Ent-kaurane diterpenoids from the pericarps of Datura metel L. acted on the vascular endothelial cells via TRPC6 and NF-κB protein

Article abstract of DOI:10.1007/s00044-017-2046-z

Two new ent-kaurane diterpenoid glycosides, namely kaurane acid glycosides B–C (1–2), together with three known compounds, 16β,17-dihydroxy-ent-kauran-19-oic acid (3), paniculoside-IV (4), and kaurane acid glycoside A (5), were isolated from the n-butyl a

Full text of DOI:10.1007/s00044-017-2046-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2046-z
ORIGINAL RESEARCH
Ent-kaurane diterpenoids from the pericarps of Datura metel L.
acted on the vascular endothelial cells via TRPC6 and NF-κB
protein
1 ●
1 ●
1 ●
1 ●
1
Bing-you Yang Yong-qiang Zhou Yan Liu Zhen-kun Lu Hai-xue Kuang
Received: 7 February 2017 / Accepted: 20 August 2017
©
Springer Science+Business Media, LLC 2017
Abstract Two new ent-kaurane diterpenoid glycosides,
namely kaurane acid glycosides B–C (1–2), together with
three known compounds, 16β,17-dihydroxy-ent-kauran-19-
oic acid (3), paniculoside-IV (4), and kaurane acid
glycoside A (5), were isolated from the n-butyl alcohol-
soluble fractions of ethanol extracts of dried pericarps
of Datura metel L. Compounds 3–5 were isolated from
Solanaceae for the first time. The structures of these com-
pounds were identified by the analyses of spectroscopic
data. All isolates were screened for their bioactivities by
acting on the vascular endothelial cells. It was determined
that compounds 1–4 exhibited potential inhibiting activities
of vascular endothelial cells with IC50 > 79.5 μg/mL.
Moreover, compound 5 at the concentration of 5 μg/mL
could promote the proliferation of vascular endothelial
cells by increasing the expression of TRPC6 protein, and
reduce the expression of NF-κBp65 protein in a dose-
dependent manner.
Introduction
Datura metel L. is an erect branched undershrub belonging
to Solanaceae with long white flowers and spiny spherical
fruits (Nisar et al. 2011). The whole plant is considered to
be as narcotic, anodyne, and antispasmodic (Chinese
Pharmacopoeia Commission 2010). Importantly, some
researchers have reported that fruits are used for the treat-
ment of catarrh, rheumatic pains, skin-ulcers, bronchitis,
and wounds (Zia-Ul-Haq et al. 2011, Cowan 1999). Other
studies have also demonstrated that its fruits possess anti-
oxidant and antimicrobial abilities (Anju and Ratan 2011,
Okwu and Igara 2009). These bioactivities are often closely
related with different kinds of chemical constituents from
the fruit of D. metel.
Recently, researchers have extensively investigated the
active constituents of the D. metel flower, which mainly
contain withanolides (Kuang et al. 2008), flavonoids
(
Mohammad et al. 2014), and alkaloids (Siddiqui et al.
●
●
●
Keywords Solanaceae Ent-kaurane diterpenoids TRPC6
NF-κB HUVECs
1986). In the literatures, chemical researchers have only
reported that the fruits contain some extra chemical ingre-
dients, such as β-sitosterol, triterpene, and daturadiol (Oseni
et al. 2011, Mann et al. 1984). However, there is little
information reported regarding whether fruits have the other
important chemical constituents and pharmacological
activities.
●
In this report, in order to expand the available
resources and search for new bioactive components of
D. metel, we describe the isolation and structural
elucidation of ent-kaurane diterpenoids from pericarps
of D. metel. All isolates were evaluated for their bioactiv-
ities by acting on the vascular endothelial cells via TRPC6
and NF-κB.
Electronic supplementary material The online version of this article
https://doi.org/10.1007/s00044-017-2046-z) contains supplementary
material, which is available to authorized users.
(
*
Hai-xue Kuang
hxkuang@126.com
1
Key Laboratory of Chinese Materia Medica of Ministry of
Education Heilongjiang University of Chinese Medicine 150040
Harbin, China

Med Chem Res
Materials and methods
C18 column (250 × 10 mm i.d., 5 μm) (55% MeOH/H O,
2
flow rate, 3 mL/min).
General experimental procedures
7
β,16α,17-trihydroxy-ent-kauran-19-oic acid 19-O-β-D-
NMR spectra were recorded in methanol and pyridine with
glucopyranoside ester (1)
tetramethylsilane as an internal standard on Bruker DPX
1
1
4
00 instrument (400 MHz for H-NMR and 100 MHz for
H-NMR (CD OD, 400 MHz): δ 0.91(1H,m,H-1a), 1.86
3
H
1
3
C-NMR). Coupling constants (J) were given in Hz.
(1H,m,H-1b), 1.41(1H,m,H-2a), 1.96(1H,m,H-2b), 1.08
(1H,dd,4.3,13.5,H-3a), 2.17(1H,br.s,H-3b), 1.78(1H,
Chemical shifts (δ) were expressed in ppm with reference to
the solvent signals. HR-ESI-MS was measured by a Waters
Xevo-TOF-MS™ instrument. Semi-preparative high-per-
dd,1.6,13.2,H-5), 1.95(1H,m,H-6a), 2.21(1H,m,H-6b), 3.61
(1H,br.s,H-7), 1.39(1H,d,6.3,H-9), 1.51(1H,m,H-15a), 1.74
(1H,m,H-15b), 1.57(1H,m,H-11a), 1.64(1H,m,H-11b), 1.57
(1H,m,H-12a), 1.68(1H,m,H-12b), 2.02(1H,m,H-13), 1.71
(1H,m,H-14a), 1.19(1H,s,H-18), 0.97(1H,s,H-20), 1.85(1H,
d,11.3,H-14b), 3.61(1H,d,11.4,H-17a), 3.70(1H,d,11.4,H-
17b), 5.38(1H,d,7.9,H-1′), 3.38(1H,m,H-2′), 3.41(1H,m,H-
3′), 3.39(1H,m,H-4′), 3.36(1H,m,H-5′), 3.70(1H,d,11.3,H-6′
formance liquid chromatography (HPLC) (Shimadzu, LC-
TM
6
AD) was performed using Waters SunFire C18 column
(
250 × 10 mm i.d., 5 μm). Gas chromatography (GC) ana-
lysis was performed on Agilent 7890A-5975C gas chro-
matograph equipped with a DB-1701 column (60 m × 0.25
mm × 0.25 μm, film thickness). Bio-Rad electrophoresis
apparatus, containing a silica gel of 200–300 mesh size, was
purchased from Qingdao Marine Chemical Ltd., Qingdao,
China. All the solvents used were of analytical grade and
obtained from Tianjinfuyu Company Ltd., Tianjin, China.
1
3
a), 3.82(1H,d,11.3,H-6′b). C-NMR (CD3OD, 400 MHz):
δH 41.6(C-1), 20.1(C-2), 39.1(C-3), 44.6(C-4), 49.8(C-5),
30.3(C-6), 78.1(C-7), 49.6(C-8), 51.0(C-9), 40.5(C-10),
19.2(C-11), 27.6(C-12), 46.1(C-13), 37.3(C-14), 50.1(C-
1
5), 82.9(C-16), 66.7(C-17), 28.8(C-18), 178.6(C-19), 16.2
(C-20), 95.7(C-1′), 74.1(C-2′), 78.7(C-3′), 71.1(C-4′), 78.7
C-5′), 62.4(C-6′).
Plant material
(
Pericarps of D. metel L. were collected in September 2014
from Harbin, Heilongjiang Province of China and identified
by Prof. Lian-Jie Su of Heilongjiang University of Chinese
Medicine. Voucher specimens (number 20140905) have
been deposited at Heilongjiang University of Chinese
Medicine.
16α,17-dihydroxy-ent-kauran-19-oic acid 19-O-β-D-
glucopyranosy-(6′ → 1′′′) -β-D-glucopyranosy-(2′ → 1′′) -β-D-
glucopyranoside (2)
1
H-NMR (CD OD, 400 MHz): δ 0.79 (1H,m,H-1a), 1.88
3
H
(1H,m,H-1b), 1.41 1H,m,H-2a), 1.92 (1H,m,H-2b), 1.01
Extraction and isolation
(1H,m,H-3a), 2.28 (1H,d,13.5,H-3b), 1.08 (1H,dd,3.1,11.4,
H-5), 1.85 (1H,m,H-6a), 1.91 (1H,m,H-6b), 1.44 (1H,m,H-
7a), 1.64 (1H,m,H-7b), 1.01 (1H,m,H-9), 1.57 (1H,m,H-
11a), 1.62 (1H,m,H-11b), 1.54 (1H,m,H-12a), 1.64 (1H,m,
H-12b), 2.03 (1H,br.s,H-13), 1.64 (1H,m,H-14a), 1.96 (1H,
m,H-14b), 1.43 (1H,m,H-15a), 1.54 (1H,m,H-15b), 3.58
(1H,d,11.3,H-17a), 3.68 (1H,d,11.3,H-17b), 1.23 (1H,s,H-
18), 0.94 (1H,s,H-20), 5.53 (1H,d,8.0,H-1′), 3.86 (1H,m,H-
2′), 3.54 (1H,m,H-3′), 3.53 (1H,m,H-4′), 3.36 (1H,m,H-5′),
3.81 (1H,d,4.5,H-6′a), 4.11 (1H,d,10.6,H-6′b), 4.81 (1H,
d,7.8,H-1′′), 3.20 (1H,m,H-2′′), 3.67 (1H,m,H-3′′), 3.27
(1H,m,H-4′′), 3.27 (1H,m,H-5′′), 3.68 (1H,m,H-6′′a), 3.87
(1H,m,H-6′′b), 4.35 (1H,d,8.0,H-1′′′), 3.20 (1H,m,H-2′′′),
The air-dried pericarps of D. metel (35 kg) were extracted
with 70% ethanol (350 L) for 2 h × 3 times. The ethanol
extract was evaporated to dryness using a rotary evaporator
and the residue (7.1 kg) was suspended in water, and
extracted with petroleum ether, EtOAc, and n-BuOH. The
n-butyl alcohol-soluble fraction (200 g) was chromato-
graphed on a silica gel column and eluted with a CH CI /
2
2
MeOH (20:1 → 1:1, v/v) gradient, to yield five fractions
(
A–E) according to thin layer chromatography. Fraction A2
(
11.6 g) was subjected to octadecylsilane (ODS) column
chromatography with MeOH/H O (2:8–1:0) to afford sub-
2
fractions A1–A12. Sub-fraction A3 (2.4 g) was finally
purified by semi-preparative HPLC using Waters SunFire
3.27 (1H,m,H-3′′′), 3.27 (1H,m,H-4′′′), 3.36 (1H,m,H-5′′′),
TM
13
3.68 (1H,m,H-6′′′a), 3.87 (1H,m,H-6′′′b).
C-NMR
C18 column (250 × 10 mm i.d., 5 μm) (58% MeOH/H O,
(CD OD, 400 MHz): δH 41.8 (C-1), 20.4 (C-2), 38.9 (C-
2
3
flow rate, 3 mL/min) to yield compound 1 (12 mg) and
compound 4 (62 mg). Compound 3 (43 mg) was obtained
from fraction A4 (4.1 g) by ODS column chromatography
3), 45.8 (C-4), 58.5 (C-5), 23.1 (C-6), 43.4 (C-7), 45.3 (C-
8), 57.3 (C-9), 40.9 (C-10), 19.6 (C-11), 27.2 (C-12), 46.2
(C-13), 38.6 (C-14), 53.8 (C-15), 82.9 (C-16), 66.9 (C-17),
29.5 (C-18), 177.8 (C-19), 17.2 (C-20), 94.1 (C-1′), 78.8
(C-2′), 77.8 (C-3′), 71.0 (C-4′), 77.9 (C-5′), 69.8 (C-6′),
103.9 (C-1′′), 75.9 (C-2′′), 78.6 (C-3′′), 72.0 (C-4′′), 78.3
with MeOH/H O (2:8–1:0). Compound 2 (15 mg) and
2
compound 5 (35 mg) were isolated from sub-fraction A5
TM
(
2.7 g) by semi-preparative HPLC using Waters SunFire

Med Chem Res
(
7
C-5′′), 62.8 (C-6′′) 104.9 (C-1′′′), 75.2 (C-2′′′), 78.0 (C-3′′′),
1.6 (C-4′′′), 77.9 (C-5′′′), 63.3 (C-6′′′).
ELISA assay. The ELISA kit is commercially available and
the manufacturers’ instructions are followed.
Acid hydrolysis of compound 1 and GC analysis
Western blotting
Compounds 1 and 2 (2.0 mg) were dissolved in 1 M HCl
The cells were treated with compound 5 for 24 h and washed
twice with ice-cold PBS, and proteins were then extracted
with radioimmunoprecipitation assay buffer (200 mM NaCl,
1% Triton X-100, 20 mM Tris–HCl and 1 mM dithio-
threitol), containing 1 mM phenylmethylsulfonyl fluoride
and 1% protease inhibitor cocktail (Sigma). The total protein
concentration was measured through a bicinchoninic acid
protein assay and the proteins were stored at −20 °C. Fifty
micrograms of proteins were subjected to SDS-
polyacrylamide gel electrophoresis, and the separated pro-
teins were transferred to nitrocellulose filter membranes. The
membranes were blotted with 5% fat-free milk for 1 h at
room temperature and then incubated with the following
primary antibodies: anti-β-actin, anti-TRPC6, and anti-NF-
κBp65. Subsequently, the blots were washed with Tris
Buffered Saline with Tween-20 (10 mM Tris–HCl, 50 mM
NaCl, and 0.25% Tween 20), incubated with an horse radish
peroxidase-conjugated secondary antibody, visualized with
enhanced chemiluminescence and exposed to photographic
film. β-actin was used as a loading control.
(
2.0 mL) and hydrolyzed at 90 °C for 3 h. After this period,
NaHCO was added into the reaction mixture for adjusting
3
pH to neutral. Then the reaction mixture was extracted with
ethyl acetate (3 × 5 mL). The aqueous layer was evaporated
to dryness. The residue of sugar was dissolved in 1 mL
anhydrous pyridine and treated with L-cysteine methyl ester
HCl (1.5 mg) and stirred at 60 °C for 1 h, then 0.6 mL
hexamethyldisilazane and 0.3 mL trimethylchlorosilane
were added, and the mixture was stirred at 60 °C for another
3
0 min. The residue was partitioned between n-hexane and
H O (0.1 mL each), and the hexane layer (1 μL) was ana-
2
lyzed by GC (Wu et al. 2012, Qin et al. 2012). The con-
figuration of D-glucose for compounds 1 and 2 was
determined by comparison of the retention times of corre-
sponding derivatives with those of standard D-glucose,
giving a single peak at 17.06 min.
Cell proliferation assay
Human umbilical vein ECs (HUVECs) were maintained in
Dulbecco’s modified eagle medium (DMEM) supplemented
with 10% fetal bovine serum, 100 U/mL of penicillin, and
Statistical analyses
1
00 μg/mL streptomycin. Compounds 1–5 were dissolved
Data are presented as mean ± SD from three independent
experiments. Statistical significance was carried out using
one-way analysis of variance. P < 0.05 was considered
statistically significant.
in 0.1% dimethyl sulphoxide (DMSO) to get final con-
centrations of 5, 10, 20, 40, and 80 μg/mL, respectively.
The cells (5 × 10 cells/well in a 96-well plate) were
3
maintained in growth media for 24 h at 37 °C in the pre-
sence of 5% CO . Once they reached 80% confluence, the
2
cells were treated with various concentrations of com-
Results and discussion
pounds 1–5 for 24 h. Cell proliferation was determined by
3
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
Identification of compounds’ structures
mide (MTT) assay. After treatment, 10 μL of MTT (5 mg/
mL) was added to each well and the samples were incubated
in the dark for 4 h at 37 °C. The medium was discarded and
the precipitated formazan was dissolved in 100 μL of
DMSO per well. The absorbance at 570 nm was measured
by microplate reader. The cell proliferation in DMEM
without compounds 1–5 was used as the control and con-
sidered 100%, and the degree of cell proliferation level with
compounds 1–5 treatments was expressed as a percentage
of the control.
The structures of three known ent-kaurane-type diterpe-
noids 3–5 were identified by comparing spectroscopic data
with previously reported results on 16β,17-dihydroxy-ent-
kauran-19-oic acid (Chou and Wang 2006), paniculoside-IV
(Cai et al. 2003), and kaurane acid glycoside A (Xian and
Qian 2010) (Fig. 1).
Compound 1 was obtained as a white powder. HR-ESI-
MS analysis revealed the molecular formula to be
+
C H O based on the [M + Na] signal at m/z 537.2643
(
2
6 42 10
1
calcd. for 537.2676). The H-NMR spectrum of compound
Cytokine analysis by enzyme-linked immunosorbent
assay (ELISA)
1 showed the signals for protons of two methyl groups at δH
0.97 (3H, s) and 1.19 (3H, s), one oxymethine at δ 3.61
H
(
br.s), and one oxymethylene group at δ 3.61 (d, J = 11.4
H
The concentration of vascular endothelial growth factor
Hz) and 3.70 (d, J = 11.4 Hz). One anomeric proton at δH
5.38 (1H, d, J = 7.9 Hz) suggested the presence of a sugar
(
VEGF) in cell-free culture supernatants was detected by

Med Chem Res
Fig. 1 Structure of compounds 1–5 from the pericarp of D. metel L
Fig. 2 Selected HMBC and
Correlation Spectroscopy
correlations of compounds 1
and 2
1
3
moiety. The C-NMR and distortionless enhancement by
polarization transfer (DEPT) spectra revealed the presence
of 26 carbons, including one carboxyl, four quaternaries
H-6 (δ 1.95 and 2.21) to C-7 (δ 78.1). The HMBC cor-
H
C
relations between H-20 (δ 0.97) and C-1 (δ 41.6)/C-5 (δ
C
H
C
49.8)/C-9 (δ 51.0)/C-10 (δ 40.5) confirmed the position
C
C
(
(
including an oxygenated carbon at δ 82.9), nine methines
including six oxymethines), ten methylenes (including two
of methyl group at C-10. The configuration of the methyl
group at C-4 was confirmed to be β by the observation of
Nuclear Overhauser Enhancement Spectroscopy (NOESY)
correlations between H-18 (δ 1.19) and H-5 (δ 1.78) and
C
oxymethylenes), and two methyl carbons. A comparison of
1
3
the C NMR data of 1 to those of 7β,16α,17-trihydroxy-
ent-kauran-19-oic (Nhiem et al. 2015) suggested that com-
pound 1 possessed the same ent-kaurane skeleton except for
the additional glucose at C-19. The 1H detected hetero-
nuclear multiple bond correlation (HMBC) between H-18
H
H
the NOESY correlation of H-7 (δH 3.63)/H-14 (δH 1.70)
indicated the configuration of the hydroxyl group at C-7 to
be β (Fig. 3). The configuration of the hydroxyl group at C-
16 was confirmed to be α by comparing the chemical shifts
of C-16 (δ 82.9) and C-17 (δ 66.7) in compound 1 with
(
δ 1.19) and C-3 (δ 39.1)/C-4 (δ 44.6)/C-5 (δ 49.8)/C-
H C C C
C
C
1
9 (δ 178.6) confirmed the positions of both methyl and
the data of the 7β,16α,17-trihydroxy-ent-kauran-19-oic
(Nhiem et al. 2015). Acid hydrolysis of 1 provided D-glu-
cose. In addition, the coupling constant of glc H-1′ and glc
H-2′, J = 7.9 Hz indicated the configuration of hydroxyl
group at anomeric carbon in sugar to be β. The position of
this sugar at C-19 of aglycone was confirmed by HMBC
C
carboxylic groups at C-4 (Fig. 2). The HMBC correlations
from H-17 (δ 3.61 and 3.70) to C-16 (δ 82.9)/C-13 (δ
C
H
C
4
6.1)/ C-15 (δ 50.1) confirmed two hydroxyl groups at C-
C
1
6 and C-17. The position of the hydroxyl group at C-7 was
determined by the HMBC correlations from H-5 (δ 1.78)/
H

Med Chem Res
Fig. 3 Key NOESY correlations
of compounds 1 and 2
correlations between glc H-1′ (δH 5.38) and C-19
(
δC 178.6). Based on the above evidences, the structure of
compound 1 was identified as 7β,16α,17-trihydroxy-
ent-kauran-19-oic acid 19-O-β-D-glucopyranoside ester
(
Fig. 1).
Compound 2 was obtained as a white powder. HR-ESI-
MS analysis revealed the molecular formula to be
+
C H O based on the [M + Na] signal at m/z 845.3746
(
3
8 62 19
1
calcd. for 845.3783). The H-NMR spectrum of compound
2
showed the signals for protons of two methyl groups at δH
0
.94 (3H, s) and 1.23(3H, s), and one oxymethylene group
at δ 3.58 (d, J = 3.4 Hz) and 3.68 (d, J = 5.5 Hz). Three
H
anomeric protons at δH 5.51 (1H, d, J = 8.0 Hz), δH 4.81
Fig. 4 Effects of compounds 1–5 on the viability of HUVECs (treated
(
1H, d, J = 7.8 Hz), and δ 4.35 (1H, d, J = 7.8 Hz), sug-
H
for 24 h). The viability of HUVECs was assessed by MTT assay. The
1
3
★
*
gested the presence of three sugar moieties. The C-NMR
and DEPT spectra revealed the presence of 38 carbons,
including one carboxyl, four quaternaries (including an
results were expressed as means ± SD (n = 4). P < 0.05 and P <
0.05 vs. control group
8
2.9) and C-17 (δ 66.7) in compound 2 with the data of the
C
oxygenated carbon at δ 82.9), 18 methanes (including 15
C
7
β,16α,17-trihydroxy-ent-kauran-19-oicb (Nhiem et al.
oxymethines), 13 methylenes (including four oxymethy-
1
13
2015). Acid hydrolysis of 2 provided D-glucose. In addition,
the coupling constant of glc H-1′ and glc H-2′, J = 8.0 Hz,
glc H-1′′ and glc H-2′′, J = 7.8 Hz, and glc H-1′′′ and glc H-
lenes), and two methyl carbons. The H and C NMR data
of 2 were similar to those of kaurane acid glycoside A (Xian
and Qian 2010), except for the additional glucose at C-6′
2
′′′, J = 7.8 Hz, indicated the configuration of hydroxyl
(
(
6′ → 1′′′) was confirmed by HMBC cross peaks from H-6′
δ 3.79 and 4.10) to C-1′′′ (δ 104.9) (Fig. 2). The HMBC
group at anomeric carbon in sugar to be β. The position of
the sugar at C-19 of aglycone was confirmed by HMBC
correlation between glc H-1′ (δ 5.53) and C-19 (δ 177.8).
H
C
correlations between H-18 (δ 1.23) and C-3 (δ 38.6)/C-4
H
C
H
C
(
δC 45.8)/C-5 (δC 58.5)/C-19 (δC 177.8) confirmed the
The glucose at C-2′ (1 → 2) was confirmed by HMBC cross
peaks from H-2′ (δ 3.86) to C-1′′ (δ 103.9). Based on the
positions of both methyl and carboxylic groups at C-4. The
H
C
HMBC correlations from H-17 (δ 3.58 and 3.68) to C-16
H
data above, the structure of compound 2 was elucidated to
be 16α,17-dihydroxy-ent-kauran-19-oic acid 19-O-β-D-glu-
(
δC 82.9)/C-13 (δC 46.2)/ C-15 (δC 53.8) confirmed two
hydroxyl groups at C-16 and C-17. The HMBC correlations
copyranosy-(6′ → 1′′′)-β-D-glucopyranosy-(2′ → 1′′)
-β-D-
between H-20 (δ 0.94) and C-1 (δ 41.8)/C-5 (δ 58.5)/C-
H
C
C
glucopyranoside (Fig. 1).
9
(δ 57.3)/C-10 (δ 40.9) confirmed the position of methyl
C C
group at C-10. The configuration of the methyl group at C-4
was confirmed to be β by the observation of NOESY cor-
relation between H-18 (δ 1.23) and H-5 (δ 1.08) (Fig. 3).
The configuration of the hydroxyl group at C-16 was con-
firmed to be α by comparing the chemical shifts of C-16 (δC
Biological activity
H
H
As a kind of plant secondary metabolite, the pharmacological
activities of ent-kaurane diterpenoids were demonstrated to
possess anti-angiogenic, anti-cancer, anti-Alzheimer’s

Med Chem Res
Furthermore, we investigated whether HUVECs pro-
liferation or inhibition is associated with NF-κBp65 and
TRPC6 protein expressions by Western blot analysis.
Compared with untreated cells, compound 5 (5 μg/mL)
could upregulate the TRPC6 levels and downregulate NF-
κBp65 levels (Fig. 5).
In addition, the effect of compound 5 on VEGF cyto-
kines production was detected by ELISA assay. An exciting
finding of the present study is that VEGF cytokines
production can be promoted by compound 5 (5 μg/mL)
(Fig. 6).
Conclusions
In conclusion, our data provided the evidence that ent-
kaurane diterpenoids from the pericarps of D. metel might
be used as effective constituents for the treatment of
HUVECs-related diseases. This will provide theoretical
basis and scientific evidence for development of new
pharmaceutical drugs by making full use of D. metel.
Fig. 5 Western blots analysis of NF-κBp65 and TRPC6 protein
expression (treated with compound 5 at 5 μg/mL, 10 μg/mL, and 20
μg/mL) in HUVECs for 24 h. Each bar represents the mean ± SD from
*
★
three independent experiments, P < 0.05 and P < 0.05 vs. control
group
Acknowledgements This study was supported by the Research
Innovation Project for Graduates of Heilongjiang University of Chi-
nese Medicine (No. yjscx2016010) and sponsored by Chang Jiang
Scholar Candidates Program for Provincial Universities in Hei-
longjiang (No. 2013CJHB006).
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
Ethical approval Prior approval from Institutional Biosafety Com-
mittee was obtained to carry out cytotoxicity study.
References
Anju D, Ratan L (2011) Phytochemical and pharmacological status of
Datura fastuosa Linn. Int J Res Ayurveda Pharm 2:145–150
Cai XF, Shen GH, Tien N, Kang OH, Lee YM, Lee JJ, Kim YH
(
2003) Inhibitory effect of kaurane type diterpenoids from
Fig. 6 Effects of compound 5 on VEGF Cytokine of cell-free culture
Acanthopanax koreanum on TNF-α secretion from trypsin-
stimulated HMC-1 cells. Arch Pharmacal Res 26:731–734
Chinese Pharmacopoeia Commission (2010) Pharmacopoeia of the
People’s Republic of China version. Chinese Medical Science
Press, Beijing.
supernatants of HUVECs (treated for 24 h). Data are expressed as
*
mean ± SD, n = 3. P < 0.05 vs. control group
Chou GX, Wang ZT (2006) Study on chemical constituents of herba
Siegesbeckiae. Chin Pharm J 41:1854–1857
Cowan MM (1999) Plant products as timicrobial agents. Clin Microb
Rev 12:564–582
Huang WH, Liang YY, Wang JJ (2016) Anti-angiogenic activity and
mechanism of kaurane diterpenoids from Wedelia chinensis.
Phytomedicine 23:283–292
Kuang HX, Yang BY, Xia YG, Feng WS (2008) Chemical con-
stituents from the flower of Datura metel L. Arch Pharm Res
311:094–1097
disease, and anti-inflammatory activities (Huang et al. 2016,
Li et al. 2006, Kim et al. 2009, Wu and Wang 2015). In the
present study, the effects of compounds 1–5 on the pro-
liferation of HUVECs were measured by the MTT assay.
Most of the tested materials inhibited proliferation in a dose-
dependent manner compared with the control group, except
compound 5 (5 μg/mL), which could promote proliferation of
the cells (Fig. 4).

Med Chem Res
Kim HS, Lim JY, Sul D, Hwang BY, Won TJ, Hwang KW, Park SY
Okwu DE, Igara EC (2009) Isolation, characterization and anti-
bacterial activity of alkaloid from Datura metel Linn leaves. Afr J
Pharm Pharmacol 5:277–281
Oseni OS, Igbe F, Olagboye SA (2011) Distribution of antinutrients
and antioxidant properties of the plants of Throne apple (D.
Stramonium). J Agric Biol Sci 2:136–140
Qin XJ, Sun DJ, Ni W, Chen CX, Hua Y, He L, Liu HY (2012) Steroidal
saponins with antimicrobial activity from stems and leaves of Paris
polyphylla var. yunnanensis. Steroids 77:1242–1248
Siddiqui S, Sultana N, Ahmed SS, Haider SI (1986) Isolation and
structure of a new alkaloid datumetine from the leaves of Datura
metel. J Nat Product 3:511–513
(
2009) Neuroprotective effects of the new diterpene, CBNU06
against beta-amyloid-induced toxicity through the inhibition of
NF-kappaB signaling pathway in PC12 cells. Eur J Pharmacol
6
22:25–31
Li X, Xiao WL, Pu JX, Ban L, Shen Y, Weng Z, Li S, Sun H (2006)
Cytotoxic ent-kaurene diterpenoids from Isodon phyllostachys.
Phytochemistry 13:1336–1340
Mann J, Michalodimitrakis M, Koutselinis A (1984) Discussion of
Datur stramonium: a fatal poisoning. J Forensic Sci 29:961–962
Mohammad AH, Marwah SAK, Shaima AJAF (2014) Comparative
study of total phenolics, flavonoids contents and evaluation of
antioxidant and antimicrobial activities of different polarities
fruits crude extracts of Datura metel L. Asian Pac J Trop Dis
Wu HY, Wang WG (2015) Six new cytotoxic and anti-inflammatory
11, 20-epoxy-ent-kaurane diterpenoids from Iso Isodon wik-
stroemioides. Chinese. J Nat Med 5:383–389
5
:378–383
Nhiem NX, Hien NTT, Tai BH, Anh HLT, Hang DTT, Quang TH,
Kiem PV, Minh CV, Ko W, Lee S, Oh H, Kim SH, Kim YH
Wu SB, Meyer RS, Whitaker BD, Litt A, Kennelly EJ (2012) Anti-
oxidant glucosylated caffeoylquinic acid derivatives in the inva-
sive tropica soda apple. Solanum viarum. J Nat Prod 75:2246–50
Xian LN, Qian SH (2010) A new ent-Kaurane Glycoside from the
Stems of Acanthopanax gracilistylus. Chin Herb Med 3:168–169
Zia-Ul-Haq M, Ahmad M, Mehjabeen J, Marwat IK (2011) Anti-
microbial screening of selected flora of Pakistan. Arch Biol Sci
63:691–695
(
2015) New ent-kauranes from the fruits of Annona glabra and
their inhibitory nitric oxide production in LPS-stimulated
RAW264.7 macrophages. Bioorg Med Chem Lett 25:254–258
Nisar M, Kaleem WA, Qayum M, Hussain A, Zia-Ul-Haq M, ALI I,
Choudhary MI (2011) Biological screening of Zizyphus oxyphylla
Edgew stem. Pak J Bot 43:311–317