ORIGINAL ARTICLES
extract, 375 lethanoland125 lofa1 mMfreshlypreparedDPPHsolution
in ethanol. Different concentrations of test samples were prepared while the
final concentration of DPPH in the reaction mixture was 0,125 mM. After
◦
incubation of the mixture at 37 C for 30 min in the dark the absorbance was
measured at 517 nm. Blank samples contained the same amount of methanol
and DPPH solution. All experiments were carried out in triplicate. Ascorbic
acid was used as positive control. Percentage radical scavenging activity of
samples was calculated using the following formula:
ꢀ
ꢃ
ꢁ
ꢂ
ABlank − ASample
Radical Scavenging Activity(%) =
100
ABlank
ED50 values, the concentration of the substrate that causes 50% loss of the
DPPH activity (colour), were calculated for the standard and the extract from
a graph plotted for the % inhibition against the concentration in g/ml.
3.5.2. Oxygen radical absorbance capacity
Reactive oxygen species, ROS are generated by the thermal degradation
of 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) and quench the
¨
signal of the fluorescent probe fluorescein. The subsequent addition of
antioxidants reduces the quenching by preventing the oxidation of the fluo-
rochrome. A vitamin E derivate, 6-hydroxy-2,5,7,8-tetra-methylchroman-2-
carboxylic acid (Trolox), was used as positive control. Test compounds 5
and 6 were dissolved in phosphate buffered saline (10 mM, pH 7.4) and
investigated for their antioxidant capacity. Experiments were done in black
96-well plates. In each well of a 96-well Plate 150 l fluorescein (final
concentration: 2.5 nM), 25 l Trolox (final concentrations: 0.78 – 25 M)
or 25 l test compound were pipetted in quadruplicate. Plate was allowed
◦
to equilibrate at 37 C for 30 min. After this time, fluorescence measure-
ments (Ex. 485 nm, Em. 520 nm) were taken every 90 s; first to determine
the background signal. After three cycles 25 l AAPH (final concentration:
60 mM) were added manually in each well with a multi-channel-pipette.
This was done as quickly as possible since the ROS generator displays
immediate activity after addition. Fluorescence measurements were contin-
ued for 90 min. Half life time of fluorescein was determined using MS Excel
software.
Fig. 3: Cell cycle of HaCaT keratinocytes determined 24 h (A) and 72 h (B) after
culture of cells in the presence of 2,3-digalloyl-O-(␣/)-glucopyranose (2)
[nilocitin] or2,3-HHDP-(␣/)-glucopyranose (1). Cells were incubated at
3.5.3. Cytotoxicity assay
concentrations which corresponded to the IC50 values of the isolates. Results
are given as mean ± SD of 3 independent experiments. Significance was
checked by One-way Anova with Tuckey’s Multiple Comparison test:
HaCaT cells were obtained from CLS Cell Lines Service (Eppelheim,
Germany). Cells were cultured in RPMI 1640 medium (BioWhittaker,
Lonza, Verviers, Belgium) supplemented with 8 % fetal bovine serum
*
#
p < 0.05; **p < 0.01; ***p < 0.001 vs. control; #p < 0.05; # #p < 0.01; # #
p < 0.001 vs. vehicle.
(Sigma Aldrich, Taufkirchen, Germany) and antibiotics (100 U/ml peni-
cillin/100 g/ml streptomycin; Sigma Aldrich, Taufkirchen, Germany) at
◦
9
5% humidity, 5% CO2 and 37 C. HaCaT cells were sub-cultured twice
cose (CoPC) and gallic acid (CoPC and 1H NMR). On controlled acid
a week and regularly tested for mycoplasma contamination. Cytotoxicity
of test samples against both cell lines was investigated using the neu-
tral red uptake (NRU) assay. After 24 h cultivation in 96 well plates (3
◦
hydrolysis (41 mg in 15 ml aq. 0.5 N HCI, l00 C. 2 hrs] it yielded 3,6-
di-O-galloyl-(␣/)-glucose (8a). Compound 8a: R, values: 62 (H2O), 69
(
[
AcOH), 43 (BAW). UV data: max, (nm): 275. Mr 484; negative ESIMS,
3
or 8 × 10 cells/well) medium was removed and cells were exposed for
−
M−H] = 483; HRESI-MS negative mode: m/z = 483.079, corresponding
72 h to various concentrations (max. 500 g/ml) of test samples. After
to a molecular formula of C20 H19 O14 (calc. 483.3572). On complete acid
hydrolysis of 8a (7 mg, worked up as usual) it yielded gallic acid and glu-
cose (CoPC). On controlled acid hydrolysis it (9 mg) yielded 6-monogalloyl
removal of the medium wells were washed with HBSS (Hanks Balanced
Salt Solution, PAA). Cells were than incubated for 3 h with 100 l 3-amino-
7-dimethylamino-2-methylphenazine hydrochloride (neutral red, Merck,
1
glucose (CoPC). H NMR of 8a: ␣-glucose moiety: 5.01 (d, .I = 3.5 Hz, H
Darmstadt, Germany, stock solution 3.3 g/ml; working solution 33 ng/ml).
Medium was removed and wells were washed twice with HBSS. Afterwards
cells were lysed with 100 l of 1% acetic acid in 50% EtOH. Finally, after
1
) 3.5-3.9 (m, H-2 and H-4), 5.3 (t, J = 9 Hz, H-3), 3.88 (m, H-5), 4.38 (d,
J = 12.5 Hz, H-6), 4.25 (dd, J = 12.5 and 4.5 Hz, H-6’); -glucose moiety:
.05 (d, J = 9 Hz, H-l), 3.5-3.9 (m. H-2 and H-4), 4.98 (t, J = 9 Hz, H-3), 3.9
m, H- 5), 4.42(d, J = 12.5 Hz, H-6), 4.29 (dd, J = 12.5 and 4.5 Hz, H-6’);
5
(
45 min optical density was measured at 450 nm in a plate reader (Fluostar
Omega, BMG Labtech, Offenburg, Germany). The IC50 values were defined
from obtained dose-response curves and expressed in mean ± SD. All com-
pounds were tested in duplicate. Etoposide (Alexis Biochemicals, ≥ 98 %
purity) was used as positive control.
1
galloyl moieties: 6.94 (s), 6.96 (s), 6.99 (s), 7.01 (s). HNMR of 21: ␣-
glucose moiety: 5.37 (d, 5 = 3.5 Hz, H-l), 4.95 (dd, J = 8 and 3.5 Hz, H-2).
5
.70 (t, 5 = 8 Hz, H-3). 3.70 (m, H-4), 3.95 (m, H-5), 4.38 (d, .I = 12.5 Hz,
H- 6), 4.25 (dd, J = 12.5 Hz and 4.5 Hz, H-6’); -glucose moiety: 5.05 (d,
I = 8 Hz. H-l), 5.10 (t, J = 8 Hz, H-2), 5.33 (t, J = 8 Hz, H-3), 3.8 (m, H-4).
.92 (m, H-5). 4.42 (d, J = 12.5 Hz, H-6)‘, 4.29 (dd. J = 12.5 and 4.5 Hz, H-
’); galloyl moieties: 7.0 (s), 6.99 (s), 6.98 (s), 6.95 (s), 6.93 (s), 6.88 (s).
C NMR of 21: ␣-glucose moiety: 91.2 (C-l), 72.2 (C-2), 72.8 (C-3), 69.3
C-4)‘, 72.3 (C-5). 64.3 (C-6); -glucose moiety: 95.3 (C-l), 73.5 (C-2), 75.5
C-3). 70.8 (C-4)‘, 74.9 (C-5). 64.4 (C-6); galloyl moieties: 121.6, 121.7,
21.8 (C-l), 109.8, 109.9, 110.2 (C-2 and C-6), 146.1, 146.0, 145.9, 145.4
C-3 and C-5). 139.3, 138.6, 138.8 (C-4), 167.0, 166.8, 166.6, 165.0 (C = O).
.
3
3.5.4. Cell cycle analysis
6
The distribution of HaCaT cells in different cycle stages was analyzed using
flow cytometry 24 h and 72 h after the cells were cultured in the presence
1
3
(
(
1
(
5
of the extract of A. auriculata and six of the isolates. Briefly, 1 × 10 cells
were seeded into 24 well plates with 500 l RPMI 1640 medium, 8% FCS.
◦
After overnight culture at 37 C medium was removed and 500 l extract or
isolates (IC50) were added. Control cells received 500 l medium or vehicle.
Cells were than further cultured for 24 h or 72 h. After culture the cells were
5
3
.5. Biological assays
detached and counted using a Neubauer chamber. 5 × 10 cells were incu-
◦
◦
bated in 1 ml cold (−20 C) 70 % ethanol at least for 1 h (4 C) and washed
3
.5.1. Radical scavenging effect
2+
2+
twice with 2 ml PBS (without Ca /Mg ). Subsequently, the cells were
◦
The estimation was done according to the method of Brand-Williams and
Cuvelier (1995). DPPH (2,2-diphenyl-1-pycrylhydrazyl), a stable radical,
is reduced after reaction with an antioxidant compound and its absorbance
at 517 nm is than reduced. The reaction mixture contained 500 l of test
incubated for 30 min in RNase (50 g/mL in PBS) at 37 C. After washing
with 2 ml FACS buffer (1 % FCS, 8 g/mL sodium azide in PBS), the cells
were resuspended in 500 L FACS buffer and stained with 25 L propidium
iodide (50 g/mL). 5 × 10 cells per sample were analyzed with a MAC-
3
Pharmazie 69 (2014)
863