incubated
for 20 min (37 ◦ C). Then, the reaction was initiated by the addition of 40
μL of solution containing DTNB (0.625 mM) and acetylthiocholine io­
dide (0.625 mM) for AchE inhibitory activity assay, respectively. The
hydrolysis of acetylthiocholine was monitored at 405 nm every 30 s for
1 h. Tacrine was used as a positive control with final concentration of
0.333 μM. All the reactions were performed in triplicate. The percentage
inhibition was calculated as follows: % inhibition = (E − S)/E × 100 (E
is the activity of the enzyme without test compound and S is the activity
of enzyme with test compound).
4.6.4. Measurement of NO production
The NO level in culture media was determined using a Griess reagent
for NO kit. Briefly, the cytotoxicity of compound 8 was evaluated by
using a CCK8 assay kit (TargetMol, USA) as described previously. Then,
the cells were treated with or without various concentrations of com­
pound 8 in the presence and absence of LPS (20 ng/mL) for 24 h. The
supernatant medium was collected and mixed with same volume of
Griess reagent, and the absorbance was determined by a SynergyMx
Multi-Mode Microplate Reader (Biotek, Winooski, VT) at 550 nm. The
experiments were performed three times independently.
4.6.5. RNA extraction and quantitative real-time polymerase chain
reaction (RT-qPCR)
The total RNA was isolated from the cell using an RNA Extraction Kit
(Solarbio Science & Technology, Beijing, China) in accordance with the
manufacturer’s procedure. Complementary DNA (cDNA) was synthe­
sized from 1 μg total RNA using SuperScript® VILO cDNA Synthesis Kit
(Invitrogen). The RT-qPCR amplification was conducted with One-Step
SYBR PrimeScript Plus RT-PCR kit on LightCycler®480 Real-Time PCR
System (Roche, Swiss). The primer sequences for qRT-PCR were as fol­
lows: TNF-α-Forwards 5′ -CCCTCACACTCAGATCATCTTCT-3′ , Reverse
5′ -TGAGGCCATAGTAGAGTGTCCT-3′ , IL-6-Forwards 5′ - CCAA­
GAGGTGAGTGCTTCCC-3′ , Reverse 5′ - CTGTTGTTCAGACTCTCTCCCT-
3’. α7nAChR-Forwards 5′ - AATGCTGCAAAGAGCCATACC-3′ , Reverse
5′ -TGAGGCCATAGTAGAGTGTCCT-3′ , GAPDH-Forwards 5′ - CTTCATT­
GACCTCAACTACATGGTCTA-3′ , Reverse 5′ - GATGA CAAGCTTCCC
ATTCTCAG-3’. The fold change of mRNA was calculated using the
2− ΔΔCT method, and GAPDH was set as internal reference. The experi­
ments were conducted in triplicate.
4.6.6. ELISA assay for TNF-α and IL-6
The RAW 246.7 cells were treated with or without different con­
centration of compound 8 in the presence or absence of LPS for 24 h. The
culture supernatant was collected and used to detect TNF-α and IL-6
secretion following with the instruction provided by the manufacturer.
The absorbance was measured using a SynergyMx Multi-Mode Micro­
plate Reader (Biotek, Winooski, VT) at 450 nm. Three experiments were
conducted independently.
4.6.7. Western blotting assay
The cells with or without indicated treatment were collected and
lysed RIPA buffer containing phenylmethylsulfonyl fluoride and prote­
ase inhibitor cocktail. Equal amount of protein was electrophoresed in
10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels
(SDS-PAGE) and then the separated proteins were transferred to PVDF
membrane (Millipore, USA). After blocking with 5% BSA, the
8
Chemico-Biological Interactions 362 (2022) 109998
membranes were incubated with primary anti-bodies overnight at 4 ◦ C,
following by incubating with HRP-conjugated with secondary anti­
bodies at room temperature for 1.5 h. After that, the bands were visu­
alized using the enhanced chemiluminescence detection system. The
quantitative data of blots were measured with ImageJ software (NIH,
NY). And three independent experiments were conducted.
4.6.8. Statistical analysis
The data were analyzed using GraphPad Prism 8.0 software (San
Diego, USA) and presented as the mean ± standard deviation (SD).
Comparisons of data among three or more groups were analyzed by one-
way ANOVA multiple comparisons. Comparisons of data between two
groups were evaluated using a two-tailed unpaired t-test. P < 0.05 was
considered significant difference.
Author statement
Conceptualization, Jun-Cheng Su, Xueping Lei, and Peng Zhang;
Experiment implementation, Jun-Cheng Su and Xueping Lei; Data pro­
cessing, Qianrong Pan, Xingyuan Xu, and Xia Wei; Writing—original
draft preparation, Jun-Cheng Su; Writing—review and editing, Xueping
Lei and Peng Zhang. All authors have read and approved the final
manuscript.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This work was financially supported by the National Natural Science
Foundation of China (nos. 82003605 and 32070391), Science and
Technology Program of Guangzhou (202002030026), and Central
Public-interest Scientific Institution Basal Research Fund (Y2022QC32
and Y2021XK25).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.cbi.2022.109998.
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