Neuroprotective Aurones from Sophora japonica

Article abstract of DOI:10.1007/s10600-019-02663-1

A new aurone, named (Z)-4,5-methylenedioxy-6-hydroxybenzene-2[(2′,3′,4′-trimethoxy)-2-methyl-2(4-methyl-3-penten-1-yl)-2H-1-benzopyran-5-yl)methylene]-3-benzofuranon (1), and nine known aurones (2–10) were isolated from Sophora japonica L. for the first time. Compounds 1–10 were evaluated for their neuroprotective activities against 6-OHDA-induced cell death in SH-SY5Y cells. Compounds 1, 3, 8, and 10 exhibited significant neuroprotective activities with IC₅₀ values in the range of 2.89–6.02 μM.

Full text of DOI:10.1007/s10600-019-02663-1

DOI 10.1007/s10600-019-02663-1
Chemistry of Natural Compounds, Vol. 55, No. 2, March, 2019
NEUROPROTECTIVE AURONES FROM Sophora japonica
1*
1*
2
Qinge Ma, Rongrui Wei, and Zhipei Sang
A new aurone, named (Z)-4,5-methylenedioxy-6-hydroxybenzene-2[(2′,3′,4′-trimethoxy)-2-methyl-2(4-methyl-
3-penten-1-yl)-2H-1-benzopyran-5-yl)methylene]-3-benzofuranon (1), and nine known aurones (2–10) were
isolated from Sophora japonica L. for the first time. Compounds 1–10 were evaluated for their neuroprotective
activities against 6-OHDA-induced cell death in SH-SY5Y cells. Compounds 1, 3, 8, and 10 exhibited
significant neuroprotective activities with IC values in the range of 2.89–6.02 μM.
50
Keywords: Sophora japonica L., aurone, neuroprotective.
Sophora japonica L. (Fabaceae) is a well-known herbal medicine that has been widely used in traditional Chinese
medicine for many years [1]. Modern pharmacological studies show that S. japonica possesses antihemostatic [2], antioxidant
[3], antiplatelet [4], aldose reductase inhibitory [5], tyrosinase inhibitory [6], and antimicrobial [7] activities, and that it can
effectively alleviate pancreas and kidney injury in the streptozotocin-induced diabetic mice model [8]. Previous phytochemical
studies indicated that S. japonica contained flavanones [9], alkaloids [10], saponins [11], organic acids [12], volatile oils [13],
and so on.
To the best of our knowledge, there are few reports on aurones and on the neuroprotective activities of S. japonica,
which prompted us to investigate its active compounds further. We carried out a bioassay-guided investigation of S. japonica
in order to evaluate its neuroprotective constituents. As a result, a new aurone (1) and nine known aurons (2–10) were obtained
from the active fractions (EtOAc fraction) of S. japonica on the basis of extensive spectral data and references, and the known
aurones 2–10 were isolated from this plant for the first time. Meanwhile, compounds 1–10 were evaluated for their
neuroprotective activities by the rotenone model and the free-serum model. Among them, compounds 1, 3, 8, and 10 showed
significant neuroprotective activities. In this paper, we mainly report the isolation and structural elucidation of compounds 1–10,
together with their neuroprotective activities.
+
Compound 1 was obtained as a yellow solid and showed the quasimolecular ion at m/z 545.1853 [M + Na] ,
corresponding to the molecular formula C H O in HR-ESI-MS. The IR spectrum of compound 1 showed absorptions of
29 30
–1
9
–1
–1
–1
hydroxyl (3408 cm ), carbonyl (1670 cm ), aromatic ring (1614 cm ), and methyl (1386 cm ) functionalities. Compound 1
was characterized as an aurone on the basis of color reactions and UV (MeOH, λ , nm): 208, 245, 268, and 340 [14].
max
1
The H NMR spectrum of compound 1 showed two singlets at δ 6.52 (1H, s, H-7) and 5.68 (2H, s, H-11) corresponded
to a single proton and a methylenedioxy group, respectively [14]. The H NMR spectrum of compound 1 showed two
1
cis olefinic protons at δ 6.87 (1H, d, J = 10.0 Hz, H-1′′) and 5.76 (1H, d, J = 10.0 Hz, H-2′′), two isolated olefinic protons at
13
δ 7.08 (1H, s, H-10) and 5.06 (1H, t, J = 7.0 Hz, H-7′′) corresponding to δ 106.9 (C-10) and 123.8 (C-7′′) of the C NMR and
C
HSQC spectra (Table 1), together with three methyls at δ 1.44 (3H, s, H-4′′), 1.64 (3H, s, H-9′′), and 1.59 (3H, s, H-10′′) and
H
two methylenes at δ 1.76 (2H, m, H-5′′) and 2.12 (2H, m, H-6′′), which indicated a geranyl aurone skeleton in compound 1
H
according to the biogenetic considerations and literature [15]. Additionally, there was a characteristic methoxyl peak at δ 3.77
1
(9H, s, 2′, 3′, 4′-OCH ) in the high H NMR spectrum of compound 1, corresponding to a carbon resonance at δ 56.3 (2′, 3′,
3
13
4′-OCH ) of the C NMR and HSQC spectra (Table 1).
3
1) Key Laboratory of Modern Preparation of TCM of Ministry of Education, Research Center of Natural Resources
of Chinese Medicinal Materials and Ethnic Medicine, Jiangxi University of Traditional Chinese Medicine, 330004, Nanchang,
P. R. China, e-mail: maqinge2006@163.com; weirongrui2011@163.com; 2) College of Chemistry and Pharmaceutical
Engineering, Nanyang Normal University, 473061, Nanyang, P. R. China, e-mail: sangzhipei2006@163.com. Published
in Khimiya Prirodnykh Soedinenii, No. 2, March–April, 2019, pp. 228–231. Original article submitted January 30, 2018.
0009-3130/19/5502-0265 ©2019 Springer Science+Business Media, LLC
265

1
13
TABLE 1. H (400 MHz) and C NMR (100 MHz) Data of Compound 1 (DMSO-d , δ, ppm, J/Hz)
6
C atom
C atom
δH
δC
δH
δC
2
3
4
5
6
7
8
9
10
11
1′
2′
–
–
–
–
159.6
175.9
135.1
129.4
148.2
98.9
156.2
100.1
106.9
92.4
–
–
138.6
112.8
119.1
131.4
78.4
25.6
40.4
22.6
123.8
131.4
25.3
17.5
56.3
5′
′
6
6.87 (1H, d, J = 10.0)
5.76 (1H, d, J = 10.0)
–
1.44 (3H, s)
1.76 (2H, m)
2.12 (2H, m)
5.06 (1H, t, J = 7.0)
–
1′′
′′
2
–
3′′
4′′
5′′
6′′
7′′
8′′
9′′
10′′
6.52 (1H, s)
–
–
7.08 (1H, s)
5.68 (2H, s)
–
–
–
–
110.8
141.2
147.8
146.5
1.64 (3H, s)
1.59 (3H, s)
3.77 (9H, s)
′
′ ′ ′
2 , 3 , 4 -OCH3
3
4′
OCH
H CO
3
3
3'
1'
4''
5'
H CO
O
CH
3
3
7
8
HO
O
3''
O
1
5''
9''
CH
10
5
3
3
1''
9
8''
7''
O
CH
3
O
11
10''
1
1
1
HMBC
2D NOESY
H- H COSY
1
1
Fig. 1. Key HMBC, 2D NOESY, and H– H COSY
correlations of compound 1.
In the HMBC spectrum of compound 1, the connectivity within the fragments of compound 1 was established on the
HMBC correlations of H-7/C-5, C-9; H-10/C-3, C-2′, C-6′; H-11/C-4, C-5; H-1′′/C-5′, C-3′′; H-7′′/C-5′′ (Fig. 1), the 2D NOESY
1
1
correlations of H-7′′/9′′-CH , H-7′′/10′′-CH (Fig. 1), and the H– H COSY correlations of H-1′′/H-2′′; H-5′′/H-6′′; H-6′′/H-7′′
3
3
(Fig. 1). According to above spectral evidences, compound 1 was determined to be (Z)-4,5-methylenedioxy-6-hydroxybenzene-
2[(2′,3′,4′-trimethoxy)-2-methyl-2(4-methyl-3-penten-1-yl)-2H-1-benzopyran-5-yl)methylene]-3-benzofuranon.
Additionally, another nine known aurones (2–10) were identified by comparison of their spectroscopic data
with those reported in the references. Their structures were determined as cephalocerone (2) [14], altilisin I (3) [15],
(Z)-6,7,3′,4′-tetrahydroxyaurone (4) [16], (Z)-4,6,4′-trihydroxyaurone (5) [17], (Z)-6-O-β-D-glucopyranosyl-7,3′,4′-
trihydroxyaurone (6) [16], (Z)-4,6,3′,4′-tetrahydroxyaurone (7) [17], (Z)-4-O-β-D-glucopyranosyl-7,3′,4′-trihydroxyaurone
(8) [17], (Z)-4-methoxy-6,4′-dihydroxyaurone (9) [18], and (Z)-3′-O-β-D-glucopyranosyl-4,5,6,4′-tetrahydroxy-7,2′-
dimethoxyaurone (10) [19]. Moreover, the known compounds (2–10) were isolated from this plant for the first time.
Neuroprotective Activity. Compounds 1–10 were evaluated for their neuroprotective activities against 6-OHDA-induced
cell death in SH-SY5Y cells with curcumin as the reference compound. It was found that the selective compounds exhibited
significant neuroprotective activities with IC 2.89–6.02 μM. Among them, compound 1 showed the strongest neuroprotective
50
activities with IC values of 2.89 μM, and compounds 3, 8, and 10 showed moderate neuroprotective activities with
50
IC values of 3.56, 6.02, and 4.72 μM, respectively. However, compounds 2, 4, 5, 6, 7, and 9 exhibited no neuroprotective
50
activities compared with the standard drug curcumin with IC value greater than 6.51 μM.
50
266

OH
H CO
3
OCH
3
H CO
O
O
H CO
3
O
O
CH
3
CH
3
3
HO
HO
O
CH
CH
CH
CH
3
3
3
O
O
3
O
3
1
HO
O
OH
OH
HO
OH
O
OH
HO
O
OH
H CO
3
H CO
3
O
O
HO
HO
O
OH
O
OH
O
OH
10
8
OH OH
EXPERIMENTAL
General Procedures. The UV spectral data were measured by an Australian GBC UV-916 spectrophotometer,
and the IR spectral data were measured by a Nicolet 5700 FT-IR spectrometer with KBr pellets. The HR-ESI-MS data
were recorded on an Agilent 1100 series LC/MSD ion trap mass spectrometer, and the ESI-MS data were recorded on an
LTQ Orbitrap XL spectrometer. The 1D and 2D NMR spectra were run on a Bruker-400 with TMS as internal standard.
Column chromatography was performed on silica gel (100–200 mesh or 200–300 mesh), Sephadex LH-20, and Toyopearl
HW-40. TLC was performed on precoated silica gel GF254 plates, and the spots were visualized under UV light (254 or
365 nm) or by spraying with 10% H SO in 95% EtOH followed by heating [20].
2
4
Plant Material. S. japonica was harvested from Nanyang, Henan Province, China, in April 2017. This plant was
identified by Dr. Su Zhang of Wuyang Weisen Biological Medicine Co., Ltd. The voucher specimen (No. YHH-201704) has
been deposited in Nanyang Normal University.
Extraction and Isolation. The air-dried S. japonica (10.0 kg) was extracted with 90% EtOH (12 L × 3) three times
under reflux conditions to obtain a black residue (0.9 kg). The combined extract was successively suspended in H O and
2
partitioned with petroleum ether, EtOAc, and n-butanol to obtain the petroleum ether soluble fraction (58.6 g), EtOAc soluble
fraction (234.2 g), and n-butanol soluble fraction (301.5 g). According to the screening results of the bioactivity-guided
investigation, it was found that the EtOAc soluble fraction exhibited potential neuroprotective activity.
The EtOAc soluble fraction was applied to a column (100–200 mesh) and eluted with petroleum ether–acetone
(10:1, 8:1, 6:1, 4:1) to obtain four fractions: A (42.6 g), B (84.5 g), C (59.7 g), and D (22.8 g). According to the results of
pharmacological screening, the B part exhibited potential neuroprotective activity. Therefore, the B part was purified on the
Sephadex LH-20 column (eluted with MeOH in H O, 85%, 95%, 100%, in a step gradient manner) to afford three subfractions,
2
B1 (19.8 g), B2 (34.5 g), and B3 (21.7 g). The separation of B2 was performed on a silica gel (100–200 mesh) column eluting
with a gradient of CHCl –CH OH–H O (10:1:0.5, 7:1:0.5) to obtain two fractions: B2a (12.4 g) and B2b (20.6 g). The B2a
3
3
2
part was repeatedly chromatographed over silica gel (200–300 mesh), Sephadex LH-20, and Toyopearl HW-40 to yield 1
(8.69 mg), 2 (10.54 mg), 3 (9.64 mg), and 9 (12.57 mg). Similarly, the B2b part was chromatographed on silica gel
(100–200 mesh or 200–300 mesh), Sephadex LH-20, and Toyopearl HW-40 eluting with suitable mobile phases repeatedly to
yield 4 (11.24 mg), 5 (9.77 mg), 6 (10.25 mg), 7 (8.98 mg), 8 (11.35 mg), and 10 (11.47 mg).
Acid Hydrolysis of Compounds 6, 8, and 10. Compounds 6, 8, and 10 (4 mg each) were treated in 5% HCl (0.5 mL)
and heated at 90°C for 2 h. Each reaction mixture was extracted with EtOAc after cooling, and the aqueous layer was neutralized
with 0.1 M NaOH. The glucose was obtained from compounds 6, 8, and 10, which was detected by thin-layer chromatography
(TLC) with authentic sugars. Finally, the type of glucose was identified by TLC with authentic sugars. With CHCl –MeOH–H O
3
2
(14:6:1, Kieselgel 60 F 254 plate), the R value of glucose was 0.14. Meanwhile, with n-BuOH–pyridine–H O (6:4:3, cellulose
f
2
60 F plate), the R value of glucose was 0.38 [21]. Therefore, the type of sugar from compounds 6, 8, and 10 was identified
f
as glucose.
267

Neuroprotective Assay. Compounds 1–10 were assayed for their neuroprotective activities against 6-OHDA-induced
cell death in SH-SY5Y cells, and the pharmacological screening results were typically expressed as IC with curcumin as
50
4
positive control [22]. The SH-SY5Y cells were cultured in 96-well plates at a density of 2 × 10 cells/well in 200 μL for 24 h
and treated with 100 μM 6-hydroxydopamine and various concentrations of compounds 1–10 for an additional 24 h. The
viability of the cell was obtained by treating it with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT),
which was dissolved in 0.5 mg/mL of phosphate-buffered saline (PBS) at 37°C for 4 h. The formazan crystals were dissolved
using dimethyl sulfoxide (DMSO) after removing the PBS. The absorbance of individual wells was measured with a microplate
reader (BioTek, VT, USA) at 540 nm. The neuroprotective activity against 6-OHDA-induced cell death was calculated by a
semilogarithmic graph depicting the relationship between at least four different concentrations of compounds and their percentage
effects [23].
In this work, 10 aurones (1–10) from S. japonica were isolated and identified by their spectral data and from references.
Among them, compound 1 was a new aurone, named (Z)-4,5-methylenedioxy-6-hydroxybenzene-2-[(2′,3′,4′-trimethoxy-2-
methyl-2(4-methyl-3-penten-1-yl)-2H-1-benzopyran-5-yl)methylene]-3-benzofuranon, and the known compounds (2–10) were
obtained from this plant for the first time. Moreover, compounds (1–10) were evaluated for neuroprotective activities against
6-OHDA-induced cell death in SH-SY5Y cells with curcumin as the standard drug. Compounds 1, 3, 8, and 10 showed
significant neuroprotective activities with IC = 2.89–6.02 μM. These research results may guide the search for new natural
50
products with neuroprotective attributes.
ACKNOWLEDGMENT
This work was supported by the Key Scientific Research Project of Colleges and Universities in Henan Province
(No. 19A350006), the National Natural Science Foundation of China (No. 81803843), and the Standard Revision Research
Project of National Pharmacopoeia Committee (No. 2018Z090). We are grateful to the Department of Instrumental Analysis
of Nayang Normal University and Jiangxi University of Traditional Chinese Medicine for measurements of UV, IR, NMR, and MS.
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