Gastro-protective effect of capsaicin in Anguilla anguilla (Linnaeus, 1758) : evidence from an experimental study on gastric bags

ITTIOPATOLOGIA, 2006, 3: 263-271
Gastro-protective effect of capsaicin in
Anguilla anguilla (Linnaeus, 1758): evidence
from an experimental study on gastric bags

Effetto gastro-protettivo della capsaicina in
Anguilla anguilla (Linneo, 1758):
studio sperimentale su sacchetti gastrici

Maria Gabriella Denaro1*, Gabriella Caruso2, Lucrezia Genovese2
1 Dpt. di Fisiologia Generale e Farmacologia, Facoltà di Scienze Matematiche, Fisiche e Naturali, Università di
Messina, Salita Sperone, 31 - 98166 S. Agata (ME); 2 Istituto per l’Ambiente Marino Costiero (IAMC), Consiglio
Nazionale delle Ricerche, Spianata S. Raineri, 86 - 98122 Messina
_______________________________



SUMMARY - An experimental, macroscopical, study was undertaken in european eel (Anguilla anguilla) to
assess the gastro-protective effect of capsaicin, the active component contained in pepper. Gastric bags were
prepared from specimens of Anguilla anguilla and used for in vitro study of the ability of capsaicin to recover
ulcers induced by aspirin or ethanol. Gastric ulcers were experimentally induced and comparison was made with
the same experiment repeated in presence of capsaicin at different concentrations (10-6, 10-5, 10-4 mol/l).
Macroscopical observations showed that capsaicin at low doses (10-6 mol/l) was effective in repairing the
mucosal layer injured, thus proving in vitro its protective effect on the gastrointestinal tract of this fish species.
Moreover, the similar physiological behaviour of the protective effect found between our study and other in vivo
studies performed on rats supported the possibility of using the gastro-intestinal tract of eel as a model substrate
for pharmacological and toxicological studies.


RIASSUNTO – Uno studio sperimentale, macroscopico, è stato effettuato in anguilla (Anguilla anguilla) per
valutare l’effetto gastro-protettivo della capsaicina, un principio attivo contenuto nel peperoncino. Sacchetti
gastrici sono stati preparati da esemplari di anguilla ed utilizzati per studiare in vitro la capacità della
capsaicina di prevenire la formazione di ulcere indotte dall’aspirina o dall’etanolo. Sono state indotte
sperimentalmente ulcere gastriche; in parallelo lo stesso esperimento è stato ripetuto in presenza di capsaicina a
differenti concentrazioni (10-6, 10-5, 10-4 mol/l). L’osservazione macroscopica ha evidenziato come la capsaicina
a basse dosi (10-6 mol/l) sia efficace nel prevenire l’alterazione dello strato mucosale, dimostrando in vitro il suo
effetto protettivo sul tratto gastrointestinale di questa specie di pesci. Inoltre, il comportamento simile fra quanto
riscontrato in vitro ed altri studi condotti in vivo su ratti, suggerisce la possibilità di utilizzare il tratto
gastrointestinale di anguilla come modello sperimentale per saggi farmacologici e tossicologici.







Key words: Anguilla anguilla, Gastric ulcer, Capsaicin, Aspirin, Ethanol.
_______________________________


* Corresponding Author: c/o Dpt. di Fisiologia Generale e Farmacologia, Facoltà di Scienze Matematiche,
Fisiche e Naturali, Università di Messina, Salita Sperone, 31 – 96166 S. Agata (ME), Italia; Tel.: 090-6765210;
Fax: 090-394030; E-mail: mgdenaro@unime.it

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INTRODUCTION

Peptic ulcer represents one of the most spread human diseases in the world; generally, in
healthy conditions, mucosal damaging agents and gastric mucosal defences are well
balanced (Grossman, 1980). Among these latter, the mucus, covering with a thin layer the
surface epithelial cells of the gastrointestinal tract, represent a first line of defence against the
acidic environment created by pepsin. Also, mucosal prostaglandins are reported to be
involved in modulating gastric mucosal defense mechanisms (Mózsik et al., 1977a; 1977b);
their reduction has been considered as a predisposing factor in the genesis of gastric ulcer
(Miller, 1983). Other factors which play a role in the mucosal protection include the tight
junctions between the surface epithelial cells and the cell turnover, bicarbonate secretion,
gastric mucus phospholipids, gastric mucosal blood flow (Miller, 1988).
Different drugs have been tested to experimentally induce gastric ulcer and to study their
pathogenesis (Szabò & Goldberg, 1990). For some chemicals (acetic, acetylsalicylic acids,
bile salts and ethanol) the capability of breaking the gastric mucosal barrier has been
demonstrated (Davenport, 1969). On the other hand, several other compounds such as
atropine (Mózsik et al., 1980), antacids and cimetidine (MacKercher et al., 1977), or some
coating agents, barrier agents or mucosally active drugs have been used in the treatment of
acid peptic disorders (Hollander & Tarnawski, 1987). Particular interest is also addressed to
compounds of natural origin, showing significant pharmacological properties for the therapy
of peptic ulcer disease, such as compounds derived from peppers. Since ancient times, this
fruit (Capsicum annuum, belonging to the family of Solanaceae) has been known for the
therapeutical properties of its active components, particularly concerning its content in
capsaicin (8-methyl-N-vanillyl-6-nonenamide). This alkaloid, responsible for the pungent
nature of pepper, is detected in this fruit in amounts ranging from 0.01 to 0.22%, while in
form of essential oil (capsicol) it is present in concentrations ranging from 1 to 1.5%. This
substance induces pyrexia (Szolcsànyi, 1982), but a lot of studies have suggested its
application in medicine for its therapeutic potential (Szallasi & Blumberg, 1993; Abdel-
Salam et al., 1997). Capsaicin is known to have protective effects not only in the stomach
but also in the neonatal pre-treatment of chronic colitis induced in the rat by trinitrobenzene
sulfonic acid (Evangelista & Meli, 1989). Concerning the mechanism of action of capsacin,
its receptor is a cationic channel which, during its activation, allows a flux of Ca2+ and Na2+
ions (Winter, 1987; Wood et al., 1988) which induces the depolarisation of neuronal
membranes of fibres involved in pain generation; then this sensation is transmitted to the
brain via the dorsal ganglion.
Previous research (Faggio et al., 2000) showed that the gastric mucosa of Teleosts exhibits
physiological responses similar to those observed in superior Vertebrata; therefore, it is
possible to use fish as experimental models in pharmacological and toxicological
experimentation.
A study was carried out in order to evaluate the protective effect of capsaicin on gastric
ulcers experimentally induced on a Teleost species, Anguilla anguilla (European eel), with
the objective of verifying whether, even in in vitro condition (in absence of any nervous
regulation and blood flow) this effect is still present.


MATERIALS AND METHODS

Studied specimens
The specimens under experimentation were European eel (Anguilla anguilla) of average
age 48 months, showing a mean weight 133 ± 2.3 g and length of 26 cm. They were

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maintained in the aquaculture plant of the Istituto per l’Ambiente Marino Costiero (IAMC)
of Messina, in circular tanks of PVC (capacity 300 l) under natural photoperiod.
After a period (thirty days) of acclimatisation, during which fish were not feed, eels were
sacrificed after treatment with MS 222 (final concentration 10 g/l), their gastrointestinal tract
were removed and the stomach isolated (Figure 1). This organ, Y-shaped, includes two
regions (cardial and fundal) with gastric glands, and a third one, the pyloric, folded and
without glands. After its removal, the stomach underwent to depletion of the muscle and
connectival layers in Petri dishes containing physiological Ringer solution, buffered with
HCO -
3 , under proper oxygenation, then filled with a not-buffered solution and enclosures
were made in order to obtain some “gastric bags”.




Figure 1 - Removal of the gastrointestinal tract from Anguilla anguilla.
Figura 1 - Prelievo del tratto gastrointestinale di Anguilla anguilla.



During a first series of experiments, these bags were incubated for 6 h in a beaker
containing Ringer solution buffered with HCO -
3 or with HEPES NaOH. The chemical
composition of the solutions used is reported in Table 1; the incubation media (Ringer-
HCO -
3 or Ringer-HEPES buffers) were also added with histamine 10-4 mol/l, a substance
which stimulates the acid secretion by the gastric mucosa of eel (Trischitta et al., 1988).
The pH of all the solutions was 8.0 ± 0.1. Tissue oxygenation was ensured by the addition
to the solutions of a gas mixture containing 99 ml/l O
-
2 + 1 ml/l CO2, for Ringer–HCO3
buffer solution, and containing 100 ml/l O2, for Ringer-HEPES solution.
During the second series of experiments, the formation of ulcers (both acid and not-acid)
on the gastric mucosa was experimentally induced by treatment with ethanol 10% or aspirin
(10-3 mol/l) (used as control gastric bags). Each of the compounds tested during the
experiments were added to the incubation beaker at the beginning of the experiment, except
for ethanol 10%, which was dissolved in the not-buffered solution and tested by direct
inoculation inside the gastric bag. Aspirin was dissolved in methanol (final concentration in
the incubation medium, 1 g/l).
In order to test the gastro-protective potential of capsaicin, both the experiments were
repeated, including as a final step the treatment with this ingredient, dissolved in methanol,
and tested at decreasing concentrations (10-4, 10-5, 10-6 mol/l).

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Ions Ringer-HCO (1)
3
Ringer-HEPES (2)
Solution without
buffer (3)
Na+
153 153 153
K+
4 4 4
Cl-
144 144 144.8
Mg2+
1.4 1.4 1.4
Ca2+
2.5 2.5 2.5
HCO3- 20
/
/
H2PO4
0.8 0.8 /
Glucose 20
20
20
Gluconate /
15
20
HEPES
/ 4.5 /
1T Solutions (1) and (2) were added with 99 ml/l O2 and 1ml/l CO2, solution (3) was added
with 100 ml/l O2


Table 1 - Composition of the solutions (concentration in mmol/l).
Tabella 1 – Composizione delle soluzioni (concentrazione in mmol/l).




After incubation, the gastric bags were opened and underwent to macroscopic observation,
in order to detect the presence of gastric ulcers and their recovery after treatment with
capsaicin. Each kind of experiment was repeated 6 times.





Plate 1: Figure 2 - Gastric mucosa of Anguilla anguilla in normal conditions; Figure 3 - Gastric mucosa of
Anguilla anguilla after incubation for 6h in Ringer-Hepes and histamine (10-4 mol/l); Figure 4 - Gastric mucosa
of Anguilla anguilla after incubation for 6h in Ringer-Hepes, histamine (10-4 mol/l) and capsaicin (10-6 mol/l);
Figure 5 - Gastric mucosa of Anguilla anguilla after incubation for 6h in Ringer-HCO -
3 histamine (10-4 mol/l) and
ethanol. The gastric bag is supported by a plastic sheet due to the tissue damage induced by ethanol; Figure 6 -
Gastric mucosa of Anguilla anguilla after incubation for 6h in Ringer-HCO -
3 histamine (10-4 mol/l), ethanol and
capsaicin (10-6 mol/l); Figure 7 - Gastric mucosa of Anguilla anguilla after incubation for 6 h in Ringer-HCO -
3
histamine (10-4 mol/l) and aspirin (10-3 mol/l); Figure 8 - Gastric mucosa of Anguilla anguilla after incubation for
6h in Ringer-HCO -
3 histamine (10-4 mol/l), aspirin (10-3 mol/l) and capsaicin (10-6 mol/l).

Tavola 1: Figura 2 - Mucosa gastrica di Anguilla anguilla in condizioni fisiologiche normali; Figura 3 - Mucosa
gastrica di Anguilla anguilla dopo incubazione per 6h in Ringer-Hepes e istamina (10-4 mol/l); Figura 4 -
Mucosa gastrica di Anguilla anguilla dopo incubazione per 6 h in Ringer-Hepes, istamina (10-4 mol/l) e
capsaicina (10-6 mol/l); Figura 5 - Mucosa gastrica di Anguilla anguilla dopo incubazione per 6h in Ringer-
HCO -
3 istamina (10-4 mol/l) ed etanolo. Il sacchetto gastrico è adagiato su un supporto di plastica a causa del
danno tissutale prodotto dall’etanolo; Figure 6 - Mucosa gastrica di Anguilla anguilla dopo incubazione per 6h
in Ringer-HCO -
3 istamina (10-4 mol/l), etanolo e capsaicina (10-6 mol/l); Figura 7 - Mucosa gastrica di Anguilla
anguilla dopo incubazione per 6h in Ringer-HCO -
3 istamina (10-4 mol/l) e aspirina (10-3 mol/l); Figura 8 -
Mucosa gastrica di Anguilla anguilla dopo incubazione per 6h in Ringer-HCO -
3 istamina (10-4 mol/l), aspirina
(10-3 mol/l) e capsaicina (10-6 mol/l).


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2 3


2

3

4 5





6 7
8







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RESULTS

Figure 2 shows the aspect of the stomach of eel in normal conditions, with mucosal folding
and clear border (boundaries, margins) of the gastric tissue. After incubation in presence of
HCO -
3 , the gastric tissue did not show any change (not shown in Figure), while in Ringer-
HEPES a wide erosion of the surface mucosal layer was observed, with a flattening of the
folded surface (Figure 3). Under this condition, the effects of capsaicin addition were
different according to its concentration. In fact, following the incubation with capsaicin at a
10-6 mol/l concentration, the gastric mucosa remained undamaged, with clear borderlines and
folds (Figure 4), while at higher doses (10-5 and 10-4 mol/l) this protective effect was mostly
undetectable.
The treatment with ethanol 10% resulted in a wide erosion of the gastric mucosa, with deep
ulcers (Figure 5). Following the treatment with capsaicin, particularly at the 10-6 mol/l
concentration a regeneration of the mucosal layer was observed (Figure 6), and ulcers
disappeared. The same effect, but not so evident, was recorded after the addition of capsaicin
at the highest concentrations (10-5 and 10-4 mol/l).
The addition of aspirin (10-3 mol/l) caused erosion and ulcers in the mucosal layer (Figure
7). Capsaicin determined a protective effect similar to that one previously described, with a
greater efficacy at the lowest concentration (10-6 mol/l) (Figure 8).


DISCUSSION AND CONCLUSIONS

Results of the experiments performed provided evidence that capsaicin, even “in vitro”
studies, such as those performed in our laboratory, played a protective role on the gastric
mucosa. Following the treatment with this compound, in fact, it was possible to prevent the
erosion of the gastric mucosa caused by the incubation of the gastric bag in solutions
containing histamine, while lacking of bicarbonate. This latter is known for its buffer and
protective properties on gastric mucosa; therefore, the lacking of bicarbonate and the
presence of histamine are equally important for gastric tissue integrity (Trischitta et al.,
1988).
In rat, Alfoldi et al. (1987) have also shown that capsaicin is able to reduce gastric
secretion induced by histamine.
The protective effect of capsaicin was mostly detected at the lowest concentration (10-6
mol/l), confirming what found in in vivo experiments carried out in mammals (Mózsik et al.,
1997c). A similar, gastro-protective, effect of capsaicin was also observed after ethanol
treatment of the gastric bags, causing a deep tissue damage and ulceration; this was also in
agreement with other studies (Holzer & Lippe, 1988; Esplugues & Whittle, 1990; Gyìres &
Barna, 2002) performed “in vivo” in rats treated with the same experimental procedures. The
mucosal microvasculature has been reported to be not only an initial site of damage after
ethanol application, but also the site where prostaglandins likely exert their protective effects
(Trier et al., 1987).
Capsaicin was also effective in recovering gastric damage produced by aspirin; this
compound is an inhibitor of ciclo-oxigenase, enzyme required for the synthesis of
prostaglandins (Faggio et al., 2000), which have a protective action on the gastric mucosa. A
similar effect was observed by application of resiniferatoxin, an analogous of capsaicin, in
rats showing mucosal damage following aspirin and ethanol (Szolcsànyi, 1990; Abdel-
Salam et al., 1995); both substances (capsaicin and resiniferatoxin) also increased the blood
flow in the gastrointestinal tract of rats (Abdel-Salam et al., 1996). Cimetidine is recognised

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to exert a protective effect on aspirin- induced gastric damaged mucosa (MacKercher et al.,
1977; Szolcsànyi & Mózsik, 1984); this effect was also observed by Kang et al. (2004).
In our experiment, capsaicin was able, at low concentrations, to repair the ulcers induced
by aspirin, in agreement with what found during in vivo experiments by Holzer et al. (1989)
through intragastric application of capsaicin.
The physiological mechanisms involved in the gastric protection during our in vitro
experiments can be supposed to be related to the production of bicarbonate or mucus, both
acting as a protective barrier inside the gastric bag. Above all consideration, it is interesting
to note that the mechanisms involved in the mucosal protection or regeneration remain
substantially unchanged along the evolutionary scale. As the response of the eel examined to
the capsaicin treatment was similar to the one observed in rat and other superior vertebrates,
then it is likely to propose this fish as a model species for animal experimentation in
pharmacological and toxicological studies. This consideration is in agreement with previous
findings (Trischitta et al., 1988; Faggio et al., 2000), which confirmed the suitability of this
fish, also in relation to its wide commercial distribution and long resistance to be kept in
aquaria, as an animal model for improving knowledge of physiological mechanisms.


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