Treatment of gastric ulcers and diarrhea with the Amazonian
herbal medicine sangre de grado
Mark J. S.
Miller1,2,
Wallace K.
MacNaughton3,
Xiao-Jing
Zhang1,2,
Jane H.
Thompson1,2,
Randi M.
Charbonnet1,2,
Paul
Bobrowski1,2,
Juan
Lao4,
Ann Marie
Trentacosti5, and
Manuel
Sandoval1,2
1 Department of Pediatrics and Center for Cardiovascular
Sciences, Albany Medical College, Albany, New York 12208;
2 Department of Pediatrics, Louisiana State University Medical
Center, New Orleans, Louisiana 70112; 3 Department of
Physiology and Biophysics, Gastrointestinal Research Group, University
of Calgary, Calgary, Alberta, Canada T2N 4N1; 4 Universidad
Nacional Agraria de la Selva, Tingo Maria, Peru; and
5 Rainforest Phytoceuticals, Delmar, New York
12054
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ABSTRACT |
Sangre de grado is an
Amazonian herbal medicine used to facilitate the healing of gastric
ulcers and to treat gastritis, diarrhea, skin lesions, and insect
stings. This study was designed to evaluate the gastrointestinal
applications. Gastric ulcers were induced in rats by brief serosal
exposure of the fundus to acetic acid (80%). Sangre de grado was
administered in drinking water at 1:1,000 and 1:10,000 dilutions from
the postoperative period to day 7. Guinea pig ileum
secretory responses to capsaicin, electrical field stimulation, and the
neurokinin-1 (NK-1) agonist [Sar9,Met(O2)11]substance P were
examined in Ussing chambers. Sangre de grado facilitated the healing of
experimental gastric ulcer, reducing myeloperoxidase activity, ulcer
size, and bacterial content of the ulcer. The expression of
proinflammatory genes tumor necrosis factor-
, inducible nitric oxide
synthase (iNOS), interleukin (IL)-1
, IL-6, and cyclooxygenase-2 was
upregulated by ulcer induction but reduced by sangre de grado
treatment, particularly iNOS and IL-6. In Ussing chambers, sangre de
grado impaired the secretory response to capsaicin but not to
electrical field stimulation or the NK-1 agonist. We conclude that
sangre de grado is a potent, cost-effective treatment for
gastrointestinal ulcers and distress via antimicrobial,
anti-inflammatory, and sensory afferent-dependent actions.
cytokine; inflammation; neuropeptides; Croton sp.; complementary medicine
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INTRODUCTION |
SANGRE DE
GRADO (Zangrado), also known as sangre de drago or
dragon's blood, is a viscous red tree sap that is used extensively by
indigenous cultures of the Amazon River basin for its remarkable healing properties (4, 12, 16).
Dragon's blood is a loose term used for any red tree sap and does not
always represent this Amazonian herbal medicine, and so we confine our
nomenclature to sangre de grado, the name used in Peru, where the
material was collected. Applied to the skin for abrasions, cuts,
scratches, and blisters, sangre de grado forms a seal, a long-standing
barrier, over the lesion (21). This appears to be due to
its ability to coprecipitate with proteins or other components of the
underlying matrix. For bites and stings in particular, sangre de grado
halts the symptoms of pain and itching within minutes, with a
subsequent reduction in swelling and redness. The mechanisms for these
beneficial effects have remained elusive. Sangre de grado is believed
to foster wound healing at a rate that is superior to natural processes and does so with reduced pain, inflammation, and scarring
(3, 19, 21), although much of
this information is anecdotal.
Sangre de grado's applications are not limited to cutaneous disorders.
Sangre de grado is also taken orally, in a dilute form, for severe
gastrointestinal distress, e.g., gastritis, gastric ulcer, intestinal
infections, and inflammation (4, 12,
19). These applications are the focus of this study.
Gastric ulcers and cancer are common afflictions in South America,
primarily because of the high incidence of Helicobacter
pylori infection. In addition, other microbial and parasitic
infections are common and place a great burden on gastrointestinal
health in the Amazonian communities. Sangre de grado and uña de
gato (cat's claw) represent the major ethnomedicines for these
complications and remain as such, in view of the high costs of western pharmaceuticals.
Derived from several Croton species (Croton
dracanoides, Croton palanostigma, Croton lecheleri), sangre de
grado is easily available throughout the Amazon, with the highest
quality material originating in the upper jungle of Peru and Ecuador.
The tree is fast growing, reaching heights of 30-45 feet in 3 years. Although the sap can be harvested like rubber (the sap flows
better in the morning but flows less freely than rubber or maple
syrup), repeated tapping of the tree can lead to fungal infections in the tree, thereby diminishing productivity. Current experimental farming techniques are focusing on growing and felling the trees in a
2- to 3-yr cycle. At this time a tree will produce ~1.5 l of sap, a
large quantity considering that sangre de grado is applied drop by drop.
The combination of antimicrobial, antioxidant, antiviral, and
cicatrizant properties makes sangre de grado a fascinating yet complex
herbal remedy for study, one with tremendous opportunities for
improvements in health care delivery in the developing world. The
experiments detailed in this article are intended to examine the
validity of the herbal medicine in its ethnomedical form. We have
deliberately attempted not to isolate "single active chemicals" but
to evaluate the medicinal as a whole. Our goal was to evaluate and
explore the mechanisms underlying the use of this ethnomedicine. By
providing this information for the peoples of the Amazon we hope that
it will stimulate commercialization of their own natural resources in a
sustainable manner, as well as improve local health care delivery in
Amazonian communities that cannot afford the luxury of western medicines.
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MATERIALS AND METHODS |
Gastric ulcer formation.
Male Sprague-Dawley rats (200-250 g) were acclimated to the
housing facilities for 5 days before initiation of the study. Free
access to standard pellet chow was allowed throughout the experimental
protocol, with the exception of overnight fasting before induction of
the ulcer. All protocols were approved by the Animal Care and Use
Committee of the Louisiana State University Medical Center, New
Orleans, where the study was conducted.
Ulcers were induced experimentally with acetic acid as we have
previously described (5). Briefly, while rats were
anesthetized (ketamine, xylazine, and acepromazine) a midline
laparotomy incision was performed and the stomach was gently
exteriorized. The barrel of a 3-ml syringe, which had been cut, heated,
and filed smooth, was placed on the serosal surface of the stomach in
the corpus region. Acetic acid (0.5 ml, 80% vol/vol) was instilled
into the barrel of the syringe and allowed to remain in contact with
the surface of the stomach for 30 s, after which time it was
aspirated and the area was rinsed three times with sterile saline. This procedure yields an exposed region of a defined size (60 mm2) and a corresponding hemispherical ulcer of a
comparable size. The stomach was then returned to the abdominal cavity,
and the wound was sutured. The rats were then divided into various
treatment groups with sangre de grado administered in drinking water.
Preliminary studies indicated that sangre de grado had no effect on
water intake by rats when delivered in the dilution range of
1:300-1:30,000. The total daily intake of dried sangre de grado
latex was 60-600 µg/day in the present study
(1:10,000-1:1,000 dilution). After 7 days of treatment the animals
were euthanized, and the stomach tissue was collected for determination
of ulcer size, myeloperoxidase activity, bacterial content in the
ulcer, histopathology, and gene expression.
Bacterial content of ulcers.
To determine the bacterial content in gastric ulcers, animals were
killed under aseptic conditions. Tissue (~150 mg) was washed in
sterile PBS and transferred to sterile, preweighed containers (to
determine sample weight), sterile PBS was added, and then the sample
was homogenized. Serial dilutions were then plated on MacConkey agar
and tryptic soy agar plates and incubated for 18-24 h under
aerobic conditions, and the colony-forming units (CFU) were determined
using a Leica colony counter. Results are expressed as CFU per gram of
tissue (5).
In vitro assessment of epithelial secretion.
Experiments were conducted using standard Ussing-type diffusion
chambers to determine the effect of sangre de grado on sensory afferent-evoked chloride secretion. Guinea pigs were killed by an
overdose of pentobarbital sodium. A segment of ileum was removed 3 cm
proximal to the ileocecal junction, flushed with cold (4°C) Krebs
buffer and stripped of external muscle. Segments of stripped ileum were
opened along the mesenteric border and mounted between two halves of a
diffusion chamber, which exposed 0.6 cm2 of the mucosal
surface (Navicyte, San Diego, CA). Ileal segments prepared in this
manner retain intact submucosal secretomotor neurons and axon
collaterals of primary sensory afferent neurons and respond to
capsaicin with chloride secretion (24). Tissues were
bathed with Krebs buffer (maintained at 37°C, pH 7.4). Guinea pig
experiments were approved by the University of Calgary Animal Care Committee.
The serosal bathing solution contained 10 mM glucose, and the mucosal
bathing solution contained 10 mM mannitol. The electrical potential
difference across the tissue was maintained at 0 V with a voltage-clamp
apparatus (EVC4000, World Precision Instruments, Sarasota, FL). The
short-circuit current (Isc) required to clamp the tissue was taken as the measure of active ion transport by the
intestinal epithelium and was recorded using a digital data acquisition
system (MP-100, BioPac, San Diego, CA). Analysis of recordings was
conducted using AcqKnowledge software (version 3, BioPac).
Tissues were allowed to equilibrate with respect to basal
Isc and were paired on the basis of basal tissue
conductance. Only pairs of tissues with conductances within 20% of
each other were included in subsequent experiments. Once a stable
baseline was established (~20 min), one member of the pair was
exposed on the serosal side to sangre de grado (1:1,000 dilution) while
the other member of the pair received the vehicle (1:1,000 dilution of
80% ethanol). Ten minutes later both tissues of the pair were exposed on the serosal side to 100 nM capsaicin. The Isc
response was measured and calculated as area under the curve. Once
baseline was reestablished, tissues were challenged with electrical
field stimulation (EFS) to confirm tissue viability and responsiveness.
To determine the site of action of sangre de grado, experiments were
conducted as above with the exception that the neurokinin-1 (NK-1)
agonist [Sar9,Met(O2)11]substance
P (Sar-Met-SP, 100 nM; Bachem, Torrance, CA) was added in place of
capsaicin. This dose of Sar-Met-SP had previously been shown to
stimulate chloride secretion through selective activation of NK-1
receptors (15). The guinea pig ileum was chosen for evaluation specifically because capsaicin is thought to promote secretion only through the release of substance P and the activation of
NK-1 receptors (24). Comparing the effects of capsaicin
and the NK-1 agonist Sar-Met-SP, we can distinguish between actions of
sangre de grado at the level of the sensory nerve itself vs. an effect
on substance P-dependent processes. EFS was used to evaluate any
nonspecific effect on cholinergic nerves or the secretory apparatus itself.
Gene expression.
RNA was extracted from frozen rat stomach by the guanidine thiocyanate
extraction method immediately after tissue collection (22). First-strand cDNAs were synthesized from 0.5 µg of
total RNA using oligo(dT) and SuperScript II Reverse Transcriptase
System (GIBCO BRL, Grand Island, NY). First-strand cDNA templates were amplified by the polymerase chain for glyceraldehyde-3-phosphate dehydrogenase, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-1, COX-2, interleukin (IL)-1
, and IL-6 using a hot start and
Taq polymerase (Ampliwax and Amplitaq, Perkin-Elmer, Foster City, CA). Denaturation, annealing, and elongation temperatures for
iNOS were as follows: 95°C for 3 min, followed by 94°C for 30 s, 60°C for 45 s, and 72°C, for 1.5 min, each for 30 cycles, with a final cycle of 72°C for 4 min. The negative control was from a
cDNA reaction that used water in place of RNA. Oligonucleotide primers
were based on the sequence of a conserved region of mouse and human
iNOS. PCR products were separated in a 2% agarose gel and visualized
by ethidium bromide staining. Gels were visualized under ultraviolet
(UV) light and photographed using a Kodak Electrophoresis Documentation
and Analysis system 120 (Eastman Kodak, Rochester, NY). Essentially
similar techniques with appropriate adjustments in cycles and annealing
and elongation temperatures were used for the other
transcription-regulated products evaluated in this proposal. Details of
the sense and antisense primer sequences for these gene products are
given in Table 1.
Materials.
Sangre de grado (Zangrado) was supplied by Rainforest Phytoceuticals
(Delmar, NY) in its pure form. Zangrado was collected from the highland
jungle of the Huallaga River Valley in tropical Peru (a tributary of
the Amazon River) and verified by Eng. Warren Rios (Universidad
Nacional Agraria de al Selva, Tingo Maria, Peru). For the
current purposes, the sangre de grado that was used was a mixture of
the subspecies Croton lecheleri and Croton
palanostigma, as wild-harvested sangre de grado was obtained from
indigenous peoples and both of these subspecies grow in the Upper
Huallaga valley of Peru. We are not aware of any difference in
bioactivity attributed to these subspecies at this time. All other
materials were of research grade and were obtained from Sigma Chemical
(St. Louis, MO), except as noted.
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RESULTS |
Gastric ulcers.
The acetic acid model of gastric ulceration produces an ulcer of
reliable, reproducible dimensions that persists for 7 days followed by
gradual healing (5). We chose a treatment schedule from
day 0 to day 7 to determine whether sangre de
grado promotes ulcer healing, and if so, to identify the underlying
mechanisms. Ulcer size (length × thickness, determined
histologically) was reduced by sangre de grado at both 1:1,000 and
1:10,000 dilutions (P < 0.05, Fig.
1). This healing was of similar magnitude
to what we had described with a combination of penicillin and
streptomycin (5, 6). Related to this
antibacterial therapeutic action, we noted that the bacterial content
of the ulcer was greatly reduced by sangre de grado treatment at both
concentrations (P < 0.01, Fig.
2).

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Fig. 1.
Alterations in gastric ulcer size (as determined by
length and depth) with sangre de grado (SdG) treatment. Control ulcers
(solid bar) were significantly larger (*P < 0.05) than
in rats treated with sangre de grado at either 1:1,000 or 1:10,000
dilutions administered via drinking water. All groups were evaluated 7 days after ulcer induction.
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Fig. 2.
Myeloperoxidase (MPO) activity in ulcers of rats with
experimental gastric ulcers. Treatment with sangre de grado in drinking
water at dilutions of 1:1,000 or 1:10,000 significantly reduced ulcer
MPO levels (*P < 0.05). All groups were evaluated 7 days after ulcer induction.
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In contrast to the results of our previous studies with antibiotics and
probiotics (5), sangre de grado also reduced the granulocyte content of gastric ulcers (Fig.
3, P < 0.05). Thus experimental ulcers were smaller and less inflamed and had reduced bacterial colonization. Examples of this healing are shown in Fig.
4. The ability of sangre de grado to kill
bacteria in vitro has already been described (3) and was
confirmed in this in vivo model. However, we readdressed the
antibacterial potency of sangre de grado in vitro using
Escherichia coli. Undiluted sangre de grado was completely
effective in killing E. coli. At 1:10 dilution sangre de
grado was still 90% effective, but at 1:100 dilution its ability to
reduce CFU was indistinguishable from control values. These results are
depicted in Fig. 5. with ampicillin noted
as a positive control.

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Fig. 3.
Levels of bacteria in the ulcer bed, as denoted by colony
forming units (CFU). Nontreated ulcers had significantly more bacteria
than those treated with diluted sangre de grado (1:1,000 or 1:10,000,
*P < 0.05) administered via drinking water. The
horizontal line depicts the bacterial content of normal rat stomach.
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Fig. 4.
The antibacterial actions of sangre de grado in vitro.
Different concentrations of sangre de grado were streaked onto plates,
allowed to dry, and then treated with E. coli. Bacterial
colonies were absent in plates treated with pure sangre de grado. With
dilution, the antibacterial effects of sangre de grado were reduced,
with the 1:100 dilution being indistinguishable from control. The
effects of ampicillin are included for comparative purposes.
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Fig. 5.
Gross morphology of experimental gastric ulcers. In the
top panel from control rats, a round ulcer is clear. In the bottom two
panels are examples of rats treated with sangre de grado in drinking
water at dilutions of 1:1,000 and 1:10,000, respectively. In
sangre de grado-treated rats, the smaller ulcers are evidence of
healing.
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On histopathological examination, the untreated ulcers displayed
profound necrosis of the gastric epithelia with cellular debris mixed
with bacteria (Fig. 6). In rats that
received sangre de grado in drinking water, areas of ulceration
remained but regions of regenerating epithelia were evident. The mucosa
was still inflamed, but it was clear that the healing process had been
initiated (Fig. 6).

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Fig. 6.
Low-power photographs (×40) of hematoxylin and
eosin-stained sections of experimental gastric ulcers. Note that in the
control, untreated ulcer (top) the mucosa has been lost and
replaced by a layer of necrotic tissue with an underlying region of
muscularis that has a heavy leukocyte infiltration. An example of an
ulcer treated with sangre de grado, in which regeneration of the mucosa
and epithelialization are apparent, is shown at bottom.
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Gastric ulceration establishes a local inflammatory response that may
also retard the healing process and is driven by the exposure of the
mucosa to gastric contents after disruption of the epithelial barrier
and colonization of the ulcer bed. To characterize this inflammatory
response we collected tissue for RT-PCR. Figure 7 depicts the RT-PCR results of
inflammatory gene expression in normal rats and rats with gastric
ulcers with and without sangre de grado treatment. COX-1 was the only
gene product evident in normal rats. Furthermore, the expression of
COX-1 was not affected by the induction of gastric ulceration, as
expected. COX-1 expression also remained steady with sangre de grado
treatment (Fig. 7). On the other hand, the expression of iNOS, tumor
necrosis factor-
(TNF-
), IL-1
, and IL-6 was minimal or absent
in normal rats and markedly upregulated in gastric ulceration.
Treatment with sangre de grado reduced the expression of these genes,
in particular iNOS and IL-6 and, to a lesser extent, TNF-
, COX-2,
and IL-1
.

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Fig. 7.
Gel depicting RT-PCR products for cyclooxygenase (COX)-1,
COX-2, inducible nitric oxide synthase (iNOS), tumor necrosis
factor- (TNF- ), interleukin (IL)-1 , and IL-6 from rat stomach.
Lane 1 is derived from an ulcer-free naïve rat;
lane 2 is from a control, untreated ulcer; lane 3 is from an ulcer treated with sangre de grado (1:1,000 dilution);
lane 4 is from an ulcer treated with sangre de grado
(1:10,000 dilution). A: COX-1 (left) and COX-2
(right). B: iNOS (left) and TNF-
(right). C: IL-1 (left) and IL-6
(right). Note that COX-1 expression was consistent in all
groups. In contrast, COX-2, iNOS, TNF- , IL-1 , and IL-6 were
absent in normal animals and elevated in ulcer-bearing stomachs but
reduced by sangre de grado treatment. GAPDH, glyceraldehyde-3-phosphate
dehyrogenase.
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Intestinal epithelial secretion.
The ability of sangre de grado to provide relief from cutaneous stings
and bites as well as a therapy for diarrhea may be caused by an action
on sensory afferent neurons (10). To determine whether
sangre de grado acted on sensory afferent nerves, we used a previously
described bioassay of capsaicin-evoked chloride secretion from isolated
segments of guinea pig ileum mounted in Ussing chambers (24). Exposure to capsaicin caused an immediate increase
in Isc indicative of chloride secretion. The
peak response occurred within 5 min and returned to baseline within
10-15 min. The other agonists used to promote chloride secretion,
EFS and Sar-Met-SP, produced qualitatively similar changes in
Isc to capsaicin. Pretreatment with serosal
sangre de grado, at a dilution of 1:1,000, was noted to selectively
attenuate by ~70% the secretory responses to capsaicin (Fig.
8, P < 0.01), quantified
as a change in Isc (µA/cm2). In
contrast, the Isc responses to EFS and the NK-1
agonist Sar-Met-SP were unaffected by sangre de grado. The
Isc response to capsaicin in this preparation is
mediated by the release of substance P from sensory afferents with a
subsequent activation of NK-1 receptors (15,
24). Thus the selective effect on capsaicin-evoked responses suggests that suppression of epithelial secretion was caused
by a direct effect on the sensory afferents and not by an action on the
neurotransmitter, its receptor, or its cellular target.

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Fig. 8.
Epithelial secretion as determined by changes in
short-circuit current (Isc,
µA/cm2) in guinea pig ileum. Secretion was induced by
capsaicin, electrical field stimulation (EFS), and the neurokinin-1
agonist
[Sar9,Met(O2)11]-substance P
(Sar-Met-SP). Serosal application of sangre de grado (1:1,000 dilution)
reduced by ~70% the secretory response to capsaicin
(*P < 0.05), whereas the responses to NK-1 activation
or EFS were not significantly altered.
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DISCUSSION |
Ethnomedicines like sangre de grado are the mainstay of health
care delivery in tropical South America, in part because of their rich
history and confidence in their use but also because western
pharmaceuticals are beyond the financial reach of these communities.
This is not a unique problem; similar hurdles face most of the
developing world. Although the use of traditional medicines may be
widespread, there is little direct evidence as to the efficacy of these
therapeutic approaches, and this lack of data is particularly apparent
for Amazonian medicinals. Certainly, pharmacognosy studies have been
performed (19) with a number of unique chemicals isolated
from sangre de grado. However, we are left with an informational void
as to how these phytoceuticals work, the breadth of their applications,
and how they can be combined with other therapeutic approaches
(traditional or pharmaceutical). This information is required not only
for the peoples of tropical South America but also for the public of
western nations who are embracing herbal medicines as a component of
their own health maintenance.
Sangre de grado is widely available in Amazonia, where it is used for a
large variety of conditions
for insect bites and stings, for wound
healing, to limit the blood loss in childbirth, and, relevant to this
investigation, to heal gastric ulcers and for diarrhea (4,
12, 16). In this study we treated
experimental gastric ulcers in rats with sangre de grado in a fashion
that was consistent with ethnomedical traditions. Sangre de grado is taken orally in a highly diluted form, e.g., three drops in a beverage
two or three times a day or, alternatively, a teaspoon in a liter of
water with a cup of this stock drunk two or three times a day. We
duplicated this approach by introducing sangre de grado in the drinking
water of rats at dilutions of 1:1,000 or 1:10,000. At these
concentrations, sangre de grado was particularly effective in healing
gastric ulcers, with an estimated intake of dry sangre de grado latex
of 60-600 µg · rat
1 · day
1.
The mechanisms for ulcer healing appear to involve the antimicrobial
effects of sangre de grado. Gastric ulcer healing is determined by the
bacterial content of the ulcer bed as we have previously described
(5). Antibiotics or the promotion of lactobacillus colonization lead to ulcer healing. In this study, sangre de grado had
similar effects. Sangre de grado markedly reduced the bacterial content
of the ulcer concomitant with healing and reduced inflammation. A
causal link between reduced bacterial load and sangre de grado is
likely, because in vitro sangre de grado was a highly effective antibacterial agent (Fig. 5). However, sangre de grado was administered to the rats in a highly dilute form, and in vitro the bactericidal activities were evident at higher concentrations. The reasons for this
discrepancy are unclear. It appears that sangre de grado makes the
microenvironment either unsuitable for bacterial growth or more
susceptible to other means of clearance, as has been suggested for
epidermal growth factor (EGF) (6). When sangre de grado is
mixed with rat gastric mucus there is a change in UV absorbance, suggesting an interaction that may alter the suitability of this microenvironment for colonization (data not shown).
The healing effects of sangre de grado on ulcer size and bacterial
numbers rivaled the combination of penicillin and streptomycin (5) as well as EGF (6, 14), but,
in addition, sangre de grado also reduced the mucosal inflammatory
response, an effect not seen with antibiotics. Myeloperoxidase (MPO)
activity is used as an index of granulocyte content/infiltration in
tissues, and ulcer MPO levels were greatly reduced by sangre de grado
treatment. In our previous study, MPO values were unaffected by
antibiotic administration. This suggests that sangre de grado may
promote ulcer healing through additional mechanisms, and these may
involve a direct effect on the inflammatory response. Inhibition of
neurogenic inflammation, as indicated by the ability of sangre de grado
to inhibit the activation of sensory afferent nerves, is a possible explanation.
Expression of proinflammatory genes was used to evaluate ulcer-induced
gastritis and the anti-inflammatory effects of sangre de grado. In
normal rat stomach COX-1 was expressed, whereas the genes for TNF-
,
iNOS, IL-1
, IL-6, and COX-2 remained in their quiescent state.
Induction of gastric ulcers resulted in the expression of these latter
proinflammatory genes, whereas the expression of COX-1 remained
unaltered. Sangre de grado suppressed the activation of these
proinflammatory genes, particularly iNOS. This effect may be due, in
part, to a reduction in the bacterial content of the ulcer, thereby
reducing the signals activating the transcription of these gene
products. The reduced infiltrate of inflammatory cells may also
contribute to the reduced expression of these proinflammatory genes in
the ulcer tissue. Together, these results support an action of sangre
de grado to promote ulcer healing, as indicated by Amazonian shamans.
The ethnomedical use of sangre de grado for intestinal distress and
diarrhea was evaluated in vitro for a potential suppression of
secretagogue responses. Sangre de grado had no effect on EFS- or
NK-1-induced secretion. This suggests that it does not compromise submucosal secretor motor neuron-induced epithelial secretion, cholinergic or substance P-dependent processes. On the other hand, the
responses to capsaicin were greatly attenuated. In the guinea pig ileum
preparation this indicates that sangre de grado directly compromised
sensory afferent activation. Vanner and MacNaughton (24)
showed that acute application of capsaicin to these preparations causes
chloride secretion that is entirely dependent on the activation of
extrinsic sensory afferent neurons. The response to capsaicin was
abolished by pretreatment with tetrodotoxin, confirming the neural
dependence of the response. Secretory responses to capsaicin in this
preparation are caused by the activation of NK-1 receptors (15, 24), and to address the potential
interaction at the NK-1 receptor level we evaluated the selective NK-1
agonist Sar-Met-SP. Epithelial secretory response to NK-1 activation
was not altered by sangre de grado. Another control was EFS; EFS
depolarizes all neurons in the preparation to elicit a net chloride
secretion (2). Sangre de grado did not alter the secretory
response to EFS, indicating that it did not act as a general inhibitor
of nerve activation. Not shown were the secretory responses to
carbachol, which directly activates epithelial cells to promote
secretion; responses to carbachol were also unaltered by sangre de grado.
Thus it appears that the ethnomedical reports of sangre de grado's
utility in the treatment of diarrhea and intestinal distress involve a
selective suppression of nonmyelinated sensory nerves and thereby
neuropeptide-dependent responses. This is a unique therapeutic
approach. Sangre de grado therapy should not only reduce the secretory
response (as quantified in this study) but also other sensory
afferent-dependent mechanisms
cramping and pain perception. These data
offer compelling evidence to support the evaluation of sangre de grado
for therapeutic utility in other states of neurogenic inflammation.
An extract of sangre de grado, SP-303, consisting of proanthocyanidins,
has been shown to block intestinal secretory responses via
cAMP-mediated mechanisms (2), including cholera toxin. An
additional study in AIDS-associated diarrhea indicated that sangre de
grado reduces stool weight and frequency (8). The doses of
SP-303 required to suppress intestinal secretion appear to be higher
than those described for sangre de grado in its pure ethnomedical form.
This leads us to conclude that proanthocyanidins are not the only
chemical constituents responsible for the efficacy of sangre de grado
in treating diarrhea. We are unaware of any reports of effects of
SP-303 on sensory afferents. However, we postulate that the ability of
sangre de grado to block capsaicin-activated sensory afferents may
indicate that it attenuates the cramping and pain associated with
intestinal distress as well as secretory processes. Sensory afferent
activation has long been associated with intestinal secretory responses
to a diversity of stimuli. More recently, rotavirus-induced secretory
diarrhea has been linked to the enteric nervous system
(13). Lundgren et al. (13) postulated that
agents that inhibit neurally driven intestinal secretion may offer a
means of limiting secretory diarrhea in response to numerous
initiators. It appears unlikely that cholinergic antagonists with
sufficient selectivity will be available to achieve that goal, but
sangre de grado with its selectivity for sensory afferents could be the
prototypical therapeutic agent. Its low cost and the high incidence of
diarrhea in the developing world make its applications quite attractive.
Although the inhibition of sensory afferent mechanisms may explain the
utility of sangre de grado in diarrhea and intestinal cramping, it is
less clear whether this mechanism is involved in ulcer healing.
Neuropeptides (mainly calcitonin gene-related peptide) are protective
of the gastric mucosa under acute protocols (9). In models
of chronic colitis, neurokinin receptor antagonism failed to promote
healing (25). Thus the importance of suppression of
sensory afferent activation in ulcer healing remains unclear. However,
this mechanism is a likely explanation for the ethnomedical use of
sangre de grado for the treatment of insect bites, stings, and burns.
Suppression of sensory afferent activation would block the pain and
symptoms associated with these conditions. Ulcer healing in this model
has been achieved by either antibiotic administration (5,
6) or EGF (6). Reductions in EGF from
salivary glands by cigarette smoking retards ulcer healing
(14). However, neither antibiotics nor EGF reduces the MPO
activity of the ulcer bed despite reductions in the bacterial load. In
contrast, sangre de grado does reduce ulcer MPO activity. The reasons
for additional anti-inflammatory action of sangre de grado is not
clear, but it is interesting to speculate that it may result from an
inhibition of neurogenic inflammation.
Ulcer healing by sangre de grado may also be due to other chemical
constituents. Although proanthocyanidins are the major chemical class
present in sangre de grado, there are a number of other chemicals that
have been isolated and are thought to be involved in the diverse
effects exhibited by sangre de grado. Crolechinol, crolechinic acid,
korberin A and B, 3',4-O-dimethylcedrusin, and taspine
(3, 19) have received considerable attention, although only a few studies have evaluated this herbal medicine. 3',4-O-dimethylcedrusin and the polyphenolic fraction have
been suggested to be the chemicals responsible for wound healing via fibroblast activation (1, 3,
23). Taspine is present in Peruvian sap but is less
evident in Ecuadorian sap and has been implicated in its use in
inflammation and cancer because it readily kills tumor cells
(3, 20). In studies in cell culture, sangre de grado inhibits cell proliferation yet protects against cell death
initiated by media starvation (11, 18,
20). This suggests a critical action at the level of cell
cycle regulation and apoptosis, which we have explored. Taspine has
been touted as a principal component of the wound healing actions of
sangre de grado, on the basis of its early stimulation of wound repair
(3, 11, 23). However, the
taspine content varies with geographical location, although the
ethnomedical use of sangre de grado for wound repair is widespread
throughout Amazonia, leading many to contemplate that other chemicals
are important, including the polyphenols (1). The efficacy
of sangre de grado, including the cicatrizant effect, is perhaps better
explained by the array of chemicals acting in concert rather than a
single chemical. For example, beyond the antiviral actions of
proanthocyanidins, antimicrobial actions may be critical, an effect
thought to be due to 1,3,5-trimethoxybenzene and
2,4,6-trimethoxyphenol, which are present in trace amounts but are 30 times more potent than penicillin (3, 19).
In summary, sangre de grado is an effective treatment for the healing
of gastric ulcers well as an antidiarrheal agent. The ability of sangre
de grado to reduce intestinal fluid secretion appears to be due to its
ability to selectively inhibit epithelial electrolyte movement driven
by sensory afferent nerves and not to a direct effect on the secretory
apparatus at the doses tested. In terms of ulcer healing, its
effectiveness was equivalent to either antibiotics or EGF, with the
added benefit of reducing the degree of granulocyte infiltration. With
further research the potential of this medicinal plant in managing
gastrointestinal disease will be placed in proper perspective, but its
low cost, effectiveness, and wide clinical experience in Amazonia are encouraging.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
M. J. S. Miller, Dept. of Pediatrics, MC-8, Albany Medical
College, 47 New Scotland Ave., Albany, NY 12208 (E-mail:
millermj{at}mail.amc.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 17 December 1999; accepted in final form 9 February 2000.
 |
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