This Issue is Dedicated to the Memory of Professor Ivano Morelli

1104 Natural Product Communications Vol. 1 (12) 2006 Simões-Pires et al.
in ppm as δ rel. to Me 4 Si (int. std.). LC/MS was
performed directly after UV-DAD measurements. A
Finningan LCQ ion trap (Finningan MAT, San Jose,
CA, USA) with APCI interface was used with the
following conditions: capillary temp. 150°C;
vaporizer temp. 370°C; positive mode; sheath gas
flow: 60 arb, corona needle current 5 µA; spectra
(150-900 mu). HRESIMS was performed using a
Bruker FTMS 4.7T. TLC: silica gel 60 F 254 Al sheets
(Merck), detection at 254 nm and with vanillinsulfuric
acid reagent. LC/UV-DAD analysis of the
alkaloid extract was performed on a Hewlett-Packard
(Waldbronn, Germany) Series 1100 photodiode array
detector (DAD) liquid chromatograph system. The
separation was achieved on a Nucleosil 100-5 C 18 AB
column (125 x 4.6 mm i.d., 5 µm; Macherey-Nagel)
with MeOH/H 2 O (containing Et 3 N 2 mM) in the
gradient mode (10% of MeOH to 100% in 40 min).
The flow rate was 1 mL/min; the UV traces were
measured at 210 and 254 nm and UV spectra (DAD)
were recorded between 200 and 500 nm. HPLC
microfractionation: Fractions were collected, after the
LC/UV analysis, every 1 min (1 mL) in Eppendorf
tubes by a Gilson collector (FC204). After collection,
all fractions were evaporated to dryness on a
Speedvac system (RCT 90, Jouan). The content of
each fraction was suspended in 1 mL of PMN
suspension and then used for the antichemotactic
assay. Semi-preparative HPLC was carried out with
a Shimadzu LC-8A pump equipped with a Knauer
UV detector using a Symmetry-Prep column (7 µm,
19x150 mm, Waters).
Plant material: P. myriantha was collected in
Reserva Estadual do Turvo, Derrubadas Rio Grande
do Sul, Brazil and identified by M. Sobral. A voucher
specimen (M. Sobral et al., 8913) was deposited in
the ICN Herbarium (Universidade Federal do Rio
Grande do Sul, Porto Alegre, Rio Grande do Sul,
Brazil).
Extraction and isolation: Dried leaves (823 g) were
extracted with EtOH (3.5 l) at room temperature. The
extract was concentrated under vacuum at 40 °C and
an alkaloid extract was obtained by classical
acid/base extraction. In addition to the alkaloid rich
CH 2 Cl 2 extract, it was noticed that the aqueous
fraction was also positive for alkaloids with
Dragendorff reagent. In order to extract these
alkaloids, a second partition was conducted between
the residual aqueous fraction and n-BuOH. The
butanolic extract (1 g) was purified by semipreparative
HPLC using Symmetry-Prep column
(7 µm, 19 x 150 mm, Waters), MeOH-H 2 O (30:70)
with Et 3 N 2 mM, flow rate 10 mL/min, UV 254 nm)
providing alkaloids 1 (33 mg) and 2 (12 mg).
Acid hydrolysis: Compounds 1 and 2 were submitted
to hydrolysis with 20 mL of HCl 0.05 N at 65°C
during 24h. Sugars were extracted by partition with
n-BuOH and were compared by TLC (solvent:
AcOEt/Formic acid/H 2 O 100:20:30; detected with
p-anisaldehyde-H 2 SO 4 reagent) with reference
compounds: glucose (R f 0.40), arabinose (R f 0.45),
fructose (R f 0.41), rhamnose (R f 0.64), and galactose
(R f 0.33), all 1 mg/mL in H 2 O. Compounds 1 and 2
gave glucose (R f 0.40).
Enzymatic hydrolysis: Compounds 1 and 2 were
treated with β-D-glucosidase in 1 mL NaOAc buffer
(pH 5.0) for three days at 40°C. The aglycones were
extracted by partition with n-BuOH, and submitted to
LC/UV-DAD analysis hydrolysis in order to confirm
hydrolysis.
Antichemotactic assay: Chemotaxis was measured in
a Boyden chamber by the method previously
described [24]. Prior to the chemotactic assay, rat
leukocytes were treated with 100 μg/mL of each
sample (alkaloids and extract), at 37°C for 1 h.
Plasma collected from rats was incubated at 37°C for
30 min with 65 μg/mL of lipopolysaccharide (LPS)
from Escherichia coli, and then diluted in Hanks
buffer 1:5 (v/v). Chemotactic migration of leukocytes
through an 8.0-μm cellulose nitrate filter, towards the
chemotactic stimulant (LPS treated plasma) was
measured after incubation for 1 h at 37°C using the
micrometer on the fine-focus knob of a Nikon
Alphaphot-2 YS2 microscope. The distance from the
upper surface of the filter to the lower surface of
focus still containing two cells allowed the evaluation
of leukocyte migration in five microscopic fields per
filter. The assay was carried out in duplicate and
measurements were statistically analyzed by
Student’s t-test, using genistein as positive control.
Strictosidinic acid (1)
Amorphous powder.
[α] 25 D: +143.14° (c 0.1, MeOH).
Rf: 0.30: TLC system: ethyl acetate / acetic acid /
formic acid / water (100:11:11:10).
1 H NMR (500 MHz, DMSO-d 6 ): 4.10 (1H, d,
J = 10.4 Hz, H-3), 2.92-3.47 (2H, m, H-5), 2.73-2.92
(2H, m, H-6), 7.41 (1H, d, J = 7.81 Hz, H-9), 6.97
(1H, dd, J = 7.33 and 7.81 Hz, H-10), 7.05 (1H, dd,