Mechanisms involved in the gastro-protective effect of STW 5 (Iberogast) and its components against ulcers and rebound acidity.

Abstract

The protective effect of a commercial preparation (STW 5, Iberogast), containing the extracts of bitter candy tuft, lemon balm leaf, chamomile flower, caraway fruit, peppermint leaf, liquorice root, Angelica root, milk thistle fruit and greater celandine herb, against the development of gastric ulcers was previously reported in an earlier publication (Khayyal et al., 2001). All extracts produced a dose dependent anti-ulcerogenic effect associated with a reduced acid output, an increased mucin secretion, an increase in prostaglandin E(2) release and a decrease in leukotrienes. The effect on pepsin content was not uniform and did not seem to bear a relationship with the anti-ulcerogenic activity. The best effects were observed with the combined formulation, STW 5. Furthermore, the effect of the latter in protecting against the development of rebound gastric acidity was examined experimentally in rats and compared with the effect of some commercial antacid preparations (Rennie, Talcid and Maaloxan). A model of testing rebound acidity was developed by inducing a marginal increase in gastric acidity through the administration of indomethacin, in such a way that it could be easily neutralized, allowing any eventual secondary increase in acidity to be measured within a few hours of administration. In addition, the serum gastrin level was measured after drug treatment to establish any correlation between it and any rebound acidity. The results obtained demonstrated that STW 5 did not only lower the gastric acidity as effectively as the commercial antacid, but it was more effective in inhibiting the secondary hyperacidity. Moreover, STW 5 was capable of inhibiting the serum gastrin level in rats, an effect which ran parallel to its lowering effect on gastric acid production.

Full text

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Phytomedicine 13 (2006) SV 56–66
Mechanisms involved in the gastro-protective effect of STW 5
(Iberogasts) and its components against ulcers and rebound acidity
M.T. Khayyala, M. Seif-El-Nasra, M.A. El-Ghazalyb, S.N. Okpanyic,
O. Kelberc,?, D. Weiserc
aDepartment of Pharmacology, Faculty of Pharmacy, Cairo University, Egypt
bNational Centre for Radiation Research and Technology, Nasr City, Cairo, Egypt
cSteigerwald Arzneimittelwerk GmbH, Darmstadt, Germany
Abstract
The protective effect of a commercial preparation (STW 5, Iberogasts), containing the extracts of bitter candy tuft,
lemon balm leaf, chamomile flower, caraway fruit, peppermint leaf, liquorice root, Angelica root, milk thistle fruit and
greater celandine herb, against the development of gastric ulcers was previously reported in an earlier publication
(Khayyal et al., 2001). All extracts produced a dose dependent anti-ulcerogenic effect associated with a reduced acid
output, an increased mucin secretion, an increase in prostaglandin E2release and a decrease in leukotrienes. The effect
on pepsin content was not uniform and did not seem to bear a relationship with the anti-ulcerogenic activity. The best
effects were observed with the combined formulation, STW 5. Furthermore, the effect of the latter in protecting against
the development of rebound gastric acidity was examined experimentally in rats and compared with the effect of some
commercial antacid preparations (Rennies, Talcidsand Maaloxans). A model of testing rebound acidity was
developed by inducing a marginal increase in gastric acidity through the administration of indomethacin, in such a way
that it could be easily neutralized, allowing any eventual secondary increase in acidity to be measured within a few
hours of administration. In addition, the serum gastrin level was measured after drug treatment to establish any
correlation between it and any rebound acidity. The results obtained demonstrated that STW 5 did not only lower the
gastric acidity as effectively as the commercial antacid, but it was more effective in inhibiting the secondary
hyperacidity. Moreover, STW 5 was capable of inhibiting the serum gastrin level in rats, an effect which ran parallel to
its lowering effect on gastric acid production.
r 2006 Elsevier GmbH. All rights reserved.
Keywords: STW 5; Iberogast; Iberis amara; Gastric ulcer; Indomethacin; Gastric mucosa; Mucin; Prostaglandin E2; Leukotriene
D4; Gastrin; Acid rebound; Rebound acidity
Introduction
Initially, the potential anti-ulcerogenic effect of STW
5 and its individual components were studied (Khayyal
et al., 2001). The pathogenesis of peptic ulcer involves a
disturbance of the natural balance between aggressive
factors in the stomach, e.g. the production of acid and
pepsin, and defensive mechanisms involving the elabora-
tion of mucus and bicarbonate as well as mucosal
turnover (Piper and Stiel, 1986). The primary endogen-
ous mediators of gastric acid secretion are mainly
acetylcholine, gastrin and histamine, the roles of which
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0944-7113/$-see front matter r 2006 Elsevier GmbH. All rights reserved.
doi:10.1016/j.phymed.2006.03.019
?Corresponding author. Tel.: +4961513305154;
fax: +4961513305493.
E-mail address: kelber@steigerwald.de (O. Kelber).

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are intricately interrelated (Hayward et al., 1991). The
protective mechanisms of mucus and bicarbonate
secretion depend to a large extent on prostaglandin E1
and E2 secretion, and these in turn depend on the
activity of the cyclooxygenase enzyme system. Gastric
mucosal injury could be produced by a variety of agents
which disturb that balance, including alcohol, nonster-
oidal anti-inflammatory drugs, parasympathomimetics,
and drugs with histamine-like properties.
Some plant constituents and their combination in the
form of a commercial preparation (STW 5) have been
studied to determine their usefulness as anti-ulcerogenic
agents. STW 5 (Steigerwald Arzneimittelwerk GmbH,
Darmstadt, Germany) consists of a fixed combination of
plant extracts namely bitter candytuft (Iberis amara),
Melissa leaf (Melissa officinalis), Matricaria flower
(Matricaria recutita), caraway fruit (Carum carvi),
peppermint leaf (Mentha?piperita), Angelica root
(Angelica archangelica), milk thistle (Silybum maria-
num), elan-dine herb (Chelidonium majus), in addition to
liquorice root (Glycyrrhiza glabra). The anti-ulcerogenic
effect of the individual plant extracts as well as their
combination was determined using indomethacin as a
standard ulcerogen and cimetidine as a reference anti-
ulcerogenic agent. Further experiments were then
carried out to investigate possible mechanisms involved
in their anti-ulcerogenic action. Parameters such as free
acidity, mucin and pepsin concentrations in the gastric
juice, as well as prostaglandin and leukotriene levels in
the gastric mucosa were measured.
Many agents that reduce gastric acidity often have a
tendency to induce a secondary rise in acidity within a
short time of administration. These include H2-receptor
blockers, proton pump inhibitors (Nwokolo et al., 1991)
and many antacid preparations. Rebound acid hyperse-
cretion may contribute to the high ulcer relapse rate
after discontinuation of H2-receptor antagonists (Nwo-
kolo et al., 1991; Wilder-Smith et al., 1991). A variety of
factors have been proposed to explain the earlier ulcer
relapse rate, including secondary hypergastrinemia with
rebound acid hypersecretion after discontinuation of the
drug. Secondary hypergastrinemia may also lead to
tolerance to prolonged courses of H2 antagonists
associated with decreased acid inhibition (McQuaid,
1991). Similarly, many antacids containing calcium, as
carbonate or other salts, cause an increase in gastric acid
secretion which might be explained, at least in part, to
direct stimulation by the calcium ions. Other antacids,
notably magnesium hydroxide and aluminum hydroxide
may also cause acid rebound, possibly, through antral
alkalinization with subsequent gastrin release (Hade and
Spiro, 1992). Gastrin secretion is a function of the
gastric antral mucosa and is highly dependent upon
gastric intraluminal pH. It is inhibited significantly by a
pH of less than 3.0 (McQuaid, 1991). Courses of
standard therapy with antisecretory agents such as H2
blockers or H+/K+/ATPase inhibitors cause a signifi-
cant rise in the 24h plasma gastrin levels. Since STW 5
was found to exert some beneficial properties on gastric
acidity, it was therefore of interest to investigate whether
it causes rebound hyperacidity or not. The effect of this
agent was compared in rats with conventional antacid
trade preparations. Since acid production is associated
with gastrin secretion, among other factors, serum
gastrin level of rats was estimated during the phase of
the rebound hyperacidity to illustrate the possible mode
of action of the selected drugs.
Materials and methods
Plant extracts and drugs
STW 5 (Iberogasts), and its individual components:
bitter candytuft (Iberis amara), melissa leaf (Melissa
officinalis), Matricaria flower (Matricaria recutita), car-
awayfruit(Carumcarvi),
(Mentha?piperita), Angelica root (Angelica archangeli-
ca), milk thistle (Silybum marianum), elan-dine herb
(Chelidonium majus), and liquorice root (Glycyrrhiza
glabra) were obtained either as 30% alcoholic extracts
or as lyophilized extract from Steigerwald Arzneimittel-
werk, GmbH, Darmstadt, Germany.
The commercial antacid products used were Rennies
(Roche Nicholas, Eppstein-Bremthal, Germany), Talcids
(Bayer, Leverkusen, Germany) and Maaloxans(Rho ˆ ne-
Poulenc-Rorer Casella-med, GmbH&Co. KG, Ko ¨ ln,
Germany). They were used as the powdered form of the
commercial product.
Magnesium carbonate, indomethacin and cimetidine
were obtained from Sigma Chemical Co. (St. Louis,
MO, USA).
peppermintleaf
Animals
Male Wistar rats weighing 120–150g each were used for
this study. Animals were obtained from the National
Research Centre, Dokki, Cairo, and were fed on a
standard pellet diet. They were housed at the animal
facility at the Faculty of Pharmacy, Cairo University at a
temperature of 25711C and humidity of 6075%. The
study was conducted according to the guidelines for
animal experiments set by the Faculty of Pharmacy, Cairo
University in accordance with international guidelines.
The rats were randomly divided into groups of 8–10
animals each for the experiments described below.
Induction of ulcers and assessment of anti-
ulcerogenic effect of extracts
Thirty-six hours before subjecting the rats to experi-
mentation, they were subdivided into groups of ten.
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Feed was withdrawn from them, allowing them free
access to water only, then the latter was also withdrawn
one hour before the experiment (Meshali et al., 1983;
Bhargava et al., 1973).
Ulcers were induced acutely by giving the animals
indomethacin orally in a dose of 10mg/kg (Shay et al.,
1954). Five hours later the rats were killed by decapita-
tion. The stomachs were removed, opened along their
greater curvature, and gently rinsed with cold normal
saline and pinned on a paraffin and plastic polymer
plate, before being examined under an illuminated
magnifying glass. The mucosal layer was carefully
inspected for the occurrence of ulcers and their numbers
counted. The severity of ulcers was given an arbitrary
score from 0 to 4 according to the extent of ulceration, 0
indicating a normal mucosa with no ulcers, 1–4
signifying the presence of lesions varying in intensity
from 1mm to perforation.
The alcoholic extracts were administered at three dose
levels of 2.5, 5 and 10ml/kg body weight orally one hour
before the oral administration of indomethacin to
groups of 10 rats each in order to assess the dose-
dependent nature of their anti-ulcerogenic effect and to
be able to choose the most appropriate dose of each
extract for the experiments to follow. Five hours later,
the extract-treated animals were killed and their
stomachs were examined and assessed for ulcers.
Another group of rats was given cimetidine orally in a
dose of 100mg/kg one hour before indomethacin
administration and served as reference standard. The
ulcer index for each group was calculated according to
the following formula (Robert et al., 1967):
% Incidence of
animals
with ulcersmean severity on mean
+arbitary scale+number of
ulcers
/stomach10 (0–4)
The protective anti-ulcerogenic effect of the extracts
was taken as the percentage change in the ulcer index as
compared to the indomethacin treated group.
Assessment of antisecretory and cytoprotective
parameters
For the assessment of the antisecretory and cytopro-
tective activities of STW 5, the lyophilized extracts were
reconstituted with water before use to give the same
concentration as in the original alcoholic extracts. In
this way, the possible interfering effect of alcohol
in the original alcoholic extracts was obviated. The
extracts were given orally to groups of 8 rats each, in the
dose regarded from the above experiment as showing
good anti-ulcerogenic activity. Accordingly, the recon-
stituted aqueous extracts were administered in the
following doses: 10ml/kg for STW 5, Matricaria
recutita, Angelica archangelica and Silybum marianum,
5ml/kg for Glycerrhiza glabrata and Melissa officinalis,
2.5ml/kg for Mentha?piperita, Carum carvi and Iberis
amara.
Forty-eight hours before starting the experiment, each
animal was then housed singly in a cage with a raised
bottom of wide mesh to allow passage of faeces and
prevent caprophagia. The feed, but not water, was
withheld from the animals before subjecting them to the
procedure of pyloric ligation (Shay et al., 1954). At the
end of the starvation period, the extracts were adminis-
tered orally in the above doses. One hour later, the rats
were lightly anaesthetized with ether, and a nearly 2cm
mid-line incision was made extending from the xiphoid
downwards to expose the stomach. The junction
between the pylorus and the duodenum was picked up
and pyloric ligation was carried out by passing a silk
thread below the pylorus and then tying the thread in
such a way to close the pylorus without crushing its wall.
The abdominal wall was then closed with Michel’s clips,
the abdominal wound cleansed thoroughly with physio-
logical saline, dried, and covered with a solution of
collodion. At this point, indomethacin was given
intraperitoneally in a dose of 10mg/kg and the animals
were then allowed to recover, placed in their individual
cages, and received neither food nor water during the
rest of the experiment.
Four hours later, the animals were again anaesthe-
tized with ether, the abdomen was opened and a ligature
was placed at the oesophageal-cardiac junction. The
stomach was then removed, washed in physiological
saline, and blotted dry before sacrificing the animals. An
opening was made along the greater curvature and
gastric contents drained through a funnel into a
graduated centrifuge tube. The stomach was then slit
open along the entire greater curvature and the mucosa
was scrubbed off and weighed.
The gastric contents of each animal were then
centrifuged and the following parameters were assessed
in the supernatant:
(a) Free acidity and acid output: Free acid concentration
was determined by direct titration of 100ml of gastric
juice against 0.005N sodium hydroxide using phenol
red as indicator and expressed as mEq/l. The acid
output/hour was calculated by multiplying the free
acidity by the gastric juice volume and dividing by 4,
as 4h had elapsed between ligation and sampling.
The acid output was expressed as mEq/h.
(b) Mucin concentration: Mucin was determined accord-
ing to the spectrophotometric method described by
Winzler (1955), based on the determination of
hexose component of mucin.
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(c) Pepsin concentration: Pepsin was determined accord-
ing to the spectrophotometric method described by
Sanyal et al. (1971) depending on the digestion of
serum albumin by the peptic activity of the gastric
juice, using tyrosine as a standard.
(d) Analysis of prostaglandins and leukotrienes in the
mucosal contents: The collected mucosa was divided
into two portions which were processed as follows:
One portion was accurately weighed, acidified with
3N hydrochloric acid, homogenized, and then
extracted twice with diethyl ether. The ether was
evaporated to dryness and the residue was kept for
the assay of prostaglandins as PGE2 using an
ELISA kit (Neogen corporation, USA).
The other portion was treated with 2.5ml absolute
ethanol, homogenized, and centrifuged. The super-
natant was evaporated to dryness and the residue
was used for assaying leukotrienes as LTD4using an
ELISA kit (Neogen corporation, USA).
Effect of STW 5 and some commercial antacids on
gastric acid rebound
Effect on gastric acidity
The principle for establishing a method for testing
rebound acidity was to find a suitable dose of
indomethacin to induce a measurable degree of acidity
in the stomach that would wane off within about 5h of
drug administration. Giving a drug in a dose that could
just counteract the increased acidity at its initial stages
of development would then allow any rebound acidity to
be detected 4–5h later. From pilot experiments,
indomethacin in an oral dose of 1mg/kg was found to
be suitable for that purpose.
Animals were randomly divided into several groups,
each of 10 rats. The animals were fasted for 18h before
the experiment, and each rat was kept singly as
previously described. One group served as control and
received only saline. Indomethacin was given orally in a
dose of 1mg/kg to three groups of animals, and gastric
acidity was determined at time intervals of 1, 2, and 5h,
respectively after administration. STW 5 (2.5ml/kg) and
thecommercialantacids
250mg/kg; Rennie, a dose corresponding to 250mg
magnesium carbonate/kg; Talcid, 332.5mg hydrotalcit/
kg; and Maaloxan in a dose equivalent to 26.5mg
Al(OH)3+43.5mg Mg(OH)2/kg) were given orally each
to a group of 10 animals, 1h after indomethacin
administration and the gastric acidity was determined
as described above after 2 and 5h of indomethacin
administration. These doses were selected after initial
pilot experiments to find the smallest dose necessary to
neutralize the indomethacin-induced acidity within 1h
of administration and ensuring that the effect wears off
(magnesium carbonate,
shortly, allowing for any rebound acidity to be detected
within 5h of indomethacin administration.
Effect on serum gastrin level
In another set of experiments, animals were divided
into six groups, each of 6 rats, to determine the serum
gastrin level. Animals were fasted for 18h before
subjecting them to experimentation. One group of rats
was kept as control, and the others were injected
intraperitoneally with 10mg/kg indomethacin. One hour
later, the rats were given the drugs mentioned above in
the pre-selected doses. Blood was collected from the
retro-orbital plexus of each animal using fine hepar-
inized capillary tubes, 2 and 5h after indomethacin
administration. The collected blood was centrifuged and
the serum was separated and kept frozen until assayed
for gastrin using a Gamma Dab Gastrin
immunoassay kit (DiaSorin Stillwater, Minnesota,
USA).
125I radio-
Statistical analysis
Statistical analysis was carried out using one way
analysis of variance (ANOVA) followed by the least
significant difference (LSD) test. Values are given as
means7S.D. and po0:05 was considered to be statis-
tically significant.
Results
Effect on indomethacin-induced gastric ulcers
STW 5 and its individual plant extracts produced a
dose-dependent protection against the ulcerogenic effect
induced by indomethacin (Fig. 1). STW 5 in the doses
used showed good anti-ulcerogenic effect particularly
with the highest dose of 10ml/kg amounting to nearly
60%, an effect nevertheless less than that produced by
cimetidine (100mg/kg).
With respect to the free acidity, STW 5 given 1h
before indomethacin prevented the marked rise in
gastric acidity and acid output induced by the latter.
Similar effects were obtained with the other plant
extracts, especially those of matricaria leaf, caraway
fruit and bitter candytuft, which were effective even in
reducing the acidity below normal values, their effect
approaching that of cimetidine (Figs. 2 and 3).
Gastric mucin, which was significantly reduced by
indomethacin, was not only preserved by STW 5, but its
level was even increased two-fold. All the individual
plant extracts, with the exception of that of peppermint
leaf, also showed an increase in the mucin content, thus
providing protection against the gastric ulceration. The
most significant effects were observed with the extracts
of liquorice root, matricaria flower and caraway fruit
(Fig. 4).
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Indomethacin tended to lower the gastric pepsin
content to a small but statistically significant extent,
but the effect of the individual extracts was rather
variable. STW 5 and some extracts, like Matricaria and
Silybum, tended to raise it, while others, like Angelica,
tended to lower it, and some, like peppermint and
liquorice, hardly affected it (Fig. 5).
Some of the above effects could possibly be associated
with effects on prostanoid levels. The prostaglandin
content of the gastric mucosa dropped from 696ng/g in
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0
STW 5
10
20
30
40
50
60
70
80
90
100
Iberis
Melissa
Matricaria
Carum
Silybum
Angelica
Mentha
Glycyrrhiza
Cimetidine
Percentage protection
2.5 ml/kg5 ml/kg10 ml/kg
Fig. 1. Protective effect of the different plant extracts against indomethacin-induced ulcers. Columns represent the change in ulcer
index of the extract treated groups (n ¼ 10) as a percentage of the indomethacin-treated rats.
0
50
100
150
200
250
Normal
Indomethacin
STW 5
Cimetidine
Iberis
Melissa
Matricaria
Carum
Silybum
Angelica
Mentha
Glycyrrhiza
Acidity [mEq/L]
*
*
*
*
*
*
**
*
*
*
Fig. 2. Effect of treatment with different extracts (doses mentioned in text) on acidity of the gastric juice of pyloric ligated rats.
Values are given as means7s.e.m. of eight observations. *Significantly different from the indomethacin-treated group at po0:05
[ANOVA followed b LSD test].
M.T. Khayyal et al. / Phytomedicine 13 (2006) SV 56–6660