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Abstract
When bradykinin-induced contraction of the isolated rat ileum was tested in the presence of Aloe barbadensis Mill. (Liliaceae) gel (fraction
F-1) and with the fraction obtained by precipitation of the F-1 with 55% ammonium sulfate (F-55), the maximal responses to bradykinin were
reduced by 10 and 22%, respectively. Furthermore, purification of the F-55 by filtration through a column of Sephacryl (S-500-HR) yielded
the F-SH fraction, which inhibited the bradykinin effect by 60%. Purification of the F-SH fraction, by filtration through a column of Sephadex
G-100, brought about four new fractions: F-GA, F-GB, F-GC, and F-GD. F-GB was the only one that showed the bradykinin inhibition
effect (67%). Clearly, Aloe barbadensis gel contains a material that inhibits the bradykinin effect, which might explain the anti-inflammatory
properties of Aloe barbadensis.
© 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Aloe barbadensis; Bradykinin; Antibradykinin activity; Isolated ileum; Inflammation; Anti-inflammatory
0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2004.03.030
90 R. Bautista-Pérez et al. / Journal of Ethnopharmacology 93 (2004) 89–92
Therefore, we hypothesized that Aloe barbadensis gel the Facultad de Estudios Superiores Iztacala, U.N.A.M.,
contains a substance that prevents bradykinin activity, in ad- where a specimen was deposited with accession number
dition to its capacity to inhibit the cyclooxygenase activ- RBP 9052.
ity, thereby potentiating its anti-inflammatory effect. Thus,
in the present paper we study the effect of Aloe barbaden- 2.4. Extraction and purification of antibradykinin-active
sis gel and the purified fractions on the bradykinin-induced material from Aloe barbadensis
ileum contraction.
The fresh leaves of Aloe barbadensis were cut in half
and the colorless gel was separated carefully by scraping
2. Methodology the green cortical layer. The gel was minced, homoge-
nized (4 kg), and centrifuged (centrifuge Sorvall RC-5B) at
2.1. Experimental animals 10,000 rpm for 30 min, at 4 ◦ C. The supernatant was dialyzed
in Spectrapor cellulose tubing (m.w. cut off 12,000–14,000)
Wistar rats weighing between 200 and 250 g raised in our against distilled water for 48 h. The nondialyzable fraction
animal house and fed on standard chow diet and water ad was lyophilized (Labco Freeze Dry System 4.5) to yield
libitum were used for the study. colorless, soft crude extract [fraction 1 (F-1)].
2.2. Assay method for antibradykinin activity 2.4.1. Ammonium sulfate precipitation and separation of
the polysaccharides from the F-1
Bradykinin activity was estimated by means of biological To separate and purify the components from Aloe bar-
assay on the isolated rat ileum preparation. Briefly, the ani- badensis gel, the crude extract (F-1) was dissolved in dis-
mals were sacrificed by cervical dislocation, the abdominal tilled water, and successive precipitations were performed
cavity was opened, and the ileum was removed. Segments by the addition of ammonium sulfate (Backer analized) to
of approximately 0.5 cm were obtained and suspended achieve 35, 55, 75, and 100% saturation. After standing
in an isolated organ bath between two nickel–chromium overnight, it was centrifuged at 10,000 rpm for 30 min, at
wire hooks. One of the hooks was fastened to the bot- 4 ◦ C. Precipitates were dissolved in distilled water, dia-
tom of the bath chamber and the other was attached to a lyzed against distilled water for 48 h, and then lyophilized
mechanical–electric transducer Narco Bio System (NSB) to yield the ammonium sulfate fractions F-35, F-55, F-75,
Mod. F60, connected to Physiograph Mod. DMP-4B (NBS), and F-100, and were assayed in the isolated rat ileum
and the recorder. The bath chamber contained 10 mL of Ty- preparation. As the antibradykinin activity was found in
rode solution with the following composition (mM): NaCl fraction F-55, the purification process was continued; the
138, KCl 2.7, CaCl2 1.8, NaHPO4 0.2, NaHCO3 11.9, and F-55 was dissolved in 0.05 M phosphate and 0.15 M NaCl
glucose 5.5; bubbled with O2 /CO2 (95%/5%), pH 7.4, at buffer, pH 7.0, and applied to a column of Sephacryl
30 ◦ C. The segments of ileum were subjected to a resting S-500HR (2 cm × 40 cm). The material was eluted through
tension of 0.3 g, which was kept constant throughout the the column with Tris 0.05 M buffer at pH 7.0 at a flow
experiments. The preparation was allowed to stabilize for rate of 21 mL/h, at 4 ◦ C. The eluate was concentrated
approximately 15 min, and the medium was changed ev- by precipitation with ammonium sulfate, and dialyzed
ery 5 min. Bradykinin (Sigma, St. Louis, MO), 1.6 mM, against distilled water followed by lyophilization to yield
was prepared in 10 mM phosphate buffer at pH 7.4 and fraction SH (F-SH) that was assayed for antibradykinin
was incubated with the vehicle or 1 mg of each fraction activity.
of the Aloe barbadensis for 10 min, at 30 ◦ C, and then
increasing concentrations of this incubation mixture (2.5, 2.4.2. F-SH filtration in sephadex G-100
7.5, 17.5, 37, 74.5, and 143 nM) were added to the organ F-SH was suspended in 0.05 M phosphate buffer, and
bath. Ileum contraction induced by the incubation mix- 0.15 M NaCl at pH 7.0 was passed through filtration in a
ture (bradykinin + Aloe barbadensis) was compared to the column of Sephadex G-100 (2 cm × 40 cm), which separates
ileum contraction induced by bradykinin alone. In all cases, molecules in a range of 20–100 kD. The F-SH was eluted
responses to each concentration of the agonist used were with the same solution at a flow rate of 20 mL/h, at 4 ◦ C.
expressed as percent of the maximal response obtained The eluate was monitored by measuring the absorbance at
in the initial concentration–response curve to bradykinin 260 nm for every 2 mL, in a Perkin Elmer model Lambda
alone. 11. Four major peaks were detected at elution volumes.
The peaks were clustered in four fractions: F-GA, F-GB,
2.3. Preparation of the gel F-GC, and F-GD. These fractions were assayed on the
isolated rat ileum preparation. The protein and carbohy-
Fresh Aloe barbadensis leaves were collected from the drate content of the fraction were determined by Bradford
Municipality of Apaxco, State of Mexico. The plant was (1976) and phenol–sulfuric acid (DuBois et al., 1956)
identified in the herbarium of the Botany Department of methods.
R. Bautista-Pérez et al. / Journal of Ethnopharmacology 93 (2004) 89–92 91
0.40 References
(C)
ABSORBANCE AT 260 nm