ORIGINAL ARTICLES
3.4.2. From the acetone fraction
3.5. Biological methods
The dried material of the acetone fraction (20 g) was applied to a poly-
amide 6s (250 g) (Riedel-De-Haen AG, Seelze, Hannover, Germany) col-
umn (88 ꢂ 6.5 cm) and eluted with H2O followed by H2O–MeOH mix-
tures of decreasing polarities. Compounds 3 (38 mg) and 5 (92 mg) were
individually separated pure from the material of the fraction eluted with
50% MeOH by applying polyamide column (30 ꢂ 2 cm) fractionation,
using EtOAc water saturated for elution. Crystallization (twice) from bi-
distilled H2O of the material of the fraction eluted with 20% MeOH af-
forded a pure colorless crystalline sample (120 mg) of compound 4.
3.5.1. Determination of radical scavenging activity by DPPH assay
Qualitative DPPH assay was made by TLC. Test substances (5 ml, metha-
nol extract, ferulic acid and isoferulic acid) and the positive control ascor-
bic acid were spotted on a silica gel plate and detected with 1 mM ethano-
lic DPPH solution (Sigma, Taufkirchen, Germany). Quantitative
determination was carried out according to the method of Brand et al.
(1995). Different concentrations of test samples in ethanol (10, 50, 100,
500 and 1000 mg/ml) were prepared and 500 ml of each of them were
mixed with 500 ml of a 1 mM ethanolic DPPH solution. The samples were
incubated for 30 min at room temperature in the dark and the absorbance
at 517 nm was measured (UVmini-1240 UV-VIS-Spectrophotometer, Shi-
madzu, Duisburg, Germany). Ascorbic acid was used as positive control.
Experiments were carried out in triplicate. Percent radical scavenging activ-
ity by sample treatment was determined by comparison with an ethanol
treated control. The radical scavenging activity was calculated as follows:
3.4.3. Isoferulic acid 3-O-b-4C1-glucopyranoside ð1Þ
Faint yellow amorphous powder, [a]2D5 –41.4ꢁ (c ¼ 0.5, MeOH), Rf -values:
0.33 (H2O), 0.68 (HOAc), 0.75 (BAW). UV max nm in MeOH: 235, 292,
309. HRFIMS: m/z ¼ 355.1112 [M–H]ꢀ, (C16H20O9). Normal acid hydro-
lysis gave glucose and isoferulic acid (Co-PC). Isoferulic acid: Rf-values:
0.39 (HOAc), 0.69 (BAW). UV max nm in MeOH: 241, 292, 322. EI-MS:
m/z ¼ 194 [M]þ. 1H NMR: d ppm: 7.45 (d, J ¼ 16 Hz, H-7), 7.08 (m, H-2
and H-6), 6.95 (d, J = 8 Hz, H-5), 6.24 (d, J ¼ 16 Hz, H-8) and 3.8 (s,
H-OMe). 13C NMR (Table 1). 1H NMR of 1: d ppm: glucose moiety: 4.86
(d, J ¼ 8 Hz, anomeric H-1), 3.50–3.90 (m, sugar protons overlapped with
water protons); Isoferulic moiety: 7.59 (broad s, Dn1/2 ¼ 4 Hz, H-2); 7.11
(d, J ¼ 16 Hz, H-7); 7.06 (broad d, J ¼ 8 Hz, Dn1/2 ¼ 4 Hz, H-6); 6.90 (d,
J ¼ 8 Hz, H-5); 6.18 (d, J ¼ 16 Hz, H-8). NMR data of 1 (Table 1).
scavenging effect (%) ¼ [(A0 ꢀ A1) / A0] ꢂ 100
where A0 was the absorbance of the control reaction and A1 was the
absorbance in the presence of the sample of the tested substance or extract.
3.5.2. Cell culture and assay conditions
The spontaneously transformed non-tumorigenic human keratinocyte cell
line HaCaT (kindly provided by Prof. Fusenig of the German Cancer Re-
search Centre, Heidelberg, Germany) was cultured in growth medium at
37 ꢁC with 5% CO2 in a humidified atmosphere. Growth medium (RPMI
1640) was supplemented with 8% heat inactivated fetal calf serum (FCS,
3.4.4. Tamarixetin 3,30-di-sodium sulphate ð2Þ
Rf-values: 0.80 (H2O), 0.69 (HOAc, 0.36 (BAW); Electrophoretic mobility
[buffer, pH 2 (2.5% formic acid þ 8% acetic acid), 250 V, 20 mA, room
temp. on Whatman paper No. 3MM, 90 min]: 6.0 cm; UV max nm in
MeOH (A): 242 (inflection), 266, 341; (A) þ NaOAC: 257 (inflection),
271, 360; (A) þ NaOAc þ H3BO3: 270, 342; (A) þ AlCl3: 257 (inflection),
276, 305, 365 (inflection), 402; (A) þ AlCl3 þ HCl: 259 (inflection), 276,
302, 430; (A) þ NaOMe: 276, 320, 396. Normal acid hydrolysis (2 N
aqueous HCl, 1/4 h, 100 ꢁC) yielded tamarixetin: Rf-values: 0.08 (H2O),
0.17 (HOAc, 0.83 (BAW); UV max nm in MeOH (A): 238,255, 268,
369 (A) þ NaOAC: 253 (inflection), 273, 312, 360 (shoulder);
(A) þ NaOAc þ H3BO3: 255, 265 (inflection), 368; (A) þ AlCl3: 268, 301
(inflection), 363, 430; (A) þ AlCl3 þ HCl: 268, 301 (inflection), 362, 426;
(A) þ NaOMe: 268, 422. EI-MS: m/z ¼ 316 [M]þ. 1H NMR: d ppm: 6.22
(d, J ¼ 2 Hz, H-6); 6.45 (d, J ¼ 2 Hz, H-8); 7.08 (d, J ¼ 8 Hz, H-50); 7.65
(m, H-20 and H-60); 3.81 (s, Me-40). Controlled acid hydrolysis (0.05 N
aqueous HCl, 15 min, 100 ꢁC) yielded intermediates 2a and 2b.
Sigma, Taufkirchen, Germany) and antibiotics (penicillin 100 units mlꢀ1
,
streptomycin 100 mg mlꢀ1). Medium was changed every three days. Cells
were subcultured using EDTA (0.05% in phosphate buffered saline, PBS)
and trypsin/EDTA (0.05%/0.02% in PBS). Cells were detached by gently
tapping and suspended in growth medium. An aliquot of the cell suspen-
sion obtained was incubated in a hypo-osmotic buffer. Cell nuclei were set
free and were counted using a Buerker haemocytometer. For the experi-
ments the growth medium was replaced by RPMI 1640 containing 0.01%
bovine serum albumine (BSA, Sigma, Taufkirchen, Germany) and penicil-
lin/streptomycin (BSA medium). Cell culture plastics and medium supple-
ments were obtained from Biochrom AG (Berlin, Germany) except other-
wise stated.
3.5.3. Cell viability determination
All assays were conducted between passages 60 and 70. HaCaT cells were
plated in 96 well plates in growth medium in a density of 2 ꢂ 104 cells
per well. After 24 h growth medium was replaced by BSA medium and
cells were incubated with different concentrations of Tamarix aphylla flow-
er extract, ferulic acid or isoferulic acid for 72 h at 37 ꢁC. After incubation
the cells were observed under the microscope for cell integrity and were
treated with MTT solution (BSA medium, final concentration 0.5 mg
mlꢀ1) for 3 h at 37 ꢁC. Formazan crystals were dissolved in DMSO and
optical density (OD) was measured at 550 nm using a multi well plate
reader (Anthos HTII, Salzburg, Austria). Cell viability was expressed as a
percentage of vehicle control.
2a: Rf-values: 0.46 (H2O), 0.40 (HOAc), 0.33 (BAW); Electrophoretic mo-
bility: 4.2 cm. UV max nm in MeOH (A): 252 (inflection), 267, 343;
(A) þ NaOAC: 255 (inflection), 272, 388; (A) þ NaOAc þ H3BO3: 254,
267, 350; (A) þ AlCl3: 268, 274, 300, 412; (A) þ AlCl3 þ HCl: 254, 268,
390; (A) þ NaOMe: 269, 320, 389. 1H NMR: d ppm: 6.20 (d, J ¼ 2 Hz,
H-6); 6.40 (d, J ¼ 2 Hz, H-8); 7.10 (d, J ¼ 8 Hz, H-50); 7.62 (m, H-20 and
H-60), 3.83 (s, Me-40). 2b: Rf-values: 0.40 (H2O), 0.32 (HOAc), 0.56
(BAW); Electrophoretic mobility: 4.0 cm. UV max nm in MeOH (A): 255
(inflection), 267, 343; (A) þ NaOAC: 274, 360; (A) þ NaOAc þ H3BO3:
254,267,358; (A) þ AlCl3: 274, 300, 380; (A) þ AlCl3 þ HCl: 2275, 405;
(A) þ NaOMe: 276, 310, 395. 1H NMR: d ppm: 6.18 (d, J ¼ 2 Hz, H-6);
6.40 (d, J ¼ 2 Hz, H-8); 6.92 (d, J ¼ 8 Hz, H-50); 7.85 (d, J ¼ 2 Hz, H-20);
7.93 (dd, J ¼ 8 Hz, J ¼ 2 Hz, H-60), 3.85 (s, Me-40). –ve ESI-MS of 2:
m/z ¼ 497 [M–Na]ꢀ, 417 [M–SO3Na]ꢀ, 395 [417 þ H-Na]ꢀ, 315
[417 þ H-SO3Na]ꢀ; þpv ESI-MS of 2: m/z ¼ 521 [M þ H]þ, 543
[M þ 23]þ. 1H NMR of 2: d ppm: 6.20 (d, J ¼ 2 Hz, H-6); 6.49 (d, J ¼ 2
Hz, H-8); 6.85 (d, J ¼ 8 Hz, H-50); 7.89 (d, J ¼ 2 Hz, H-20); 7.97 (dd,
J ¼ 8 Hz, J ¼ 2 Hz, H-60), 387 (s, Me-40). 13C NMR (Table 1).
Acknowledgements: We are indebted to AvH (Alexander von Humboldt)
foundation for the donation of a Schimazu UV-Visible-1601 spectropho-
tometer and a 8001- Kruess polarimeter. We thank the BMBF, Germany,
for the support of the stay of K. Hofmann at the NRC Cairo and of
S. Hussein at the University Greifswald (EGY 05/002).
3.4.5. Dehydrodigallic acid dimethyl ester ð3Þ
References
Rf-values: 0.58 (H2O), 0.70 (HOAc), 0.80 (BAW); HPLC/-ve-ESIMS: con-
ditions for HPLC/ESI-MS analysis: A binary gradient with the following
time program was used, where the solvents are (A) H2O–HOAc (98 : 2)
and (B) MeOH–H2O–HOAC (80 : 18 : 2): 0–5 min, 5% B; 5–50 min, in-
creased to 41% B, held for 15 min; 65–80 min, increased to 100% B, held
for 5 min; 85–90 min, decreased to 5% B, held for 10 min. The flow rate
was 0.2 ml min ꢄ 1 and the injection volume was 10 ml. The columns
(100 ꢂ 2 mm i.d.) were filled with 5 mm Nucleosil 120 C18 and for com-
pounds UV detection at 280 nm a Spectra-Physics detector was used. The
HPLC instrument was coupled to a MAT95 sector field mass spectrometer
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acid (Co-PC). Dehydrodigallic acid: Rf -values: 0.54 (H2O), 0.60 (HOAc),
0.72 (BAW). UV max nm in MeOH: 272; EI-MS: m/z ¼ 338 [M]þ. 1H
NMR: d ppm 7.02 (d, J ¼ 2.5 Hz, H-2); 6.5 (d, J ¼ 2.5 Hz, H-6); 6.9 (s,
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2); 6.6 (d, J ¼ 2.5 Hz, H-6); 6.98 (s, H-60); 3.63 and 3.55 both aare sing-
lets (protons of two methyl esters). 13C NMR: (Table 1).
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