SUPPLEMENTARY MATERIAL
Flavonoids from the halophyte Apocynum venetum and their
antifouling activities against marine biofilm-derived bacteria
Na-Na Kongab, Sheng-Tao Fanga*, Ying Liua, Jian-Hua Wanga, Cui-Yun
Yanga and Chuan-Hai Xiaa*
a
Key Laboratory of Coastal Biology and Biological Resources Utilization, Yantai
Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, P. R.
China; bCollege of Resources and Environment, University of the Chinese Academy of
Sciences, Beijing 100049, P. R. China
* Corresponding authors: Emails: chxia@yic.ac.cn; stfang@yic.ac.cn.
Eleven flavonoids were isolated from the leaves of the halophyte Apocynum venetum.
Among them, plumbocatechin A (1), 8-O-methylretusin (2) and kaempferol
3-O-(6″-O-acetyl)-β-D-galactopyranoside (7) were the first report of their isolation
from this plant. Their structures were identified by the means of the spectral methods,
including 2D NMR experiments, and confirmed by comparison with the data of the
literature reported. In addition, the antifouling activities against the marine fouling
bacteria, Bacillus thuringiensis, Pseudoalteromonas elyakovii and Pseudomonas
aeruginosa of these compounds had been evaluated in this paper.
Keywords: Apocynum venetum; flavonoids; halophyte; antifouling; antimicrobial
activity
1
Experimental
General
1D and 2D NMR spectra were recorded on a Bruker Avance III 500 instruments
(Bruker, Switzerland) with TMS as an internal standard. HPLC-ESI-MS were
determined on a LCQ Fleet ion trap mass spectrometer (Thermo Fisher Scientific, San
Jose, USA). Column chromatography was performed over silica gel (200-300 mesh;
Yantai Xinde Chemical Co., Ltd., Yantai, China), octadecyl silica (ODS) gel (YMC,
Kyoto, Japan), and Sephadex LH-20 (Amersham Pharmacia Biotech, Uppsala,
Sweden). Thin layer chromatography (TLC) was performed on the silica gel plates
(Yantai Xinde Chemical Co., Ltd., Yantai, China), and spots were visualized by
spraying with 10% H2SO4 in EtOH, followed by heating. Fractions were separated by
preparative medium pressure liquid chromatography (MPLC) (Puriflash 450,
Interchim Company, France) on the flash chromatographic columns (Santai
Technologies Inc., Changzhou, China). Semi-Preparative HPLC was performed on
Elite Preparative HPLC (Elite Analytical Instruments CO., Ltd., Dalian, China) with a
Thermo BDS HYPERSIL column (250×10.0 mm i.d., 5 μm, Thermo Fisher Scientific,
Waltham, MA).
Plant material
The leaves of A. venetum L. were collected from the Yellow River Delta, Dongying of
Shandong Province, China, in September 2011, and identified by Prof. Dongli Shi,
Binzhou University. The voucher specimen (No. 20110903-3) was deposited at Key
Laboratory of Coastal Biology and Biological Resources Utilization, Yantai Institute
of Coastal Zone Research, Chinese Academy of Sciences
Extraction and isolation
The air-dried leaves (3.0 kg) of A. venetum were powdered and extracted with MeOH
at room temperature (10 L×4). The combined extract was concentrated under reduced
2
pressure to give a residue (464 g). It was suspended in water and extracted by
petroleum ether (PE), EtOAc and n-BuOH, respectively. The EtOAc-soluble extract
(52.9 g) was subjected to column chromatography on silica gel with PE-Me2CO
gradients (15:1 to 1:5, v/v) on preparative MPLC to give five fractions (A–E).
Fraction B (4.26 g) was subjected to silica gel column with gradient eluting
(PE:EtOAc, 5:1 to 1:2, v/v) and Sephadex LH-20 using CHCl3:MeOH (1:1, v/v), and
then further purified on silica gel chromatography column (PE:Me2CO, 4:1, v/v) to
afford 2 (9.1 mg). Fraction C (6.48 g) was subjected to column chromatography on
silica gel and eluted with PE:Me2CO (3:1 to 1:1, v/v) to give 4 subfractions (C1-C4).
Subfraction C2 was further purified by repeated silica gel columns with PE:Me2CO
(2:1, 3:1, and 3:2) to yield 4 (55.6 mg) and 5 (31.3 mg). Subfraction C3 was
performed on Sephadex LH-20 and repeated column chromatography over silica gel
to obtain 1 (22.5 mg) and 3 (30.2 mg). Fraction D (7.16 g) was subjected to Sephadex
LH-20 with CHCl3:MeOH (1:1, v/v) to get 4 subfractions (D1-D4). Compounds 6
(6.3 mg), 7 (3.5 mg) and 8 (51.6 mg) were obtained from subfraction D2 after
purification by ODS, silica gel columns and semi-preparative HPLC. Subfraction D3
was subjected to repeated column chromatography over silica gel using CHCl3:MeOH
system to afford 9 (156.7 mg), 10 (145.2 mg), and 11 (15.2 mg).
Antimicrobial Assay
The antibacterial activity of the crude extracts and all of the isolated compounds from
A. venetum leaves were carried out using disc diffusion method against three marine
biofouling bacteria, Bacillus thuringiensis, Pseudoalteromonas elyakovii and
Pseudomonas aeruginosa. The crude extract (400 μg/disc) and the isolated
compounds (100 μg/disc) were prepared by dissolving with DMSO prior to being
placed on bacterial lawns. To compare the antibacterial activity, chloramphenicol (100
μg/disc) was used as the standard antibiotic and DMSO as the negative control. The
plates were incubated at 37 °C for 12 h, at which the diameters of inhibition zones
were measured in mm. All assays were carried out in triplicate, and the average
3
diameters (mm) of the growth inhibition zones were recorded.
Statistical analysis
The results were expressed as means ± SD. The significance of differences was
evaluated by one-way ANOVA using SPSS 16.0 software package, and the values of
P < 0.05 were considered as statistically significant.
4
Table S1. 1H- and 13C-NMR (500/125 MHz) data of compound 1 (Plumbocatechin A)
in DMSO-d6 and Pyridine-d5.
1 (in DMSO-d6)
1 (in Pyridine-d5)
Position
δH
δC
δH
δC
2
4.39 (s, H-2β)
70.7 d
4.79 (s, H-2β)
72.3 d
3
4.13 (d, J = 4.9 Hz, H-3β)
62.8 d
4.56 (d, J = 4.9 Hz, H-3β)
64.2 d
4
2.75 (dd, J = 17.3, 5.3 Hz, H-4β)
25.6 t
3.55 (d, J = 17.1 Hz, H-4α)
27.1 t
2.57 (d, J = 16.6 Hz, H-4α)
5
6
155.7 s
5.62 (d, J = 2.3 Hz)
7
8
3.32 (dd, J = 17.2, 5.3 Hz, H-4β)
94.5 d
157.3 s
6.56 (d, J = 2.2 Hz)
156.6 s
5.90 (d, J = 2.3 Hz)
95.4 d
96.0 d
158.7 s
6.64 (d, J = 2.2 Hz)
96.8 d
9
156.8 s
158.7 s
10
98.2 s
99.8 s
1′
123.6 s
125.1 s
2′
121.0 s
122.7 s
3′
142.8 s
144.9 s
4′
134.1 s
136.6 s
5′
144.5 s
146.5 s
6′
6.34 (s)
7′
108.7 d
7.07 (s)
75.1 s
110.2 d
76.3 s
8′
1.52 (s)
24.6 q
2.13 (s)
25.3 q
9′
1.47 (s)
28.9 q
2.05 (s)
29.7 q
-OH
9.21 (brs), 9.15 (brs), 8.89 (brs),
8.57 (brs), 8.29 (brs)
5
Table S2. 1H- and 13C-NMR (500/125 MHz) data of compound 2 (8-O-Methylretusin)
in DMSO-d6.
δH
δC
Position
153.6 d
10
117.9 s
3
123.4 s
1′
124.6 s
4
175.3 s
2′, 6′
7.52 (d, J = 8.8 Hz)
130.6 d
7.00 (d, J = 8.8 Hz)
114.1 d
Position
2
8.43 (s)
δH
δC
5
7.73 (d, J = 8.7 Hz)
121.2 d
3′, 5′
6
7.03 (d, J = 8.7 Hz)
115.7 d
4′
7
155.3 s
8-OCH3
3.88 (s)
61.3 q
8
135.2 s
4′-OCH3
3.80 (s)
55.6 q
9
151.2 s
7-OH
10.66 (s)
6
159.5 s
Table S3. 1H- and 13C-NMR (500/125 MHz) data of compound 6 and 7 in DMSO-d6.
6
7
Position
δH
δC
δH
δC
2
156.2 s
156.3 s
3
133.3 s
133.8 s
4
177.1 s
177.2 s
5
161.5 s
162.3 s
6
6.06 (brs)
7
8
100.5 d
6.05 (brs)
165.1 s
6.26 (brs)
94.7 d
100.5 d
165.1 s
6.26 (brs)
94.7 d
9
157.2 s
157.3 s
10
104.4 s
104.3 s
1′
121.3 s
121.5 s
2′, 6′
7.97 (d, J = 8.8 Hz)
131.1 d
8.02 (d, J = 8.8 Hz),
131.4 d
3′, 5′
6.86 (d, J = 8.8 Hz)
115.5 d
6.85 (d, J = 8.8 Hz),
116.0 d
4′
1″
160.6 s
5.32 (d, J = 7.5 Hz)
2″
102.0 d
160.7 s
5.26 (d, J = 7.8 Hz)
74.6 d
3″
71.5 d
76.7 d
3.10-3.70 (m)
102.9 d
73.2 d
3.10-3.70 (m)
4″
70.3 d
68.6 d
5″
74.3 d
73.4 d
6″
4.11 (dd, J = 11.8, 2.1 Hz)
63.3 t
3.96 (dd, J = 11.8, 6.1 Hz)
-AcO
4.07 (dd, J = 11.3, 8.0 Hz)
3.92 (dd, J = 11.3, 4.3 Hz)
170.3 s
1.77 (s)
63.7 t
20.7 q
7
170.4 s
1.75 (s)
20.7 q
Table S4. Antibacterial activity of the crude extracts and the isolated compounds of A.
venetum leaves.
Inhibition zone diameter (mm)a
Samples
B. thuringiensis
P. elyakovii
P. aeruginosa
–
–
–
18.1±0.4
13.5±0.6
22.3±0.7
n-BuOH extract
–
–
–
Compound 1
–
–
–
Compound 2
9.4±0.6A
–
10.5±0.9AB
Compound 3
9.2±0.5A
10.6±0.7B
10.9±0.7B
Compound 4
9.5±0.8A
9.3±0.6A
9.5±0.7A
Compound 5
17.4±0.9B
9.5±0.6AB
15.8.±0.8C
–
–
–
Compound 8
–
–
–
Compound 9
–
–
–
Compound 10
–
–
–
Compound 11
–
–
–
33.7±0.9C
29.2±0.7C
30.3±0.8D
PE extract
EtOAc extract
Compound 6 and 7
(2.5:1)
Chloramphenicol
Notes: (–), Inactive; (5-13), weakly active; (13-22), moderately active; (>22), highly active.
a
Values are mean inhibition zone (in millimetre) ± SD of three replicates, and the zone of
inhibition in mm including the disc diameter (5 mm). A-D Means within the same column followed
by the different superscript are statistically significant (P < 0.05).
8
Figure S1. 1H- and 13C-NMR spectra for the mixture of compound 6 and 7.
9
Figure S2. HPLC profiles of the mixture of compounds 6 and 7. (A) mobile phase:
water and acetonitrile (0−20 min, 20−30% acetonitrile); (B) mobile phase: water and
methanol (0−30 min, 40% methanol). Column: Zorbax Extend-C18, 4.6 mm × 250
mm, 5μm; flow rate: 1.0 mL/min; column temperature: 30 ℃; wavelength: 254 nm.
10
Figure S3. 1H- and 13C-NMR spectra of compound 6.
11
Figure S4. 1H- and 13C-NMR spectra of compound 7.
12