Journal of Ethnopharmacology 93 (2004) 89–92

In vitro antibradykinin activity of Aloe barbadensis gel

Rocı́o Bautista-Pérez, David Segura-Cobos, Beatriz Vázquez-Cruz∗
Laboratorio de Farmacologı́a, División de Investigación y Postgrado, Facultad de Estudios Superiores Iztacala,
Universidad Nacional Autónoma de México, Av., De los Barrios 1, Los Reyes Iztacala, Tlalnepantla, Mexico, CP 54090, Mexico
Received 1 January 2002; received in revised form 1 February 2004; accepted 18 March 2004

Available online 14 May 2004

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

1. Introduction We have demonstrated anti-inflammatory activity of

Aloe barbadensis Mill. (Liliaceae) is used in the tra-
ditional medicine of Mexico and other countries for
anti-inflammatory and cosmetic purposes (Diez-Martı́nez,
1981; Grindlay and Reynolds, 1986). From the fresh leaves
of Aloe barbadensis, two components are obtained: a bitter
yellow juice (exudate), which drains from the transversally
cut leaves, which is used as a laxative (Ishii et al., 1990),
and a mucilaginous gel from the leaf parenchyma, which
has been used as a remedy for a variety of pathological
states, such as arthritis, gout, acne, dermatitis, burns, and
peptic ulcers induced by epithelial alterations (Cappasso
and Ganginella, 1997; Reynolds and Dweck, 1999).
Previous studies have reported that Aloe barbadensis gel
accelerates wound healing in streptozotocin-diabetic rats as
a result of its capacity to stimulate fibroblast (Chitra et al.,
1998). It has also been reported that Aloe barbadensis pos-
sesses antidiabetic properties (Okay et al., 2001). Further-
more, some chemical constituents with immunomodulatory
and lectin-like properties have been isolated from the ex-
tracts of Aloe barbadensis gel (Qiu et al., 2000).
∗ Corresponding author. Tel.: +52-55-5623-1291;
fax: +52-55-5390-7604.
E-mail address: bvcruz@servidor.unam.mx (B. Vázquez-Cruz).
the Aloe barbadensis gel associated with cyclooxyge-
nase activity inhibition, namely, preventing the synthe-
sis of prostaglandins, some of the principal mediators in
the development of the inflammatory processes (Davis
et al., 1984). Thus, we suggested that Aloe barbadensis
anti-inflammatory activity was related to the inhibition of
prostaglandins synthesis (Vazquez et al., 1996).
However, it has been reported that other Aloe species (Aloe
arborescens Mill. var. natalensis Berger and Aloe saponaria
Haw.) have an anti-inflammatory effect related to the in-
hibition of bradykinin; the effect of Aloe arborescens on
bradykinin has been associated to bradykininase and car-
boxypeptidase activities (Fujita et al., 1979), whereas an un-
specific antibradykinin activity has been ascribed to Aloe
saponaria (Yagi et al., 1982). Bradykinin participates di-
rectly as a chemical mediator in a variety of inflammatory
diseases and produces pain through the stimulation of pri-
mary sensor neurons, and provokes the release of neuropep-
tides, such as substance P, neurokinin A, and calcitonin
gene-related peptide (Geppetty, 1993; Averbeck and Reeh,
2001); it also stimulates prostaglandin production and accu-
mulation in inflammatory cells as macrophages, as well as
acting as inflammatory mediators, such as IL-I and tumor
necrosis factor ␣ (TNF␣) (Dray and Perkins, 1993; Bogar
et al., 1999).

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
contains a substance that prevents bradykinin activity, in ad-
dition to its capacity to inhibit the cyclooxygenase activ-
ity, thereby potentiating its anti-inflammatory effect. Thus,
in the present paper we study the effect of Aloe barbaden-
sis gel and the purified fractions on the bradykinin-induced
ileum contraction.
2. Methodology
2.1. Experimental animals
Wistar rats weighing between 200 and 250 g raised in our
animal house and fed on standard chow diet and water ad
libitum were used for the study.
2.2. Assay method for antibradykinin activity
Bradykinin activity was estimated by means of biological
assay on the isolated rat ileum preparation. Briefly, the ani-
mals were sacrificed by cervical dislocation, the abdominal
cavity was opened, and the ileum was removed. Segments
of approximately 0.5 cm were obtained and suspended
in an isolated organ bath between two nickel–chromium
wire hooks. One of the hooks was fastened to the bot-
tom of the bath chamber and the other was attached to a
mechanical–electric transducer Narco Bio System (NSB)
Mod. F60, connected to Physiograph Mod. DMP-4B (NBS),
and the recorder. The bath chamber contained 10 mL of Ty-
rode solution with the following composition (mM): NaCl
138, KCl 2.7, CaCl2 1.8, NaHPO4 0.2, NaHCO3 11.9, and
glucose 5.5; bubbled with O2 /CO2 (95%/5%), pH 7.4, at
30 ◦ C. The segments of ileum were subjected to a resting
tension of 0.3 g, which was kept constant throughout the
experiments. The preparation was allowed to stabilize for
approximately 15 min, and the medium was changed ev-
ery 5 min. Bradykinin (Sigma, St. Louis, MO), 1.6 mM,
was prepared in 10 mM phosphate buffer at pH 7.4 and
was incubated with the vehicle or 1 mg of each fraction
of the Aloe barbadensis for 10 min, at 30 ◦ C, and then
increasing concentrations of this incubation mixture (2.5,
7.5, 17.5, 37, 74.5, and 143 nM) were added to the organ
bath. Ileum contraction induced by the incubation mix-
ture (bradykinin + Aloe barbadensis) was compared to the
ileum contraction induced by bradykinin alone. In all cases,
responses to each concentration of the agonist used were
expressed as percent of the maximal response obtained
in the initial concentration–response curve to bradykinin
alone.
2.3. Preparation of the gel
Fresh Aloe barbadensis leaves were collected from the
Municipality of Apaxco, State of Mexico. The plant was
identified in the herbarium of the Botany Department of
the Facultad de Estudios Superiores Iztacala, U.N.A.M.,
where a specimen was deposited with accession number
RBP 9052.
2.4. Extraction and purification of antibradykinin-active
material from Aloe barbadensis
The fresh leaves of Aloe barbadensis were cut in half
and the colorless gel was separated carefully by scraping
the green cortical layer. The gel was minced, homoge-
nized (4 kg), and centrifuged (centrifuge Sorvall RC-5B) at
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)
against distilled water for 48 h. The nondialyzable fraction
was lyophilized (Labco Freeze Dry System 4.5) to yield
colorless, soft crude extract [fraction 1 (F-1)].
2.4.1. Ammonium sulfate precipitation and separation of
the polysaccharides from the F-1
To separate and purify the components from Aloe bar-
badensis gel, the crude extract (F-1) was dissolved in dis-
tilled water, and successive precipitations were performed
by the addition of ammonium sulfate (Backer analized) to
achieve 35, 55, 75, and 100% saturation. After standing
overnight, it was centrifuged at 10,000 rpm for 30 min, at
4 ◦ C. Precipitates were dissolved in distilled water, dia-
lyzed against distilled water for 48 h, and then lyophilized
to yield the ammonium sulfate fractions F-35, F-55, F-75,
and F-100, and were assayed in the isolated rat ileum
preparation. As the antibradykinin activity was found in
fraction F-55, the purification process was continued; the
F-55 was dissolved in 0.05 M phosphate and 0.15 M NaCl
buffer, pH 7.0, and applied to a column of Sephacryl
S-500HR (2 cm × 40 cm). The material was eluted through
the column with Tris 0.05 M buffer at pH 7.0 at a flow
rate of 21 mL/h, at 4 ◦ C. The eluate was concentrated
by precipitation with ammonium sulfate, and dialyzed
against distilled water followed by lyophilization to yield
fraction SH (F-SH) that was assayed for antibradykinin
activity.
2.4.2. F-SH filtration in sephadex G-100
F-SH was suspended in 0.05 M phosphate buffer, and
0.15 M NaCl at pH 7.0 was passed through filtration in a
column of Sephadex G-100 (2 cm × 40 cm), which separates
molecules in a range of 20–100 kD. The F-SH was eluted
with the same solution at a flow rate of 20 mL/h, at 4 ◦ C.
The eluate was monitored by measuring the absorbance at
260 nm for every 2 mL, in a Perkin Elmer model Lambda
11. Four major peaks were detected at elution volumes.
The peaks were clustered in four fractions: F-GA, F-GB,
F-GC, and F-GD. These fractions were assayed on the
isolated rat ileum preparation. The protein and carbohy-
drate content of the fraction were determined by Bradford
(1976) and phenol–sulfuric acid (DuBois et al., 1956)
methods.
 R. Bautista-Pérez et al. / Journal of Ethnopharmacology 93 (2004) 89–92
2.5. Statistical analysis
Significance of the differences in the dose–response
curves were assessed by Student’s t-test for unpaired sam-
ples, and values of P < 0.05 were considered significant.
3. Results and discussion
In the present study, we showed that at least one frac-
tion of the extract of Aloe barbadensis contains proteins
that inhibited bradykinin-dependent contraction of the iso-
lated rat ileum. As seen in Fig. 1, bradykinin produced
a concentration-dependent contraction of the isolated rat
ileum. When bradykinin was previously incubated with
F-1 and was assayed on isolated rat ileum, it was ob-
served that the maximal contractile response of this organ
to bradykinin decreased by 10 ± 3.5. This effect did not
occur when F-1 was previously boiled for 10 min, suggest-
ing that the antibradykinin fraction contains heat-sensitive
proteins. Similar antibradykinin effect was obtained after
10 or 30 min incubation with F-1. Therefore, it was decided
to use 10 min as the incubation time for all assays. It was
also observed that the incubation of bradykinin with phos-
phate buffer did not modify the concentration–response
curve to bradykinin. Thus, this control suggested that
the antibradykinin effect was caused by the presence of
the Aloe barbadensis fraction in the incubation mixture.
Moreover, when the ileum was stimulated with the frac-
tions alone, no effect on the tone of the ileum was ob-
served.
When bradykinin was incubated with the fractions ob-
tained from F-1 by ammonium sulfate precipitation (F-35,
F-55, F-75, and F-100), only fraction F-55 decreased
the bradykinin-induced ileum contraction, shifting the
bradykinin dose response to the right (Fig. 1).
Fig. 1. Concentration–response curve of bradykinin on isolated rat ileum.
The ileum was stimulated with increasing concentrations of bradykinin
previously incubated for 10 min, at 30 ◦ C, with 10 mM phosphate buffer
(pH 7.4) (䊊). Bradykinin was previously incubated with 1 mg of F-1
from Aloe barbadensis gel (䊉) and incubated with F-55 obtained through
precipitation of the Aloe barbadensis gel using 55% ammonium sulfate
saturated (䊏). Each curve represents the mean ± S.E.M. of five different
experiments (∗ P < 0.05).
91
Fig. 2. Concentration–response curve of bradykinin on isolated rat ileum.
The ileum was stimulated with increasing concentrations of bradykinin,
which was previously incubated for 10 min, at 30 ◦ C, with 10 mM phos-
phate buffer (pH 7.4) (䊊). Alternatively bradykinin was previously incu-
bated with 1 mg of F-SH, obtained by elution of F-55, in a column of
Sephacryl (䊉), and incubated with 1 mg of F-GB, obtained by filtration
of F-SH, through a column of Sephadex (䊏). Each curve represents the
mean ± S.E.M. of five different experiments (∗ P < 0.05).
It has been reported that the viscous nature of Aloe bar-
badensis gel is due mainly to the presence of acetylated
polysaccharides composed of a mixture of glucomannans
(Yaron et al., 1992). Thus, the presence of the polysac-
charides in the incubation mixture could interfere with
the antibradykinin activity. Therefore, we purified F-55
by passing it through a Sephacryl column to remove the
free polysaccharides of high molecular weight, yielding
the fraction F-SH. This fraction decreased the maximal
response of bradykinin by 60 ± 8.5% (Fig. 2), suggest-
ing that indeed the free polysaccharides of high molecular
weight present in the F-55 interfered with the antibradykinin
activity.
In order to separate the proteins by molecular weight from
F-SH, the F-SH was filtered through a column of Sephadex
G-100. The eluate was monitored by measuring the ab-
sorbance at 260 nm for each 2-ml fraction. Four major peaks
were detected; these were clustered in four fractions, with
absorbance at 260 nm: F-GA, F-GB, F-GC, and F-GD (Fig.
3). F-GB was the only fraction that showed antibradykinin
activity. The maximal response of bradykinin was reduced
by 67 ± 8.5% (Fig. 2), suggesting that the molecular weight
of the protein with antibradykinin effect is between 20 and
100 kD.
In order to compare the effect of the different fractions of
the Aloe barbadensis gel on bradykinin activity, we set the
effect produced by crude gel on 143 nM of bradykinin as 1;
this value increased to 2.6, 4.7, and 7.2 for fractions F-55,
F-SH, and F-GB, respectively. These data suggest that se-
quential purification of Aloe barbadensis gel yields a com-
pound with higher antibradykinin activity. Our data agree
with previous results of Fujita et al. (1976), who reported
bradykininase activity in Aloe arborescens. These authors
also demonstrated that enzymes with bradykininase activ-
ity consist of a carboxypeptidase capable of hydrolyzing
92 R. Bautista-Pérez et al. / Journal of Ethnopharmacology 93 (2004) 89–92
0.40
(C)
ABSORBANCE AT 260 nm
0.32
0.24
(B)
0.16
(A)
(D)
0.08
0.00
0 22 44 66
FRACTION NUMBER
Fig. 3. Chromatogram obtained by filtration of F-SH, through a column
of Sephadex G-100. Measuring its absorbance at 260 nm every 2 min
monitored the elute from the column. Four major peaks were detected.
The peaks were clustered in four fractions: F-GA, F-GB, F-GC, and F-D.
The fractions were assayed on the isolated rat ileum.
bradykinin at C-terminus. Fujita et al. (1979) and Itto et al.
(1993) proved that it is a serine carboxypeptidase. On the
other hand, Yagi et al. (1982) reported antibradykinin activ-
ity of Aloe saponaria extract suggesting that several Aloe
species show the antibradykinin effect. However, it is im-
portant to point out that in accordance with our previous
studies (Vazquez et al., 1996), this antibradykinin effect
may be associated with the inhibition of prostaglandin
synthesis in the anti-inflammatory mechanism of Aloe
barbadensis.
4. Conclusion
We suggest that the antibradykinin activity (shown in
the Aloe barbadensis fraction) is caused by a protein,
which is labile to the heat, and this might be a bradykin-
inase. This antibradykinin effect of Aloe barbadensis pro-
vides a new scientific support for the utilization of this
plant in folk medicine in the treatment of inflammatory
conditions.
Acknowledgements
The authors wish to thank Dr. Bruno Escalante and
Ieroham Solomon Barouh for their critical review of this
manuscript. This work was funded by a grant from Consejo
Nacional de Ciencia y Tecnologı́a (CONACyT), México
(Ref. D111-903672).
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