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J. Drug Res. Egypt, Vol. 35, No.1 (2014) 81
Evaluation of Newly Formulated Antiulcer Drug on Experimental
Ulcer Model
1Nashwah I. Zaki, 2Lobna A. Hassanin and 3Abeer A.M. Khattab
1Dept. of Physiology, (NODCAR), Egypt.
2Dept. of Hormones Evaluation, (NODCAR), Egypt
3Dept. of pharmaceutics, (NODCAR), Egypt
ABSTRACT: The present study was conducted to evaluate the potential gastric protective
and therapeutic effects of ranitidine mucoadhesive hydrogel formulae against ethanol induced
gastric ulceration in rats. Adult female albino rats weighing between 200-220 g ,75 rats for
evaluation of mucoadhesiveness of hydrogel and 70 rats for evaluation of protective and
therapeutic effect of ranitidine hydrogel. The seventy rats were randomly divided into two
experiments, protective and therapeutic effects of newly developed ranitidine mucoadhesive
hydrogel formulae containing polymer mixture of Chitosan and Hydroxypropyl methyl
cellulose (HPMC) at ratio 9:1 (F1) or mixture of Sodium carboxymethyl cellulose (NaCMC)
and HPMC at ratio 9:1 (F2). Each experiment has five groups, seven rats each; group 1 serves
as control and group 2 serves as ulcer control since received a single oral dose of absolute
ethanol (5ml/kg body weight). Group 3 ulcer group received an oral dose of ranitidine
(27mg/kg), while groups 4 and 5 ulcer group received newly formulae of ranitidine F1& F2
respectively. In the present study some gastric parameters as ulcer index, total acidity, gastric
volume and pH besides some biochemical parameters as alanine aminotransferase (ALT),
aspartate aminotransferase (AST) and total antioxidant capacity (TAC), total protein (TP)
level and alkaline phosphatase (ALP) activity were carried out at the end of the experimental
period.The present study showed that F1 revealed more protective and therapeutic potency
than F2 as it was significantly reduced ulcer index, total acidity, gastric volume and pH in
comparison to ulcerated group (p<0.05). Also, the biochemical markers ALT, AST and TAC
were decreased significantly compared to ulcerated group in both experiments. TP level and
ALP activity were altered among different treatments. Moderate improvement in mucus
secretion was recorded for F1 & F2 treatments than the reference drug. The present results
were confirmed by the histopathology findings. Collectively, the current study confirmed the
better therapeutic action of formulae 1 & 2 over the pure drug and that F1 was the most potent
formula. Also, it encouraged the use of F2 as a curative agent of ulceration rather than a
protective one.
Key words: Ranitidine, mucoadhesive hydrogel formulation, liver function markers,
physiological ulcer indexes, mucin secretion
INTRODUCTION: Gastric ulcer is a major health hazard in terms of both
morbidity and mortality (Chaturvedi et al., 2007).
Ulcer therapy has progressed from vagotomy to
anticholinergic drugs, histamine H2 receptor
antagonists, antacids and to proton pump inhibitors
(Wallace & Granger, 1996). Even though the wide
range of available drugs, many of them do not fulfill all
the requirements and have many side effects. Although
histamine H2 receptor antagonist drugs as famotidine
and ranitidine are considered to be the safest one
(Bourdet et al., 2005), these drugs have been reported
to have low bioavailability and short biological half-
life (Hawkins & Hanks, 2000). Ranitidine is a
competitive, reversible inhibitor of the action of
histamine at histamine H2-receptors, including
receptors on gastric cells, with a minimal effect on H1-
receptors (Novak, 2002 and Chavda & Patel, 2010). It
Corresponding author
is one of the drugs of choice for the treatment of active
duodenal ulcers, gastric ulcers, Zollinger-Ellison
syndrome, gastroesophageal reflux disease, and erosive
esophagitis (McCarty-Dawson et al., 1996). The
indicated oral dosage of Ranitidine is 150 mg, twice
daily, or 300 mg once daily. It has been found that the
dose of 300 mg leads to fluctuations in its plasma
levels (Chavda & Patel, 2010). Also, the drug has a
short biological half-life of approximately two to three
hours, an absolute bioavailability of only 50%, and it is
absorbed only in the initial part of the small intestine
(Grant et al., 1989; Lauritsen et al., 1990 and Gramattι
et al., 1994). Colonic metabolism of Ranitidine is also
partly responsible for the poor bioavailability of
Ranitidine from the colon (Basit & Lacey, 2001).
Hence, these drugs have promising future if controlled
release formulations are developed. Several trails have
been used Ranitidine to develop many gastroretentive
drug delivery systems, but it failed in many cases
(Gnanaprakash, 2013). Theoretical and applied studies
stated that gastroretentive mucoadhesive dosage form
can remain in the gastric region for several hours and
hence significantly prolong the gastric residence time
of drugs (Hagerstorm, 2003 and Dua & Trivedi, 2013).
Prolonged gastric retention improves bioavailability,
J. Drug Res. Egypt, Vol. 35, No.1 (2014) 82
reduces drug waste. It is also suitable for local drug
delivery to the stomach and proximal small intestines
(Ben et al., 1994 &1996; Hagerstorm, 2003 and Dua,
& Trivedi, 2013). Mucoadhesive dosage forms have
been widely used for site-specific targeting for both
local and systemic drug delivery (Chen et al., 1997;
Nagahara et al., 1998; Harding et al., 1999; Wang et
al., 2000; Cuna et al., 2001 and Bardonnet et al.,
2006). Hydrogels found applications in formulating
controlled and mucoadhesive drug delivery systems
(Bansal et al., 2011). Hydrogels are cross-linked
hydrophilic polymers with a network structure.
Chitosan and Sodium Carboxymethyl Cellulose (Sod.
CMC), have attracted attention. However, these
hydrogels can swell slowly and exhibit low loading
capacities, (Henriksen et al., 1996; Burmania et al.,
2003 and Leonard et al., 2004) which restrict their use
in effective drug delivery. Chitosan, is a linear random
copolymer of D-glucosamine and N-acetyl D-
glucosamine and is obtained by N-deacetylation of
chitin (Domard & Cartier, 1992 and Roberts, 1992).
Chitosan hydrogel can be formed by covalent cross-
linking or ionic cross-linking. By definition,
mucoadhesive hydrogels are a class of polymeric
biomaterials that exhibit the basic characteristic of a
hydrogel to swell by absorbing water and to interact by
means of adhesion with the mucus that covers epithelia
(Nikalje et al., 2012). The goal of mucoadhesive drug
delivery system is to increase the residence time of
therapeutic molecules at the specific sites within the
gastrointestinal (GI) tract for absorption of the drug
into the circulation (Hassan & Gallo, 1990; Roberts,
1992; Takeuchj et al., 1994 and Needleman & Smales,
1995). The approach involves the use of bioadhesive
polymers such as Chitosan - hydroxyl propyl methyl
cellulose (HPMC) that can adhere to the epithelial
surface of the GIT (Sadgir et al., 2014). The adhesion
of the polymers with the mucous membrane may be
mediated by hydration, bonding - or receptor -
mediation (Park & Robinson, 1984). The current study
aimed to assessment the potential gastric protective and
therapeutic effects of ranitidine mucoadhesive hydrogel
formulations on ethanol induced gastric ulcer in rats
compared to ranitidine powder.
MATERIALS AND METHODS: Drugs and chemicals
Ranitidine HCl (R-HCl) was kindly donated by Amoun
pharmaceutical company. Chitosan was purchased
from Sigma, Chitosan (≥ 85% deacetylated, Mol. Wt ~
600,000 Mol. Wt.; ~400 m Pa.s, 1 % in acetic acid at
20 °C, Sigma Aldrich, Germany), Hydroxypropyl
methyl cellulose (HPMC),and sodium carboxymethyl
cellulose (NaCMC) were purchased from Alfa,
Germany. Double distilled water (DDW) was prepared
in the laboratory. All other chemicals used were of
analytical grade.
Preparation of hydrogel formulations
Two formulae were prepared using weighed amount of
polymer mixture of Chitosan and HPMC at ratio 9:1
dissolved in 1% acetic acid to prepare F1; and weighed
amount of polymer mixture of NaCMC and HPMC at
ratio 9:1 dissolved in distilled water to prepare F2.
336 mg of ranitidine HCl was added to each formula.
The formed solutions were casted into Petri dishes and
left to dry at room temperature. Then the dried
hydrogel films were cut into 2 x 2 mm square pieces
and stored in tightly closed container at room
temperature (Paloma et al., 2003).
Experimental animals
Induction of ulcer
All groups under investigation received single oral
dose of absolute ethanol (5ml/kg body weight)
according to the method described by Abdulla et al.
(2010) except the control one.
Evaluation of Mucoadhesiveness of hydrogel
Six week aged female Swiss albino rats weighing 200-
220 g were obtained from the animal house of National
Organization for Drug control and Research
(NODCAR, Giza, Egypt). The animals were
acclimatized in Kafr El Gabal animal house for 2
weeks before the experiment. The animals were fed a
standard diet (El-Nasr Company, Abou-Zaabal, Cairo,
Egypt) and tap water ad libitum, and kept under
controlled conditions of room temperature (21 ± 1 °C),
relative humidity (55±5%) and a 12-h light/12-h dark
cycle. Animals were fasted overnight prior to the
experiment, but were allowed free access to water.
Animals were divided into three groups, each group
contains 25 albino rats, as follows: Group I: (positive
control): this group contained a total of 25 albino rats
and were administrated 5 ml of ranitidine HCl solution
at concentration of 10 mg/ml, Group II: This group
contained a total of 25 rats and administrated a hard
gelatin capsule containing 2 x 2 square particles of
formula I (F1) equivalent to 50 mg ranitidine. Group
III: This group contained a total of 25 rats and
administrated a hard gelatin capsule containing 2 x 2
square particles of formula II (F2) equivalent to 50 mg
ranitidine. At 0.5, 1, 2, 3 and 4 hours following
administration, 5 rats from each group (I, II and III)
were euthanized by cervical dislocation, the stomachs
were excised, cut along the greater curvature, and
gently rinsed by 25 ml of 0.1N HCl and put in
sonicator for dissolving of existed amount of ranitidine
in rinsed solution then filtered through 0.45 µm
Millipore filter. The amount of ranitidine remained was
evaluated by high-performance liquid chromatography
using UV detector at wave length (λmax) 314 nm and C8
column, (250 x 4.6 mm; 5µ). The mobile phase
consisted of 0.05 M potassium dihydrogen phosphate
(pH 3): acetonitrile (25:75 v/v) and was delivered to
the system at a flow rate of 1.2 ml/min. All assays were
performed at ambient conditions. The remaining
percentage of ranitidine as an index of residence in the
stomach, i.e., Mucoadhesiveness, was calculated by the
following equation:
Remaining percentage = (R/T) x100
Where R represents the amount of ranitidine remaining
in the stomach and T represents the amount of
ranitidine administrated. The method was validated for
selectivity, linearity, accuracy and precision (Radi &
Mansoor, 2004).
J. Drug Res. Egypt, Vol. 35, No.1 (2014) 83
Evaluation of protective and therapeutic effect of
ranitidine hydrogel To achieve study goals, seventy healthy adult female
Swiss albino rats, weighing between 200-220 g, were
obtained from the animal house at the National
Organization for Drug Control and Research
(NODCAR). They were housed in wire cages with
natural ventilation and illumination and allowed free
water and standard diet for 10 days before beginning of
the experiment. Throughout the experiment, all
procedures, animals and the experimental protocols
were approved by the Institutional Ethics Committee at
NODCAR; and were carried out according to the
criteria outlined in the “Guide for the Care and Use of
Laboratory Animal”. Rats were fasted for 24 h prior to
the experiment but allowed free access to water except
for the last hour before the experiment. All experiments
were performed during the same time of the day to
avoid diurnal variations of putative regulators of gastric
functions.
The animals were randomly divided into two
experiments, the first to study the protective effects
(pre-treated) of newly developed formulae while the
second deals with their therapeutic effects (post-
treated). Each classified into five groups (seven rats
each); Group 1 serves as control (C) while group 2
serves as ulcer control (U), group 3 received ranitidine
(R) as a reference antiulcer drug (27 mg/kg body
weight) equivalent to human recommended dose
(Chavda & Patel, 2010), while groups 4 and 5
received newly formulae of ranitidine with the same
concentration of ranitidine F1 and F2 respectively. In
protective experiment (pre-treated) the animals in each
group were receiving the dose of the treatment as
mentioned above 45 minutes before administration of
5ml of 95% ethanol. After further one hour from
alcohol administration, blood samples were collected
from retro-orbital plexus, then the animals were
scarified by cervical dislocation; the stomach content
was collected, then it was incised along the greater
curvature and examined for gross and histological
evaluation. In therapeutic experiment (post-treated) the
animals were divided as mentioned above. The
treatment was given daily for four successive days on
empty stomach-starting one hour after induction of
ulcer. On the fifth day blood samples were collected,
then the animals were scarified by cervical dislocation
the stomach content was collected and then it was cut
along the greater curvature, washed with saline and
examined for gross and histological evaluation.
Blood sampling
Blood samples were allowed to clot for thirty minutes
at room temperature and then centrifuged at 1000 x g
and 4ºC for ten minutes. The collected serum samples
were stored at -80 ◦C for further estimation of total
protein (TP) content, alanine aminotransferase (ALT),
aspartate aminotransferase (AST), alkaline phosphatase
(ALP) activity and total antioxidant capacity (TAC).
Spectrophotometric procedures were used for their
determination using commercial kit according to
Gornall et al. (1949); Reitman & Frankel (1957); Moss
(1982) and Koracevic et al. (2001) respectively.
Gastric volume and Total acidity
The animals were sacrificed and their stomachs were
cut along the greater curvature, the content of each
stomach was collected into small tubes and centrifuged
at 3000 rpm for 5 minutes. The supernatant was
separated and its volume was measured and expressed
as ml/100 g body weight. The acid content was
determined by titration method with 0.05N NaOH.
Acidity was expressed as mEq/L/100 g of body weight
(Maity et al., 2003).
Acidity= volume of NaOH x normality / 0.1 x100
Macroscopic evaluation of stomach The macroscopic assessment of ethanol-induced gastric
lesions was performed by an independent examiner
who was blinded to the supplementation that the rats
had received. The assessment of lesions was made
according to quantitative scale described by Ismail et
al. (1999). The stomachs were opened along the greater
curvature, rinsed with saline to remove gastric contents
and blood clot then photographed to assess the
formation of ulcers. The numbers of ulcers were
counted. Mean ulcer score for each animal will be
expressed as ulcer index. The scale used was as
follows: 5 = continuous lesions that occupied almost
the entire length of the gastric fold, 4 = lesions which
occupied almost 80% of the entire fold, 3 = presence of
multiple lesions that measure 3mm in length on 80% of
the folds, 2 = presence of at least two lesions
approximately 2mm in length, 1 = presence of a single
lesion with or without generalized erythema, 0.5 =
presence of dot haemorrhage and 0 = no visible
damage.
Curative ratio The curative ratio from the ulcer was calculated
according to Begum et al. (2014) for the treated groups
by using the following equation.
Percentage (%) = [CUI-TUI] /CUI X100
Where, CUI = ulcer index of control groups, TUI =
ulcer index of treated groups.
Histological and Histochemical studies
Gastric tissue specimens from each group were fixed in
10% buffered formalin for twenty four hours. After that
the tissue specimens were washed with tap water then
serial dilutions of alcohol (methyl, ethyl and absolute
ethyl) for dehydration. Specimens were cleared in
xylene and embedded in paraffin at 56 degree in hot air
oven for twenty four hours. Paraffin blocks were
prepared for sectioning at 4 microns thickness by slide
microtome. The obtained tissue sections were collected
on glass slides, deparaffinized, stained by hematoxylin
& eosin stain for routine examination as well as by
alcian blue stain for detection of mucous through the
light electric microscope (Bancroft & Stevens, 1996).
Statistical analysis
The results are presented as the mean ± standard error
( SE) of the six animals used in each group. Statistical
significance was determined by one-way ANOVA at
p< 0.05 followed by Dunnett t (2-sided) for
comparisons between control and other groups while
Duncan posthoc test was used for comparisons among
all treated groups at p<0.05 using the Statistical
Package for the Social Sciences software (SPSS 17).
J. Drug Res. Egypt, Vol. 35, No.1 (2014) 84
RESULTS: Evaluation of gastric mucoadhesion
Figure (1) showed the remaining percentage of
ranitidine after 0.5, 1, 2, 3 and 4 hours in the different
groups (I, II and III). It was observed that the remained
amount of ranitidine 4 hours after administration of
mucoadhesive hydrogel formulae F1 and F2 was
55.8±6.5%,45.4±3.5%, respectively compared with
5.6±2.1% after administration of ranitidine powder.
Thus, the mucoadhesive hydrogel formulae seem to be
a potential as oral gastro retentive controlled drug
delivery and targeting its site of action, the stomach,
thus improving its oral bioaviability.
Evaluation of protective and therapeutic effect of
ranitidine hydrogel
Antiulcer parameters
Table (1) showed the effect of pre-treated of R, F1 and
F2 on gastric juice parameters and ulcer parameters. It
was found that ethanol administration caused a
significant increase in pH value, gastric volume and
total acidity; it also caused a remarkably high ulcer
index (2.80± 0.20) as compared with control group.
While, pretreatment with F1 produced a significant
decrease in pH value, gastric volume, and total acidity
as compared to ulcerated group. In the protective study
there was no significant difference between R & F2 in
pH value, gastric volume and total acidity, while F2
showed a significant decrease in total acidity when
compared to ulcerated group. Pre-treatment with R, F1
and F2 offered a significant protection against ethanol
induced gastric ulcer in the experimental rats. Since
Ranitidine reduced the ulcer index to (0.50±0.16)
showing 82.14 % prevention whereas F1 reduced the
ulcer index to (0.10±0.10) showing 96.43% prevention.
While F2 reduces the ulcer index to (1.1±0.24)
showing 60.71% prevention.
Table (2) showed the effect of post-treated of R, F1 and
F2 on gastric juice parameters and ulcer parameters. It
was observed that ethanol administration caused non-
significant changes in pH value and gastric volume
while total acidity was increased significantly as
compared with control group, also caused ulcer index
(2.20±0.20). Post-treatment with R, F1 & F2 resulted
in a significant reduction in pH value. Gastric volume
showed a non-significant increase in all treated groups.
Post-treatment with F1 showed a significant reduction
in total acidity as compared to ulcerated group. While
R & F2 revealed a non-significant change in total
acidity. Post-treatment with R, F1 & F2 produced a
significant healing reached to 63.64%, 81.82% and
77.27% respectively.
Biochemical parameters
It was found that oral administration of ethanol caused
a significant decrease in serum total protein (TP) in
both pre- and post-treated experiments as compared
with control group (tables 3 and 4). Pre-treatment with
F1 and F2 showed a non-significant change in total
protein as compared to ulcerated group. A significant
increase in serum total protein levels were observed in
F1 & F2 post-treated rats while non-significantly
increased in R post-treated as compared to ulcerated
group.
Ethanol administration showed a significant increase in
the activities of the hepatic enzymes ALT& AST as
compared to control group (tables 3 and 4). While the
administration of F1& F2 in both pre- and post-
treatment experiments resulted in a significant decrease
in serum activity of these hepatic enzymes when
compared to their activities in ethanol treated rats.
Ranitidine administration caused a significant decrease
in hepatic enzymes activity in the pre- treated rats and a
non-significant increase in post-treated rats as
compared to ulcerated group.
It was observed that ethanol caused a significant
decrease in ALP levels and a significant increase in
TAC level when compared with control group. Oral
administration of R, F1 & F2 in pre- and post-treated
rats significantly increased ALP level as compared
with ulcerated group. Pre-treatment with F1 revealed a
significant decrease in TAC as compared with
ulcerated group while R and F2 showed a non-
significant decrease. Whereas post-treatment with R,
F1 & F2 revealed a significant reduction in TAC when
compared with ethanol treated group tables (3 & 4).
Histopathological studies
The severity of histopathological changes in stomach
of different treatments as pre- and post-treatments was
summarized in tables (5) & (6) respectively. Table (5)
showed that pretreatment with F1 before ethanol had
alleviated the effect of ethanol. Thereby F1 was more
effective than R & F2 groups. Table (6) revealed that
post-treatment with R, F1 & F2 decrease the sever
ulcer induced by ethanol. Formula 2 was more
effective than R & F1 treatments.
Histochemistry studies
Histochemical studies Plate (1) showed that; in the
control group; the mucous secretion was localized in
the superficial surface and in the lining epithelium of
the glandular structure. Rat's stomach under the
protective (pre-treated) experiment revealed marked
alteration compared to control group where, very few
mucous was noticed in the mucosal layer among
ulcerated rats. While rats administered ranitidine, the
intact mucosa and glandular structure showed mucous
secretion. F1 treated rats revealed minor alterations
where, the blue color of the mucous was noticed in the
desquamated mucosal epithelium as well as in the
underlying intact glandular structure. In the other hand,
the mucous was localized in the deep intact glandular
structure of the mucosa among rat treated with F2. In
rats experimentally induced ulcer and sacrificed after
four days (post-treated); the mucous was absent from
the mucosa. There was very few mucous in the
ulcerated surface of Ranitidine (post-treated) group.
Rats under F1 treatment showed that mucous was
localized in the deep layer of the mucosa in the glands.
While in F2 rats group, little mucous secretion was
detected in the deep area of the mucosal epithelium.
DISCUSSION: The main target of this research was to evaluate the
healing efficacy of the two newly developed
gastroretentive drug delivery system with ranitidine
F1& F2 to meet the therapeutic needs relating to
J. Drug Res. Egypt, Vol. 35, No.1 (2014) 85
experimental pathological conditions in rats as gastric
ulcer. Also, assessed their role in augmenting the
mucosal defense against ethanol induce gastric ulcer.
Disturbances in gastric indices parameters include
ulceration; necrosis in mucosa; hemorrhage; odema in
mucosa and submucosa beside inflammatory reaction
in submucosa. As well as depletion of gastric mucus
were recorded in the present study to alcohol
administration. Ethanol–induced gastrointestinal lesion
implicates a variety of mechanisms. It has been
reported to cause disturbances in gastric secretion,
damage to the mucosa, alterations in the permeability,
gastric mucus depletion and free radical production
(Al-Howiriny et al., 2003; Vinothapooshan & Sundar,
2010; Choudhary et al., 2014 and Olaibi et al., 2014).
These may be attributed to the release of superoxide
anion and hydroperoxy free radicals during metabolism
of ethanol. In accordance, oxygen derived free radicals
has been found to be involved in the mechanism of
acute and chronic ulceration in the gastric mucosa
(Jude & Paul, 2009). Alcohol rapidly penetrates the
gastric mucosa apparently causing cell and plasma
membrane damage leading to increase intracellular
membrane permeability to sodium and water which
evident by the current histopathology finding since
odema present in all alcohol treatments. The massive
intracellular accumulation of calcium represents a
major step in the pathogenesis of gastric mucosal
injury. This leads to cell death and exfoliation in the
surface epithelium (Raju et al. 2009). In the current
study, it has been shown that pre-treatment or post-
treatment with F1 & F2 ameliorated the deleterious
effects of ethanol on ulcer index parameters with
advantage of F1. Bioadhesive dosage form (Nagahara
et al., 1998 and Wang et al., 2001) extend the
residence time of the drug in the stomach, the extended
release of the drug can maintain a higher drug
concentration in the gastric region thereby, improve the
absorption and systemic bioavailability of the drugs
that were normally poorly absorbed (Nagai & Machida,
1985) and improve the therapeutic efficacy. This may
be due to local cytoprotective enhancement by the two
formulae via their bioadhesion properties (adhering to
epithelium) and mucoadhesion properties (adhering to
mucus), since these formulations allow more
opportunity to contact the epithelium or mucus and
topically coating the injured tissue for facilitating
healing. Also, they offer significant potential for
increasing gastrointestinal tract residence time leading
to improved ranitidine bioavailability (Hemant et al.,
2010). Gastric mucus (mucin) is an important
protective factor for the gastric mucosa and consists of
a viscous, elastic, adherent and transparent gel formed
by 95% water and 5% glycoproteins that cover the
entire gastrointestinal mucosa. Moreover, mucus is
capable of acting as an antioxidant, and thus can reduce
mucosal damage mediated by oxygen free radicals
(Repetto & Llesuy, 2002). The protective properties of
the mucus barrier depend not only on the gel structure
but also on the amount or thickness of the layer
covering the mucosal surface (Penissi & Piezzi, 1999).
In the present study, decreased mucin secretion
after ethanol treatment indicating reduced ability of the
mucosal membrane to protect the mucosa from
physical damage and back diffusion of hydrogen ions.
Mucosal damage can be easily produced by the
generation of exogenous and endogenous free radicals
(Naito et al., 1995). An increase in mucus production
usually assists the healing process by protecting the
ulcer crater against irritant stomach secretions (HCl
and pepsin) thereby enhancing the rate of the
local healing process. F1 & F2 protected the gastric
mucosa from damage by increasing the mucin content
significantly as evident by the current histochemical
micrographs. The advantage of F1 may be attributed to
gastric mucus layer enhancement probably by
increasing the generation of mucosal prostaglandin E2
(Dashputre & Naikwade, 2011). Prostaglandin E2 was
known to be a strong stimulant for gastric mucus
secretion due to the presence of chitosan (Ishihara et
al., 2001), and its likely reflect an increase in the newly
secreted adherent mucus which consider to be more
important as a protective physical barrier of gastric
epithelium as supported by the current histochemical
finding. In the present study ethanol administration
revealed a significant decrease in serum total protein
levels in both experiments concurrent with significant
increase in ALT & AST enzymes activities indicating
hepatic injury (Myagmar et al., 2004). These results are
in accordance with Ahmed et al. (2002); Hornyak et al.
(2003); Nwoye, (2013) and Choudhary et al., (2014).
Improved total protein levels estimated in the current
study among F1 and F2 treatments in pre and post-
treated rats respectively than R treatment to the fact
that mucoadhesive dosage formulae increase the
absorption and systemic bioavailability of the drugs
that normally poorly absorbed as ranitidine (Hemant et
al., 2010). Also, it may be attributed to the
immunomodulatory and inflammatory properties of
ranitidine which improved by the current
mucoadhesive formulae F1 and F2 and evidenced by
the current histologic findings since the presence of
mild to moderate inflammatory cells infiltration could
motivate the immune responses. In addition, F1 & F2
had a greater protective effect than ranitidine on
hepatocytes against ethanol-induced damage and
subsequent leakage of enzymes into the circulation.
These results are in accordance with Choudhary et al.,
2014. On the other hand, a significant decrease in ALP
levels observed in ethanol treated rats was in agreement
with the study observed by Ahmed et al. (2012). Oral
administration of F1 & F2 was significantly increased
ALP level among pre- and post-treatments. Therefore,
it is possible that the differences in the effectiveness
between the two formulae may be due to the strength
and duration of adhesiveness of the developed
formulae to the mucosa or the ulcerated area with the
advantage of the local effects over the systemic one.
Conclusion: The current study throw some lights on
the role of mucoadhesive hydrogel formulations
against ethanol induced gastric ulcer in rats and
confirmed the better therapeutic action of these
formulae over the pure drug which might be a
promising drug delivery system for the ulcer treatment.
The mucoadhesive hydrogel formulations of chitosan
and Sod. CMC not only provide an excellent
J. Drug Res. Egypt, Vol. 35, No.1 (2014) 86
preventive effect in gastric ulcer models, but also
possesses a significant hepatoprotective effect. Also, it
encouraged the use of F1 formulae as protective and
therapeutic agent of ulcer since F1 was the most potent
one and formula with Sod. CMC as a therapeutic agent.
Further physiological studies needed to explore the role
of chitosan in gastric inflammation induced by
different factors.
0
10
20
30
40
50
60
70
80
90
100
%
rem
ain
ing
of
ran
itid
ine
0.5 1 2 3 4
Time(hr)
GroupI
GroupII
GroupIII
Figure (1): Remaining percentage of ranitidine in the stomachs of albino rats at different time intervals after oral
administration of F1 (group I), F2 (group II) and ranitidine solution (group III).
Table (1): Effect of different formulae on some gastric mucosal parameters as protective agents (pre-treated)
against standard ranitidine treatment.
- Results were expressed as mean±SE for each 6 rats.
-* Significance difference versus control at P < 0.05.
- Groups have the same letter mean non- significant
while groups have different letters means significant at P < 0.05.
Table (2): Effect of different formulae on some gastric mucosal parameters as therapeutic agents (post-
treatment) against standard ranitidine treatment.
- Results were expressed as mean±SE for each 6 rats.
-* Significance difference versus control at P < 0.05.
- Groups have the same letter mean non- significant
while groups have different letters means significant at P < 0.05
Treatment
Parameters
PH GV
ml
Total acidity
mEq/Lit Ulcer index
Curative ratio
C 5.56a±0.218 1.238c±0.018 25.30b±1.393 0.00a±0.00 -
U 7.48*b±0.218 3.28*b±0.102 58.00*d±2.55 2.80*c±0.20 -
R 7.60*b±0.114 2.98*ab±0.391 54.00*d±2.898 0.50*a±0.16 82.14%
F1 5.53a±0.189 2.44*a±0.178 13.00*a±1.140 0.10a±0.10 96.43%
F2 7.34*b±0.196 3.60*b±0.187 36.00*c±1.871 1.1*b±0.24 60.71%
Treatment
Parameters
PH GV
ml
Total acidity
mEq/Lit Ulcer index
Curative ratio
C 5.54c±0.220 1.244a±0.012 25.10a±1.756 0.00a±0.00 -
U 5.65c±0.022 1.320a±0.046 44.00*c ±1.696 2.20*c±0.20 -
R 4.82*b±0.107 1.320a±0.026 40.20*bc ±2.818 0.80*b±0.12 63.64%
F1 4.06*a ±0.284 1.284a±0.035 37.20*b ±2.518 0.40b±0.19 81.82%
F2 3.76*a±0.083 1.268a±0.036 46.20*c±1.855 0.50*b±0.00 77.27%
J. Drug Res. Egypt, Vol. 35, No.1 (2014) 87
Table (3): Effect of different formulae on some serum biochemical parameters as protective agents (pre-
treated) against standard ranitidine treatment .
- Results were expressed as mean±SE for each 6 rats.
-* Significance difference versus control at P < 0.05.
- Groups have the same letter mean non- significant
while groups have different letters means significant at P < 0.05.
Table (4): Effect of different formulae on some serum biochemical parameters as therapeutic agents (post-
treatment) against standard ranitidine treatment.
-Results were expressed as mean±SE for each 6 rats.
*Significance difference versus control at P < 0.05.
-Groups have the same letter mean non- significant
while groups have different letters means significant at P < 0.05.
Table (5): Severity of histopathological reaction in stomach of different treatments as protective agents(pre-
treated) . Histopathologly Alterations
-Results were expressed as mean±SE for each 5 rats.
*+++ = Sever effect; ++= Moderate effect; + Mild effect& ـــ Nil
Table (6): Severity of histopathological reaction in stomach of different treatments as therapeutic agents(pre-
treated) . Histopathologly Alterations
-Results were expressed as mean±SE for each 5 rats.
*+++ = Sever effect; ++= Moderate effect; + Mild effect& ـــ Nil
Treatment
Parameters
TP
mg/dl
ALT
U/L
AST
U/L
ALK
U/L (37°C)
TAC
mM/L
C 7.46c±0.21 20.743a±0.66 30.029b±0.43 44.382b±1.621 2.059a±0.002
U 6.86ab±0.22 26.072*c±0.39 38.399*c±0.53 22.724*a±1.856 2.129* c ±0.002
R 7.03bc±0.15 21.058ab±0.55 28.626b±0.43 49.207b±2.672 2.105b*c±0.007
F1 6.42*a±0.11 19.922a±0.54 27.067*a±0.33 54.438*c±2.735 2.079ab±0.023
F2 7.12bc±0.11 22.696b±0.73 29.427b±0.56 27.434*a±1.164 2.101bc±0.008
Treatment
Parameters
TP
mg/dl
ALT
U/L
AST
U/L
ALK
U/L (37°C)
TAC
mM/L
C 7.46c±0.21 20.743a±0.66 40.029a±0.43 44.378d±0.596 2.059b±0.001
U 6.28*a±0.19 30.704*c±0.29 55.756*c±0.49 20.616*a ±1.047 2.129*c±0.002
R 6.70*ab±0.15 23.530*b±0.63 57.416*c±0.48 28.444*b ±0.754 2.033*a±0.003
F1 7.15bc±0.12 23.673*b±0.92 45.062*b±0.45 46.558d±2.417 2.053b±0.007
F2 6.85*b±0.12 23.035b±0.61 41.461a±0.60 34.494*c ±2.954 2.075*d±0.002
Treatment Ulceration
( mucosa)
Necrosis
( mucosa)
Inflammation Reaction
(submucosa)
Haemorrhage
(mucosa& submucosa)
Degree
Of Severity
C ــــ ــــ ــــ ــــ ــــ
U ــــ ++ + + ++
R ــــ ــــ +++ ــــ ــــ
F1 ــــ ــــ ــــ ــــ ــــ
F2 ++ + + ++ ++
Treatment Ulceration
( mucosa)
Necrosis
( mucosa)
Inflammation Reaction
(submucosa)
Haemorrhage
(mucosa& submucosa)
Degree
Of Severity
C ــــ ــــ ــــ ــــ ــــ
U +++ +++ + + +++
R ــــ ــــ + ++ ــــ
F1 ــــ ــــ ++ ــــ ــــ
F2 ــــ ــــ + ــــ ــــ
J. Drug Res. Egypt, Vol. 35, No.1 (2014) 88
Photomicrograph (1) Stomach of control rats showing normal histopathological structure the mucosa(mu), submucosa
(sm), muscularis (ml) and serosa(s).H&E(A =X16); (B =X40).
Photomicrograph (2) Stomach of ulcerated rats (Pre-treated) showing necrosis (nmu) and hemorrhage (h) in mucosa
with oedema (O) and few inflammatory cells infiltration(m) in submucosa. H&E (A =X16); (B =X40)
Photomicrograph (3) Stomach of ranitidine treated rats (Pre-treated) showing inflammatory cells infiltration (m) and
congestion in blood vessel (v) of the sub mucosa. H&E (A =X16); (B =X40)
Photomicrograph (4) Stomach of formula (1) treated rats (Pre-treated) showing intact mucosa (mu) with odema(O) in
the sub mucosa. H&E (A =X16); (B =X40).
Photomicrograph (5) Stomach of formula (2) treated rats (Pre-treated) showing ulceration with haemorrhage(h) in
superficial mucosal layer (umu), lamina propria and submucosa. H&E (A =X16); (B =X40).
J. Drug Res. Egypt, Vol. 35, No.1 (2014) 89
Photomicrograph (6) Stomach of ulcerated rats (Post-treated) showing ulceration and haemorrhage (umu) in mucosa
with dilation in blood vessels (v), oedema (O) and few hemorrhage (h) in submucosa. H&E(A =X16); (B =X40).
Photomicrograph (7) Stomach of ranitidine treated rats (Post-treated) showing necrosis and desquamation of the
superficial mucosa (nmu) with inflammatory cells infiltration(m), oedema (O) and mild congestion in blood vessel (v)
in the sub mucosa. H&E(A =X16); (B =X40).
Photomicrograph (8) Stomach of formula (1) treated rats (Post-treated) showing intact mucosa (mu) with odema(O) ,
hemorrhage (h), congested blood vessel (v) and inflammatory cells infiltration(m) in sub mucosa. H&E (A =X16); (B
=X40).
Photomicrograph (9) Stomach of formula (2) treated rats (Post-treated) showing intact mucosa (mu) with odema(O)
in the sub mucosa and few inflammatory cells infiltration (m) in sub mucosa. H&E (A =X16); (B =X40).
J. Drug Res. Egypt, Vol. 35, No.1 (2014) 90
Histochemical findings:- Detection of mucous
Plate. 1: Stomach tissue of albino rat treated with different formulae using alcian blue; (Con) Stomach tissue of control
albino rat, showing mucous localized in diffuse manner all over the mucosal epithelial cells (arrow) X: 40.; (U pre-)
Ulcerated rat, showing absence of the mucus from the mucosal epithelium (arrow) X:40 . (U post-) Ulcerated rat
showing very few amount of mucus in the mucosal epithelium (arrow) X:40. (R pre-) Ranitidine treated rats, showing
very few amount of mucus in the mucosal epithelium (arrow) X:40; (R post-) Showing localization of mucus in the
epithelium of the deep mucosa (arrow), X: 40.; (F1 pre-) formula 1 treated animals showing blue color of mucus in the
desquamated mucosal epithelium as well as in the underlying intact one (arrow), X: 40.; (F1 post-) Showing localization
of mucus in the intact mucosal epithelial cells (arrow) X:40.(F2 pre-) formula 2 treated animals showing few amount of
mucus in the intact deep mucosal epithelial cells (arrow);
(F2 post-) Showing localization of mucus in the deep intact glandular mucosal structure (arrow), X: 40.
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تقييم صياغات دوائية جديدة ألحد مضادات القرح على نموذج تجريبي للقرحة
عبير مصطفى خطاب3لبنى عبد المنعم محمد حسنين, 2نشوة اسماعيل زكى, 1 الهيئة القومية للرقابة والبحوث الدوائية -شعبة الفسيولوجى -1
الهيئة القومية للرقابة والبحوث الدوائية -شعبة تقييم الهرمونات -2
الهيئة القومية للرقابة والبحوث الدوائية -شعبة الصيدالنيات -3
ى قرحة المعدة من االمراض الشائعة فى العصر الحديث ، وهى تحدث نتيجة عدم التوازن بين العصارة المعدية الهاضمة والعوامل المختلفة الت
أثير الهدام لهذه العصارة. وتعالج القرحة اما بمعادل للحموضة، او مثبط للعصارة المعدية ، او مواد تحمى االغشية تحمى جدار المعدة من الت
تزيد من فترة وجود الدواء فى المنطقة المصابة ألطول بالغشاء المخاطي المبطن للمعدة المخاطية و صياغة العقار فى أنظمة دوائية الصقة
وقد اجريت هذه الدراسة لدراسة قوه االلتصاق الحيوي و لتقييم التأثير تاحة الموضعية للعقار وزيادة فاعليتهإلامتداد ا فترة ممكنة مما يؤدى الى
لقد تم استخدام اناث الجرذان البالغة الوقائي )قبل( و العالجي )بعد( لعقار الرانيتيدين في صورة هالميات مائية الصقة علي نموذج حيواني.
جرذ لتقييم التاثير الوقائى والعالجى حيث قسمت هذة المجموعة عشوائيا 70جرذ لتقدير قوة االلتصاق( و 75جم )عدد 220-200التى تزن
خليط من الكيتوزان مع هيدروكسي بروبيل من (F1)يتركب الى تجربتين وقائية وعالجية لدراسة تأثير التحضيرات الجديدة للرانيتيدين. حيث
( علي خليط من صوديوم كربوكسي ميثيل سيليولوز مع هيدروكسي بروبيل ميثيل سيليولوز F2بينما يحتوى ) 1:9ة ميثيل سيليولوز بنسب
اعتبرت بمثابة ( 1جرذان , كل الجرعات تأخذ عن طريق الفم. المجموعة ) 6.كل تجربة بها خمس مجموعات وكل مجموعة بها 1:9بنسبة
( 3) مجم/كجم من وزن الجسم( , المجموعة 5( بمثابة مجموعة القرحة الضابطة حيث تلقت ايثانول ) 2الضابطة والمجموعة رقم )المجموعة
. وقد تم بالترتيب F2,F1 ( الصيغ الجديدة للرانيتيدين5( و )4مجم/كجم من وزن الجسم(, بينما تلقت المجموعتان )27تلقت عقار الرانيتيدين )
وقد تبين من هذه TAC و ALT و AST و حجم العصارة المعديةلهيدروجيني و الحموضة الكلية و مؤشر القرحة و االس اتقدير كل من
الهيدروجيني و الحموضة حيث انه قلل مؤشر القرحة و االس كعقار وقائي و عالجي F2 من الصياغة اكثر فاعلية F1 الدراسة ان الصياغة
مقارنة AST, ALT, TACعدية مقارنة بمجموعة االيثانول. وقد لوحظ ايضا نقصان معنوي في مستوى كال من حجم العصارة الم الكلية و
بمجموعة االيثانول في كال من التجربتين. و حدثت بعض االضطرابات في مستوي البروتين الكلي ونشاط انزيم الفوسفاتيز القاعدي. لقد تحسن
الرانيتيدين )الدواء المرجعي( حيث أكدت هذه النتائج بواسطة الدراسات اكثر من مستوى الميوسين في التحضيرات الجديدة
وهذا يدل علي انها F1. وقد وجد ان كمية الرانتيدين المتبقية في المعدة بعد اربعة ساعات هي االكثر في حالة والهستوكيميائية الهستوباثولوجية
لهم F2,F1كمية كافية من العقار وتزيد االتاحة الحيوية له. اخيرا هذه الدراسة توضح ان من افضل الصياغات التي تلتصق بجدارالمعدة وتبقي
كعالجي اكثر منه كوقائي. F2اكثر فاعلية وايضا يجب تشجيع استخدام F1تأثير عالجي افضل من الرانتيدين منفردا ولكن