A new pseudodepsidone from the Antarctic lichen Stereocaulon alpinum
and its antioxidant, antibacterial activity
Hari Datta Bhattarai1,, Taikyung Kim1,, Hyuncheol Oh2 and Joung Han Yim1,*
1
Division of Life Sciences, Korea Polar Research Institute, KOPRI, Incheon 406-840,
Republic of Korea
2
College of pharmacy, Wonkwang University, Iksan 570-749, Republic of Korea
Authors
*
have equal contribution
Corresponding author. Tel.: +82-32-260-6341, Email: jhyim@kopri.re.kr
Material and methods
General information
All reagents and solvents were purchased from Sigma-Aldrich. Optical rotation was
measured in a polarimeter (Autopol III, Rudolph, USA). Melting point was measured using
DSC Q-1000 (TA Instrument, USA). ESIMS data were obtained by using a Mariner ESI-MS
instrument (Perseptive Biosystem, USA). NMR spectra (1D and 2D) were recorded in D2O
using a JEOL JNM ECP-400 spectrometer (400 MHz for 1H and 100 MHz for
chemical shifts were referenced relative to tetramethylsilane (H/C = 0).
13
C), and
HSQC and HMBC
experiments were optimized for 1JCH = 140 Hz and nJCH = 8 Hz, respectively. Mild pressure
liquid chromatography (MPLC) was carried out using Aldrich octadecyl-functionalized silica
gel (C18). Compounds were detected by UV absorption at 254 nm.
Sample collection, extraction and isolation
Stereocaulon alpinum was collected and identified by one of us (J. H. Yim) from Barton
Peninsular around King Sejong Station (S 62o14’48’’, W 58o44’46’’) on King George Island,
Antarctica in January, 2010.
A dried sample of Stereocaulon alpinum (100 g) was extracted
with MeOH (1 L  2) for 24 h.
The resulting crude MeOH extract (10.2 g) was subjected to
C18 functionalized silica gel (5  30 cm) mild pressure liquid chromatography (MPLC),
eluting with a stepwise gradient of MeOH in H2O.
The gradient program was as follows:
50% methanol for 5 min, 75% methanol in 5 min for 30 min, 85% methanol in 5 min for 30
min, 95% methanol in 5 min for 10 min. The flow rate was fixed for 45 ml/min. The
collected broad peak (tR = 43 min to 60 min) yielded compound 2 while the other peak (tR =
62-72 min) yielded compound 1 after repeated MPLC.
Antimicrobial activity test
Disk diffusion assay was performed to evaluate the antibacterial activity of compounds
qualitatively. Escherichia coli, Bacillus subtilis, Candida albicans and Aspergillus niger were
used as target species. The test compounds were dissolved in methanol and loaded in
antimicrobial assay filter paper disk in a concentration of 100 g/disk. The test compound
loaded disks were fully dried in sterile condition for 30 min. Paper disk without test
compound but soaked with methanol was used as negative control after dried for 30 min.
Ampicillin was taken as positive control. The test microorganisms, E. coli and B. subtilis
were grown in nutrient agar at 37 C while C. albicans and A. niger were grown in potato
dextrose agar at 25 C. The zone of inhibition was measured after 24-48 h of incubation
period. Minimum inhibitory concentration (MIC) was determined as the minimum
concentration of test compound to inhibit the complete growth of 107 cells of target
microorganisms in 24 h. The MIC was calculated after the linear regression analysis of the
experimental data of various concentrations of test compounds in triplicate. Only B. subtilis
and S. aureus were used to determine MIC because the test compounds were found active
against these organisms during disk diffusion assay.
DPPH reducing assay
DPPH (1-diphenyl-2-picryl-hydazil) free radical scavenging assay was performed as
described previously9 with some modification.8 Shortly, 0.25 ml of DPPH solution (0.1 mM
of DPPH in methanol) was mixed with 0.75 ml of various concentrations (0-4000 M) of the
test samples.
The mixture was incubated at RT for 30 min and the absorbance was
measured at 517 nm in a UV-Visible spectrophotometer (SCINCO).
Reaction mixtures
without the test sample and with commercial butylated hydroxyanisole (BHA) were used as
negative and positive controls, respectively. The experiment was conducted in triplicate.
Data were taken as average and standard deviation was calculated.
Brine shrimp lethality test
Brine shrimp lethality test (BST) was used to evaluate the toxicity of various test
samples10 with slight modification. The eggs of A. salina were hatched in aerated seawater in
light at 25 °C. The hatched active larvae were attracted towards the direction of light. The
active larvae (about 100) are selected and treated with various concentrations of test samples
(0-4000 M). The effects of test samples were monitored after 24 h of treatment by observing
the live larvae. The mortality rate of the larvae indicated the toxicity of the test samples.
Berberine chloride a standard anticancer drug was taken as positive control and brine shrimp
larvae in only sea water were taken as negative control.
LC/ESI-MS analysis
Freshly prepared methanol extract of Stereocaulon alpinum was analyzed using 6310 Agilent
Ion Trap LC/MS. The condition and instrumentation of LC/MS was as follows:
Column
Model-Eclipse XDB-C18
Dimension-4.6 mm X 150 mm
Particle Size- 5m
Pore size- 100Å
Temperature-25C
Sample
Methanol extract dissolved in water (1mg/ml)
Injection volumn-10l
Mobile phase
A: water mixed with 0.1% formic acid
B: Acetonitrile
Time (min)
Flow (ml/min)
%A
%B
0
0.5
100
0
5
0.5
50
50
20
0.5
20
80
30
0.5
10
90
List of figures:
Figure 1. HRESIMS spectrum of compound 2 (negative mode).
Figure 2. FT-IR spectrum of compound 2.
Figure 3. 1H NMR spectrum of compound 2 (400 MHz, DMSO-d6)
Figure 4. 13C NMR spectrum of compound 2 (400 MHz, DMSO-d6)
Figure 5. HSQC spectrum of compound 2 (400 MHz, DMSO-d6)
Figure 6. COSY spectrum of compound 2 (400 MHz, DMSO-d6)
Figure 7. HMBC spectrum of compound 2 (400 MHz, DMSO-d6)
Figure 8. NOESY spectrum of compound 2 (400 MHz, DMSO-d6)
Figure 9. HPLC spectrum of crude methanol extract of Stereocaulon alpinum. Compound 1 is
lobaric acid and compound 2 is a new compound as verified from HPLC-MS spectrum of
individual peak against purified standards.
Figure 10. Antibacterial activity of compound 1 and compound 2 against Bacillus subtilis.
Figure 11. Antibacterial activity of compound 1 against Staphylococcus aureus.
Figure 12. Antibacterial activity of compound 2 against Staphylococcus aureus.
Figure 1. HRESIMS spectrum of compound 2 (negative mode).
10 0 *M11A
95
90
85
80
32 31.9
69 1.8
74 8.8
75
78 6.5
28 55.0
%T
70
84 2.9
88 3.0
14 62.6
65
92 9.2
14 87.1
60
17 40.2
29 57.3
55
10 19.1
10 37.7
13 34.9
29 26.0
13 65.9
11 15.4
11 46.6
50
11 94.0
12 22.1
45
40
16 03.8
35
16 83.3
40 00
35 00
30 00
25 00
20 00
W aven umb ers (c m-1)
Figure 2. FT-IR spectrum of compound 2.
15 00
10 00
50 0
Figure 3. 1H NMR spectrum of compound 2 (400 MHz, DMSO-d6)
Figure 4. 13C NMR spectrum of compound 2 (400 MHz, DMSO-d6)
Figure 5. HSQC spectrum of compound 2 (400 MHz, DMSO-d6)
Figure 6. COSY spectrum of compound 2 (400 MHz, DMSO-d6)
Figure 7. HMBC spectrum of compound 2 (400 MHz, DMSO-d6)
Figure 8. NOESY spectrum of compound 2 (400 MHz, DMSO-d6)
Figure 9. HPLC spectrum of crude methanol extract of Stereocaulon alpinum. Compound 1 is
lobaric acid and compound 2 is a new compound and compound 3 is related known compound
as verified from HPLC-MS spectrum of individual peak against purified standards.
Figure 10. Antibacterial activity of compound 1 and compound 2 against Bacillus subtilis.
Figure 11. Antibacterial activity of compound 1 against Staphylococcus aureus.
Figure 12. Antibacterial activity of compound 2 against Staphylococcus aureus.