Article
Figure 4. Effect of compounds 1−10 on the NGF-induced neurite outgrowth in PC12 cells. (Top) Morphological change of PC12 cells treated
with 2 and 9 at 20 μM. The cell morphological characteristics were observed under a light microscope. (Bottom) Percentage of neurite outgrowth
of PC12 cells after treatment with compounds 1−10 at 20 μM in the presence of NGF for 72 h.
1
0
Examination of DEPT and HSQC spectra of 5 indicated that a
Mortierella isabellina, Actinoplanes sp., Mucor recurvatus,
1
13
11
12
monohydroxylation had occurred. Comparison of H and
C
Cunninghamella bainieri, Bacillus megaterium, A. niger,
Gibberella fujikuroi, and Glomerella cingulata and Mortierella
These species have the capacity of functionalizing
1 or 2 at the C-7 or C-9 position. This is a common
15
NMR spectra of 2 and 5 revealed that the substitution of an
additional hydroxyl group in 5 might be at C-7. This coincided
25,26
elongate.
1
3
with the C NMR signals observed for downfield shifts of C-6
Δδ +9.0) and C-8 (Δδ +6.0) and upfield shifts of C-5 (Δδ
(
occurrence for other microorganisms with beyerane-type
4
,6,16
−
1.8), C-9 (Δδ −2.0), C-14 (Δδ −5.5), and C-15 (Δδ −9.9),
scaffolds.
respectively, in comparison with those of 2 (Table 2). In the
In this study, C. echinulata was accountable for the most
common biohydroxylation reactions that took place in the
microbial conversions, and several hydroxylated ent-beyerane
congeners (3−8) were prepared regio- and stereoselectively by
hydroxylation at 9β-, 12β-, 14α-, 7α- and 7α-, 7β-, 9β-positions
on the ent-beyerane skeleton by using this strain. Interestingly,
it has been reported that some xenobiotics are known to
HMBC spectrum, H-7 at δ 3.22 exhibited cross-peaks with C-
H
6
(δ 32.0), C-9 (δ 55.4), C-14 (δ 51.1), and C-15 (δ 33.7)
C C C C
(
7
Figure 1). Therefore, the hydroxylation occurred at C-7 (δC
6.6). In the H NMR spectrum, the α-configuration of the
1
OH group at C-7 was deduced from the double-doublet
6
multiplicity of the C-7 signal (1H, dd, J = 8.1, 2.8 Hz) in 5.
12,27
Furthermore, the relative stereochemistry of OH-7 was also
generate conjugates with glucose in mammalian systems.
confirmed by a NOESY experiment (Figure 1), in which H-7
Selected microbes, especially fungi, have been employed
successfully as in vitro models to predict and imitate the
metabolic fate of drugs and other xenobiotics in mammalian
(
δ 3.22) showed obvious correlations with H-5 (δ 1.01), H-
H
H
9
(δ 1.14), and H-14 (δ 0.81). Thus, the structure of 5 was
H H
28−30
established to be ent-7β,16β-dihydroxybeyeran-19-oic acid.
Metabolite 7 has a molecular formula of C H O , as
systems.
Compound 7, a conjugated fungal metabolite of
2, had been obtained previously through glucosidation by the
2
6
42
8
−
12
determined by HRESIMS at m/z: 481.2809 [M − H] . Six
additional resonances in the 13C NMR spectra of 7 (Table 2)
revealed a hexose moiety in the structure. The hexose moiety
was verified to be characteristic of ß-D-glucose by the NMR
spectra. The coupling constant of the anomeric proton at δH
bacterium B. megaterium. However, the glycoside formation
by C. echinulata has been first reported for the tetracyclic
beyerane diterpene motifs.
We next tested the neurite-outgrowth promoting, α-
glucosidase and NO production inhibiting, and phytotoxic
effects of the transformation products. Results showed that
only compounds 2 and 9 were found to promote neurite-
outgrowth effects in the presence of NGF in PC-12 cells, the
others did not display any activity (Figure 4). The prevalence
of neurodegenerative diseases (ND), such as Alzheimer’s
disease, will continue to increase steadily. Despite the
advancement of treatment, the management of these disorders
remains highly ineffective. Therefore, it is essential to discover
new biologically active natural products and analogues thereof
to mitigate ND. Over the past decades, neurotrophic factors,
for example, NGF, have attracted great interest because of their
5
.4 ppm (d, J = 8.0 Hz, H-1′) of the glucose residue in 7
disclosed the ß-configuration of the glucosidic linkage.
Comparison of the 13C NMR spectra of compounds 2 and 7
suggested that 7 was a glucopyranosyl ester of 2 at C-19 due to
the upfield shift of C-19 from 181.7 to 178.2 ppm (Table 2).
12
Based on the above evidence and literature data, the
structure of 7 was characterized as shown. This is the first
report to obtain the glucosidation of 2 by C. echinulata.
An array of microbial transformations of the two beyerane-
type diterpenoids, especially isosteviol (1), involved oxidation
reactions by different species of microorganisms, such as
4
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J. Agric. Food Chem. 2020, 68, 4624−4631