T.-H. Vo et al.
Phytochemistry 184 (2021) 112666
xylose were purchased from Sigma-Aldrich (US). Polysaccharide
Compositional Assay Kit (A, B, and H reagents) was delivered from Sugar
Light Co., Taiwan (R.O.C.). The cell line was EAhy926.
Table 3
Anti-angiogenic activity of fractions and isolated compounds from S. concinnula.
Fractions
SRB
IC50
Matrigel
(
(
μ
g/mL)
M)
AREA IC50
(
μ
μ
g/mL)
M)
4.2. Plant material
Fr. A
>64
17
18.5 ± 6.5
20.5 ± 1.1
AREA IC50
>64
Fr. B
Whole plants of Staurogyne concinnula (Hance) Kuntze (Acanthaceae)
were collected in May 2019 (during the full flowering stage of the plant)
at around 1000 m above sea level on the mountain in New Taipei City,
Taiwan (25.00636◦ N, 121.75612◦ E). The plant voucher (SC201905)
was identified by Dr. Chia-Ching Liaw, Director of Herbarium, National
Research Institute of Chinese Medicine (NRICM), and deposited at Dr.
Liaw’s Herbarium.
Compounds
IC50
>64
>64
>64
32
μ
(
1
2
>64
3
>64
4
4.0 ± 0.2
>64
5
>64
7
>64
>64
Combretastatin A-4a
0.4
b
a
Positive control.
4.3. Extraction and isolation
b
Did not conduct.
Dried whole plant (5.0 kg) of S. concinnula was extracted with
ethanol 95% (4 h x twice, 50 ◦C). The crude extract (550 g) was obtained
by evaporating the filtrate under vacuum. Suspended the crude extract
in distilled water and partitioned with n-hexane (HEX), ethyl acetate
(EtOAc), and n-butanol (n-BuOH) to obtain a HEX residue (53 g), an
EtOAc residue (150 g), and a n-BuOH residue (200 g), respectively. The
ani-angiogenic effect was investigated. The bio-active n-BuOH residue
(150 g) was fractionated by operating the Diaion HP-20 column with
MeOH – H2O mobile phase (MeOH 50% and 100%) to yield two frac-
tions Fr. A (20 g) and Fr. B (15 g). These two fractions were subjected to
the preparative HPLC using a Cosmosil C18 column (i.d. 20 × 250 mm,
pathway and its downstream effectors. The results demonstrated that
after 4 or 10 μM of 4 treatments for 24 h, FAK and paxillin levels were
decreased, whereas there was no change in the expression Akt protein
(Fig. 5). The downstream matrix metalloproteases (MMP 2 and MMP 9)
were consistently decreased in a dose-dependent manner after treating
with 4.
3. Conclusion
This is the first report that five sophoradiol saponins (1-5) and four
phenylpropane glycosides (6–9) were isolated from the ethanol extract
of Staurogyne concinnula. The chemical structures of four undescribed
saponins (1–3 and 5) were identified by spectroscopic analyses. The
anti-angiogenic effect assay revealed baptisiasaponin I (4) had the most
10 μm, Nacalai Tesque, Japan). The detection wavelength was 203 nm.
With a mobile phase consist of acetonitrile and formic acid 0.05% (5 : 5,
v/v), five pure saponins (1–5) were yielded from fraction Fr. B (500 mg):
1 (19.0 mg), 2 (10.0 mg), 3 (30.5 mg), 4 (61.0 mg), and 5 (10.0 mg).
With a mobile phase consist of MeOH and formic acid 0.05% (35 : 65, v/
v), four pure phenylpropane glycosides were obtained from fraction Fr.
A (600 mg): 6 (10.0 mg), 7 (52.0 mg), 8 (15.0 mg), and 9 (8.0 mg). The
extraction and isolation procedure had been repeated to obtain the
required amount of each compound for chemical and biological
experiments.
inhibitory effect (IC50 = 4.0 ± 0.2
μM) than the other saponins 1–3, and
5 (IC50 > 64 M). Notably, the inactive compound 2 had the same
μ
glycone moiety, and similar triterpene as 4 except that C-6 of 2 was
oxidized to a ketone. The evidence and the findings that four isolated
saponins (1, 3, 4 and 5) possessed the same triterpene with different
amounts of sugar moieties, implied that the oxidation state of C-6 and
the length of sugar chains in sophoradiol saponin would play a crucial
role in the anti-angiogenic activity. Furthermore, baptiasiasaponin I (4)
was demonstrated to inhibit integrin/FAK/paxillin signaling pathway
and its downstream effectors such as MMP2 and MMP9.
4.4. Compound 1
Amorphous white powder; C61H98O25; HR-ESI-MS [M-H]- m/z
1229.6327 (calcd for C61H97O25, 1229.6319); for 1H and 13C NMR
(methanol-d4, 600 MHz and 150 MHz) data, see Tables 1 and 2
4. Experimental
4.1. General experimental procedures
4.5. Compound 2
All NMR spectra were recorded on a Bruker Avace 600 spectrometer
at 600 MHz for 1H and at 150 MHz for 13C, with methanol-d4 as solvent
and tetrametylsilane as reference. ESI-MS data was obtained on a Fin-
nigan MAT-95XL mass spectrometer combined with UPLC system
(Thermo Fisher Scientific, Bermen, Germany). The solvent was MeOH,
and the mobile phase was 0.1% HCOOH – MeOH (2 : 8, v/v), at a 0.2
mL/min flow rate. The ESI-MS was operated in negative mode. Scanning
was performed from 150 to 2000 m/z (mass-to-charge ratio). For column
Amorphous white powder; C47H74O17; HR-ESI-MS [M-H]- m/z
909.4863 (calcd for C47H73O25, 909.4848); for 1H and 13C NMR
(methanol-d4, 600 MHz and 150 MHz) data, see Tables 1 and 2
4.6. Compound 3
Amorphous white powder; C53H86O19; HR-ESI-MS [M-H]- m/z
1025.5763 (calcd for C53H85O19, 1025.5685); for 1H and 13C NMR
(methanol-d4, 600 MHz and 150 MHz) data, see Tables 1 and 2
chromatography, Mitsubishi DIAION HP20 (particle size > 250
μm,
capacity 1.01 eq/L), MeOH and water were used. Preparative HPLC was
performed on a Shimadzu LC-8A instrument equipped with a UV de-
4.7. Compound 4 (Baptisiasaponin I)
tector, Cosmosil C18 column (i.d. 20 × 250 mm, 10 μm; Nacalai Tesque
Company, Japan). Analysis HPLC was operated on Shimadzu LC 2040C
Amorphous white powder; C47H76O16; HR-ESI-MS [M-H]- m/z
895.5000 (calcd for C47H75O16, 895.5055); for 1H and 13C NMR
(methanol-d4, 600 MHz and 150 MHz) data, see Tables 1 and 2
system, photodiode array detector, Cosmosil C18 column (i.d. 4.6 × 150
mm, 5 μm). Monosaccharides in hydrolysates were qualified by using an
Exertive Plus Mass spectrometer (Thermo Fisher Scientific, Bremen,
Germany) coupled with the online UPLC system. The work solution was
separated by an ACQUITY UPLC BETH C18 column (i.d. 2.1 × 100 mm,
4.8. Compound 5
1.7 μm; Waters). Solvent for HPLC analysis were of chromatography
Amorphous white powder; C41H66O12; HR-ESI-MS [M-H]- m/z
grade. Standards for D-glucuronic acid, D-glucose, L-rhamnose, and D-
749.4460 (calcd for C41H65O12 749.4476); for 1H and 13C NMR
6