PHYTOTHERAPY RESEARCH

Phytother. Res. 24: 764–768 (2010)

Published online 26 November 2009 in Wiley InterScience
(www.interscience.wiley.com) DOI: 10.1002/ptr.3043

Increased Vascular Permeability, Angiogenesis

and Wound Healing Induced by the Serum of
Natural Latex of the Rubber Tree
Hevea brasiliensis

Ricardo José Mendonça1*, Vanessa Beatriz Maurício1, Larissa de Bortolli Teixeira1,

João José Lachat2 and Joaquim Coutinho-Netto1
1
Department of Biochemistry and Immunology, Ribeirao Preto School of Medicine, University of São Paulo, Brazil
2
Department of Surgery and Anatomy, Ribeirao Preto School of Medicine, University of São Paulo, Brazil

Increases in vascular permeability and angiogenesis are crucial events to wound repair, tumoral growth and
revascularization of tissues submitted to ischemia. An increased vascular permeability allows a variety of cyto-
kines and growth factors to reach the damaged tissue. Nevertheless, the angiogenesis supply tissues with a wide
variety of nutrients and is also important to metabolites clearance. It has been suggested that the natural latex
from Hevea brasiliensis showed wound healing properties and angiogenic activity. Thus, the purpose of this
work was to characterize its angiogenic activity and its effects on vascular permeability and wound healing. The
serum fraction of the latex was separated from the rubber with reduction of the pH. The activity of the dialyzed
serum fraction on the vascular permeability injected in subcutaneous tissue was assayed according Mile’s
method. The angiogenic activity was determined using a chick embryo chorioallantoic membrane assay and its
effects on the wound-healing process was determined by the rabbit ear dermal ulcer model. The serum fraction
showed evident angiogenic effect and it was effective in enhancing vascular permeability. In dermal ulcers, this
material significantly accelerated wound healing. Moreover, the serum fraction boiled and treated with prote-
ases lost these activities. These results are in accordance with the enhancement of wound healing observed in
clinical trials carried out with a biomembrane prepared with the same natural latex. Copyright © 2009 John
Wiley & Sons, Ltd.

Keywords: Hevea brasiliensis; Euphorbiaceae; angiogenesis; vascular permeability; wound healing.

from the prominence of these vessels (Arnold and West,

INTRODUCTION
The search for specific agents to improve healing is as
old as the practice of medicine. Wound healing cannot
occur without the formation of new blood vessels
(angiogenesis). In response to the injury, microvascular
endothelial cells initiate an angiogenic process consist-
ing of the induction of microvascular hiperpermeability,
local degradation of their basement membrane, migra-
tion and sprouting into the local stroma, cell prolifera-
tion and formation of granulation tissue, reconstruction
of basement membrane, formation of new blood vessel,
stabilization, and eventually regression and involution
of the newly formed vasculature as tissue remodeling
(Dvorak et al., 1995; Singer and Clark, 1999; Werner
and Grose, 2003). On the other hand, the generation of
new blood vessels from pre-existing vasculature is
believed to be accompanied in almost all states by an
increase in vascular permeability (Arnold and West,
1991; Dvorak et al., 1995; Clark, 1996).
Newly formed blood vessels provide nutrition to
meet the local metabolic demands and comprise up to
60% of repair tissue, giving the name to the granulation
tissue to temporary organ of repair which is derived
* Correspondence to: Ricardo José Mendonça, University of São Paulo,
Department of Biochemistry and Immunology, Ribeirao Preto School of
Medicine, Av. Bandeirantes, 3900. 14040-900, Ribeirao Preto/ SP, Brazil.
E-mail:rjmendonca2000@yahoo.com.br
Copyright © 2009 John Wiley & Sons, Ltd.
1991; Eming et al., 2007a).
Increased vascular permeability is one of the first
stages in angiogenesis. There is no doubt that in patho-
logical angiogenesis, permeability to water and macro-
molecules is increased; vessels are leaky and solute
delivery to tissue is uncontrolled. In wound healing, the
inflammatory process and angiogenesis are responsible
for the increase permeability (Dvorak et al., 1999;
Vaquero et al., 2000; Bates and Harper, 2002).
Recently, some reports are being published about a
new material based on natural latex from the rubber
tree Hevea brasiliensis, which showed as an important
inductor of wound healing, regeneration of the esopha-
geal wall of dog and regeneration of the tympanic mem-
brane through mechanisms involving a marked increase
in vascularization (Mrué, 2000; Oliveira et al., 2003;
Mrué et al., 2004). In addition, the treatment of diabetic
and phlebopathic ulcers with the biomembrane pre-
pared from the natural latex showed the presence of a
vascular growth factor in the latex, which acts on human
tissues, permitting improved local vascularization
through its capacity for angiogenic induction and accel-
erating the repair of ulcers with consequently reepithe-
lialization (Frade et al., 2001; Frade, 2003).
In this study, we report the characterization of activi-
ties as vascular permeability, angiogenic activity, and
wound healing induced by the serum extracted from the
natural latex of a rubber tree.
Received 23 April 2009
Revised 14 August 2009
Accepted 02 September 2009
 BIOLOGICAL PROPERTIES FROM HEVEA BRASILIENSIS
MATERIALS AND METHODS
The Animal Ethics Committees of the Faculty of
Medicine of Ribeirao Preto, University of São Paulo,
Brazil, approved all experimental protocols (process
N° 261/2005), in accordance with the principles of
laboratory animal care and use in research.
Preparation of the serum from natural latex. Fresh
latex was obtained from a rubber tree (Hevea brasilien-
sis) of the clone RRhim 600. The crude natural latex
treated with NH4OH (2%) was obtained from Globbor
Inc. Corp. Rubbers LTD (Guapiacu city, São Paulo,
Brazil). The latex was mixed with a solution of acetic
acid 2.2% v/v at a 1 : 2 ratio. After the coagulation, the
serum was separated from the rubber and submitted to
dialysis and liophilization.
Miles vessel permeability assay. The Miles assay was
performed in rabbit skin as described previously (Miles
and Miles, 1952; Senger et al., 1983; Needham et al.,
1988; Senger et al., 1990; 1996). Agents under test were
freshly prepared in sterile phosphate buffered saline
(PBS) and were injected intradermally (100 μL) in 3
replicates. In all experiments, plasma exudation was
measured using the accumulation of the Blue Evans dye
in the site of injection, extracted of skin with formamide
and the absorvance determined at 625 nm (Udaka et al.,
1970).
Chorioallantoic membrane assay. Angiogenic activity
of compounds from latex was examined by chorioal-
lantoic membrane (CAM) assay, which has been
described previously (Wilting et al., 1991; 1992). Briefly,
fertilized eggs of the domestic fowl (Gallus domesticus,
strain Rhoss) were purchased from Pena Branca (São
Carlos, Brazil). Eggs were incubated at 38°C with 75%
relative humidity in an automatically turning egg incu-
bator (Brasmatic Inc. Corp. Ltd, São Paulo, Brazil).
After 5 days of incubation, the eggs were then cleaned
with 70% ethanol and a window round about 1 cm2 was
opened at the air sac using a circular electric saw
(Dremel multipro, Breda, Racine, WI, USA). The
external egg membrane was removed and a drop of
sterilized saline solution was deposited under the inner
shell membrane to help the complete detachment of the
shell from the CAM. The CAM was exposed and the
window was sealed with sellotape and the eggs were
returned to the incubator. On day 13, a dried filter
paper (Whatmann N° 1) 5 mm in diameter, with a
sample: 3 μL of serum solution (2.5 mg/mL) or 3 μL of
distilled water was applied to each egg, in a sterile way.
The chick embryo was then returned to the incubator.
Three days later, the CAM was fixed in 3.7% formalde-
hyde, cut out from eggs and was photographed at 7X
magnification with the use of a stereomicroscope
(Olympus Optical Co, Tokyo, Japan) with an attached
digital camera (DXC-107A CCD-IRIS Sony, Tokyo,
Japan). Images were captured with software (PixelView
Station v5.19 TV), analyzed and quantified with soft-
ware (ImageJ 1,32J, NIH, Milwankee, WI, USA). After,
the CAM was submitted to routine histological process-
ing as described (Wilting et al., 1991).
Copyright © 2009 John Wiley & Sons, Ltd.
765
The rabbit ear dermal ulcer model. Young adult New
Zealand white rabbits 2–3 kg were anaesthetized with
ketamine (60 mg/kg) and xylazine (5 mg/kg) injected
i.m., the ears immobilized and four 5-mm ulcers were
made to the depth of bare cartilage under sterile condi-
tions using microsurgical instruments, a 5-mm punch
and a binocular stereomicroscope (Mustoe et al., 1991).
The serum latex veiculated in a 1.0; 0.1 and 0.01% w/v
concentration in carboxymethylcellulose 2% w/v in
saline or vehicle alone were applied at days 0, 3, 6, and
9, and wounds were covered with an occlusive polyure-
thane film (Tegaderm; 3M, Minneapolis, MN, USA). At
days 0, 3, 6, 9 and 12, the ulcers were photographed,
bisected, and fixed in 3.7% buffered formalin for routine
histologic processing (Mustoe et al., 1991).
Histological analyses. Histological studies were carried
out using standard semithin sections (6 μM) stained
with haematoxilin-eosin.
Statistical analyses. Results were expressed as mean ±
SEM and analyzed for statistical significance using Stu-
dent’s t-test, where P values (p ≤ 0.05) were considered
to be significant.
RESULTS
Increased permeability vascular activity and
characterization of active(s) compound(s)
The latex was coagulated by the acidification of pH with
the purpose to separate its hydrophilic components
(serum) from the rubber. The serum was dialyzed
against distillated water and freeze dried. This material
was dissolved in NaCl 0.9% w/v in the concentration of
1.2 mg/mL and its activity in the increased induction of
the vascular permeability was analyzed through the
Miles test; the serum was also boiled and treated with
unspecific protease (proteinase K). The statistic analysis
of the data was carried out in accordance with Student’s
t-test, being values p < 0.05 or 0.01 considered signifi-
cant. (Fig. 1).
This material (serum) increased vessel permeability
in the Miles assay. The result showed that the serum
compounds were able to enhance the microvascular
permeability when compared with saline solution. The
use of denaturant protein treatment (boiled and prote-
ase) on this material demonstrated less activity on per-
meability which can suggest that a protein could be
responsible for this activity showed by serum.
Angiogenic activity on CAM
The serum was dissolved in distillated water at
2.5 mg/mL concentration. Three microliters of this solu-
tion were applied on CAM in a filter paper which was
used as a vehicle. The angiogenic activity can be evalu-
ated quantitatively by counting the number of vessels
in the graph in Fig. 2 with software ImageJ. The photo-
graphs of CAM are shown in Fig. 3 and its histological
sections in Fig. 4.
When compared with the control (Fig. 2A), serum
induces growth of blood vessels (Fig. 2B). The effects
Phytother. Res. 24: 764–768 (2010)
DOI: 10.1002/ptr
766 R. J. MENDONÇA ET AL.

can be much more prominent with 7.5 μg than other
doses assays (0.75 and 75 μg) in a pilot test (data not
shown). Figure 2B shows the macroscopic effects of
serum on vascular net of CAM. The presence of a
‘spoke wheel’ vessel pattern can be compared to the
effects observed by the bFGF (Wilting et al., 1992).
The quantification of blood vessels on photographs
of CAM confirmed a higher activity by serum com-
pounds than negative control, about 50–60%. The
quantification is expressed in percentage of blood
vessels on photographs obtained of each test, and pro-
cessed with the ImageJ software in a way to separate
the vessels of background and their quantifications. The
histological sections also confirm this result, with a high
presence of capillaries, mainly on the ectoderm. The
serum induces a thickening of the stroma which is due
to the production of extracellular matrix, and prolifera-
tion or immigration of fibrocytes.

Figure 1. Vascular permeability activity induced in rabbit skin

by intradermal injection of natural latex serum submitted to
different treatment. The samples are: Group I: Serum; Group II:
Serum boiled (100°C / 1 h); Group III: Serum treated with
proteinase K (200 μL in 0.32 mg/mL); Group IV: Saline (NaCl Figure 2. Measure of blood vessel quantities in corioallantoic
0.9% w/v) and Group V: Saline with proteinase K (200 μL in membrane of chicken eggs. The results showed percent of
0.32 mg/mL). Protein concentration 1.1 mg/mL (* p < 0.05; n = 3 blood vessels pixels compared with the total area pixels in the
rabbits/group, in triplicate). cam photography. (** p < 0.01; n = 12 eggs/sample).

Figure 3. Photographs of CAM: (A) the negative control with distillated water and (B) the serum (7.5 μg) effects. The circles indicate
where the filter paper (5 mm diameter) were applied vehiculating the samples: water or serum latex.

Figure 4. Histological sections of CAM. Semithin sections of the same CAM showed in Figs 3A and 3B. ec: ectoderme; ms: mesoderme
and en: endoderme. (A) control and (B) serum. The arrows show some blood vessels on the CAM. Barr = 16.7 μm.

Copyright © 2009 John Wiley & Sons, Ltd. Phytother. Res. 24: 764–768 (2010)
DOI: 10.1002/ptr
 BIOLOGICAL PROPERTIES FROM HEVEA BRASILIENSIS 767

Wound-healing activity of the serum in the rabbit ear
dermal ulcer model
The serum dissolved in carboximethylcellulose (2% w/v
in saline) in 1.0; 0.1 and 0.01% (w/v) and applied once
at days 0, 3, 6 and 9 on four ulcers in the right ear while
the left ear was applied the control (carboximetylcel-
lulose in saline). On days 0, 3, 6, 9 and 12, the ulcers
were photographed and areas of the wound were ana-
lyzed. The area of wound (mm2) was quantified by the
software ImageJ.
The progress of the wound healing is showed in Fig.
6 where the reduced area of ulcer treated with serum
latex in 0.01% w/v in carboxymethilcellulose gel on 12
day post-wounding (D) can be compared with control
(B), saline treated. (Fig. 5). Both, reepithelialization
and formation of granulation tissue were analyzed by
histological means (not shown) and image analysis.
Because the cartilage is an avascular tissue and the peri-
chondrium was removed during surgery, new granula-
tion tissue arose solely from the periphery of the wound.
Differences in rates of healing could be readily appreci-
ated since at day 3.

DISCUSSION

Since antiquity, plants have been used to treat many

Figure 5. Kinetics of the wound-healing process in three serum
concentrations applied once at days 0, 3, 6 and 9. The area
(mean ± SE) was analyzed by processing images software
(imageJ) (n = 4 animals, 4 ulcers each ear. Left ear = control;
right ear = treated ulcers).
ailments. In traditional medicine, many herbs are used
in the treatment of angiogenic diseases and chronic
wounds. The process of wound healing involves several
phases, including the increase of vascular permeability,
the formation of granulation tissue, reepithelization and
tissue reorganization (Singer and Clark, 1999). Among
these phases, angiogenesis plays a critical role in the
early phases of wound healing. Agents that control
angiogenesis are beneficial to the development of thera-
peutics for treatment of wound healing, tissue regenera-
tion, and others related diseases.
Natural latex biomembrane, traded as Biocure, is
prepared from the natural latex of Hevea brasiliensis by
prevulcanization at temperatures lower than 80°C; since

Figure 6. Photography of ear dermal ulcers of same rabbit ((A) and (B) are images from left ear; (C) and (D) from right ear). (A) 3
days post-wounding treated with saline in carboxymethylcellulose (control); (B) 12 days post-wounding treated with saline in car-
boxymethycellulose; (C) 3 days post-wounding treated with serum of latex (0.01% w/v) in carboxymethycellulose; and (D) 12 days
post-wounding treated with serum of latex (0.01% w/v) in carboxymethylcellulose.

Copyright © 2009 John Wiley & Sons, Ltd. Phytother. Res. 24: 764–768 (2010)
DOI: 10.1002/ptr
768 R. J. MENDONÇA ET AL.
2000 this is being used in the treatment of chronic ulcers
such as diabetic foot ulcers, flebophatic and pressure
ulcers (Frade et al., 2001; Frade, 2003) with success.
However, the mechanism of its healing activity is still
unknown. We hypothesized that released compounds
linked to the rubber content of the biomembrane stimu-
late angiogenesis, a critical process for the successful
healing of chronic wounds. To test that hypothesis we
investigated whether the serum fraction of the natural
latex could induce angiogenesis on the CAM, increase
vascular permeability and accelerate wound healing in
the ear dermal wound model.
Results showed that serum fraction actively induced
neovascularization on the CAM and increased the vas-
cular permeability of the dye Evans Blue IP injected.
These activities are lost when the serum was boiled at
100°C or treated with the unspecific proteolytic enzyme
proteinase K, suggesting that a compound with poly-
peptide structure is responsible for these activities.
Fibroplasy observed on CAM could be due to its direct
effect on fibrocytes proliferation, in accordance with
action of various growth factors, such as basic fibroblast
growth factor (FGFb). The effects of the natural serum
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DOI: 10.1002/ptr