4656
I. León-Rivera et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4652–4656
chromatographic fractions. The components of the less polar chromatographic
fraction (1 g) were purified by preparative HPLC using an Ultrasil ODS column
m, Altex), eluting with a mixture of CH3CN–H2O
adhering connective tissue and was cut into 3–5 mm length rings. In some
rings, the endothelium was removed. Then, tissue segments were mounted in
stainless steel hooks, under an optimal tension of 3 g, in 10 mL organ baths
containing warmed (37 °C) and oxygenated (O2/CO2, 19:1) Krebs solution
(composition, mM: NaCl, 118; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.2 mM; KH2PO4,
1.2; NaHCO3, 25.0; EDTA, 0.026 and glucose, 11.1, pH 7.4). Changes in tension
were recorded by Grass-FT03 force transducers (Astromed, West Warwick, RI,
USA) connected to a MP100 analyzer (Biopac Instruments, Santa Barbara, CA,
(10 mm i.d. ꢂ 300 mm, 5
l
(8:2), at a flow rate of 1 mL/min at 25 °C, and detection with UV at 210 nm.
Chromatographic peaks were collected and re-injected until pure. This
technique afforded pure compounds 1 (400 mg, tR 13.5 min) and 2 (100 mg,
tR 15.2 min). Tyrianthin A (1). Amorphous white powder; mp 146–148 °C; ½a D25
ꢀ
ꢁ22.0 (c 1.1 CH3OH); IR (KBr): 3376 (OH), 2985 (C–H), 1735 (C@O), 1090 (C–O)
cmꢁ1
;
1H and 13C NMR (C5D5N): Table 1; positive-ion FABMS m/z 2091 [M+H]+;
USA). After equilibration, rings were contracted by noradrenaline (NA, 0.3
and washed every 30 min for 2 h. The absence of endothelium was confirmed
by the lack of a relaxing response to carbachol (1 M). After precontraction
with NA, the test samples (pure compounds, vehicle, and positive control) were
added to the bath in a volume of 100 L; then cumulative concentration–
lM)
negative-ion FABMS m/z 2089 [MꢁH]ꢁ, 1055 [Mꢁunit BꢁH]ꢁ, 955
[1055ꢁC5H8O2]ꢁ, 855 [955ꢁC5H8O2]ꢁ, 561, 417, and 271; HRMALDITOFMS
m/z 2091.3775 [M+H]+ (calcd for C100H168O45H+ 2091.3781). Tyrianthin B (2).
l
Amorphous white powder; mp 140–142 °C; ½a D25
ꢀ
ꢁ20.1 (c 1.3 CH3OH); IR (KBr):
l
3376 (OH), 2985 (C–H), 1735 (C@O), 1090 (C–O) cmꢁ1
;
1H and 13C NMR
response curves were obtained for each ring. The relaxant effect of pure
compounds and positive controls was determined by comparing the muscular
tone of the contraction before and after addition of the test compounds.
16. Vanhoutte, P. M. J. Cardiovasc. Pharmacol. 2001, 38, 796.
(C5D5N): Table 1; positive-ion FABMS m/z 1977 [M + H]+; negative-ion FABMS
m/z 1975 [M
–
H]ꢁ, 1055 [Mꢁunit BꢁH]ꢁ, 955 [1055ꢁC5H8O2]ꢁ, 855
[955ꢁC5H8O2]ꢁ, 561, 417, and 271; HRMALDITOFMS m/z 1977.2354 [M+H]+
(calcd for C94H158O43H+ 1977.2361).
17. GABA and glutamate release in cortical brain slices. Mice were sacrificed by
decapitation and anterior brain cortex was dissected out and slices (250–
7. Alkaline hydrolysis. The less polar chromatographic fraction (100 mg) was
refluxed in 0.2 N NaOH (10 mL) for 60 min. The reaction mixture was acidified
to pH 5 and extracted with CH2Cl2. The organic layer was washed with H2O,
dried over anhydrous Na2SO4, and evaporated under reduced pressure. The
aqueous layer was lyophilized, the residue was dissolved with methanol, and a
white solid (glycosidic acid) was obtained after removal of solvent.
8. Noda, N.; Kogetsu, H.; Kawasaki, T.; Miyahara, K. Phytochemistry 1990, 29, 3565.
9. Enríquez, R. G.; León, I.; Pérez, F.; Walls, F.; Carpenter, K.; Puzzuoli, F.; Reynolds,
W. F. Can. J. Chem. 1992, 70, 1000.
300
slices were placed at 4 °C in 5 mL in
l
m) were cut manually using a razor blade and a cover glass guide.17 Brain
a
modified Krebs-Ringer medium
(120.0 mM NaCl, 4.7 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 1.0 mM tris–HCl
buffer (pH 7.4), and 10.0 mM glucose) pH 7.4, previously oxygenated with O2
bubbling. Amino-oxyacetic acid at a concentration of 10.0 lM, was added to
the medium to prevent GABA metabolism. After 5 min, slices were placed in
5 mL of the basal medium (Krebs-Ringer medium) in a well of microplate at
37 °C for 10 min. Next, pure compounds were added at a final concentration of
10. Escalante-Sánchez, E.; Pereda-Miranda, R. J. Nat. Prod. 2007, 70, 1029.
11. Acid hydrolysis. The glycosidic acid was refluxed in 1.0 N HCl (5 mL of water–
ethanol) for 1.0 h. The reaction mixture was taken to pH 5 with NaOH solution,
and the solution extracted with CH2Cl2. An aliquot of the aqueous phase was
20.0 lg/mL and aliquots of 200 lL were taken out at 0.5, 1.0, 1.5, 2.0, and
3.0 min. At the end of the experiment, the content of GABA and glutamate into
each collected aliquot was determined by HPLC, previous derivation with O-
phthaldialdehyde. Protein content in brain slices was determined after its
homogenization in 1 mL of water.
subjected to HPLC on
250 ꢂ 4.6 mm), with an isocratic elution of CH3CN–H2O (8:2), at a flow rate
of 1 mL/min, and a sample injection of 200 L, affording three compounds
which were identified by co-elution with standard -rhamnose (1 mg tR
-glucose (quinovose 2 mg, tR 8.9 min), and b- -glucose
a Nucleosil 100 NH2 column (Alltech; 5 lm,
18. Herrera-Ruiz, M.; Gutiérrez, C.; Jiménez-Ferrer, J. E.; Tortoriello, J.; Mirón, G.;
León, I. J. Ethnopharmacol. 2007, 112, 243.
19. Antimycobacterial activity. The in vitro antimycobacterial activity of compounds
1 and 2 was measured as the minimal inhibitory concentration (MIC), and
carried out using the modified microplate Blue Alamar assay (MABA). The
l
a-
L
7.9 min), 6-deoxy-b-
(1 mg, tR 15.2 min).
D
D
12. Lu, Y.; Luo, J.; Huang, X.; Kong, L. Steroids 2009, 74, 95.
13. Reynolds, W. F.; Enríquez, R. G. J. Nat. Prod. 2002, 65, 221.
concentrations of pure compounds ranged from 100.00 to 3.13 lg/mL.
Rifampin was used as positive controls. All evaluations were carried out in
duplicate.
14. The GC–MS system consisted of a HP 6890 gas chromatograph and a HP 5970
mass selective detector in the electron-ionization. GC conditions:
25 m ꢂ 0.2 mm HP-5 column; He, 1 mL/min; Oven temperature 40 °C, 2 min,
20. Rivero-Cruz, I.; Acevedo, L.; Guerrero, J. A.; Martínez, S.; Bye, R.; Pereda-
Miranda, R.; Franzablau, S.; Timmermann, B. N.; Mata, R. J. Pharm. Pharmacol.
2005, 57, 1117.
40–250 °C,
D 15 °C/min, 250 °C 10 min; Injector temperature 300 °C; Interfase
temperature 280 °C; split 1:40.
15. Vasorelaxant assay. Male Wistar rats (250–300 g of body weight) were
sacrificed by exposure to diethyl ether. The thoracic aorta was cleaned of
21. Barnes, C. C.; Smalley, M. K.; Manfredi, K. P.; Kindscher, K.; Loring, H.; Sheeley,
D. M. J. Nat. Prod. 2003, 66, 1457.