ABSTRACT The science of life-Ayurveda is practiced in India since time immemorial. Besides being cheap and easily available, Ayurvedic drugs are considered as safe. Moreover, there is surge in the interest in Ayurveda due to quest of alternative medicines. In Ayurvedic system of medicine, Polyherbal formulations were frequently used to enhance the activity or counteract the toxic effect of compounds, from other plants, but may also act synergistically with other constituents from the same plants. Gastro esophageal reflux disease is a disorder of defense mechanism at the esophageal junction, caused by regurgitation of the gastric contents especially of gastric acid. The purpose of the study was to investigate the effect of Poly Herbal Formulation (PHF) on experimentally induced reflux esophagitis and gastrointestinal motility in animals. The PHF consists of seven medicinal plants namely Aegle marmelos, Elettaria cardamomum, Glycyrrhiza glabra , Citrus aurantifolia, Rosa damascena, Cissus quadrangularis and Saccharum officinarum. Based on acute toxicity study the PHF was considered as safe and 3 dose (100, 200 and 400 mg/kg) levels were employed for pharmacological evaluation. The test drugs were administered orally by suspending in 1% carboxy methyl cellulose solution. The PHF exhibited (P&lt;0.001) significant decrease in lesion index and enhance the % protection of lesion in experimentally induced reflux esophagitis at all the 3 doses in rats. In charcoal meal gastrointestinal transit test, PHF dose-dependently propelled the charcoal meal travel through the small intestines in mice. The study indicates that the PHF has protective effect against surgically induced reflux esophagitis

Duraisamy Srinivasan

S Sengottuvelu

Singaravel Sengottuvelu

Subramanian Ramasamy

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Journal of Pharmacy Research Vol.1.Issue 1. July-September 2008
Research Article
     The Effect Of Polyherbal Formulation - PHF On
   Experimentally Induced Reflux Esophagitis In Rats
                        Singaravel Sengottuvelu 1*, Duraisamy Srinivasan 2, Subramanian Ramasamy 3.
1Department of Pharmacology,Nandha College of Pharmacy, Koorapalayam Pirivu, Perundurai Main Road, Erode-
638052. Tamilnadu, India.
Department of Pharmacology,Rajah Muthiah Dental College & Hospital,Annamalai University,
Chidambaram,Tamilnadu, India.
3Department of Pharmacology,Arupadai Veedu Medical College,Krimampakkam Pondicherry,Tamilnadu, India.
For correspondence:S.Sengottuvelu.,Department of Pharmacology,Nandha College of  Pharmacy,Perundurai
main road,Erode-638052,Tamilnadu.
E-mail: sengt@rediffmail.com
Received on: 02-05-2008; Accepted on :14-07-2008
ABSTRACT
The science of life—Ayurveda is practiced in India since time immemorial. Besides being cheap and easily
available, Ayurvedic drugs are considered as safe. Moreover, there is surge in the interest in Ayurveda due to quest
of alternative medicines. In Ayurvedic system of medicine, Polyherbal formulations were frequently used to enhance
the activity or counteract the toxic effect of compounds, from other plants, but may also act synergistically with other
constituents from the same plants. Gastro esophageal reflux disease is a disorder of defense mechanism at the
esophageal junction, caused by regurgitation of the gastric contents especially of gastric acid. The purpose of the
study was to investigate the effect of Poly Herbal Formulation (PHF) on experimentally induced reflux esophagitis
and gastrointestinal motility in animals. The PHF consists of seven medicinal plants namely Aegle marmelos, Elettaria
cardamomum, Glycyrrhiza glabra , Citrus aurantifolia, Rosa damascena, Cissus quadrangularis and Saccharum
officinarum. Based on acute toxicity study the PHF was considered as safe and 3 dose (100, 200 and 400 mg/kg)
levels were employed for pharmacological evaluation. The test drugs were administered orally by suspending in 1%
carboxy methyl cellulose solution.  The PHF exhibited (P<0.001) significant decrease in lesion index and enhance the
% protection of lesion in experimentally induced reflux esophagitis at all the 3 doses in rats. In charcoal meal
gastrointestinal transit test, PHF dose-dependently propelled the charcoal meal travel through the small intestines in
mice. The study indicates that the PHF has protective effect against surgically induced reflux esophagitis.
Keywords: Polyherbal formulation, esophagitis, gastrointestinal motility and Charcoal meal
INTRODUCTION
Gastro esophageal reflux disease (GERD) is a
disorder of defense mechanism at the esophageal
junction, caused by regurgitation of the gastric contents,
especially of gastric acid. GERD is associated with
decreased gastric emptying and/or increased incidence
of transient lower esophageal relaxation[1]. If left
untreated, erosion and ulceration of the esophageal
mucosa by gastric acid, chronic esophagitis, aspiration
pneumonia, esophageal strictures and Barrett’s
esophagus (a premalignant condition) may result [2].
Besides that, disturbed gastrointestinal motility and
injurious effects of the acid-peptic refluxate on the
esophageal epithelium are also responsible for GERD
symptoms. Hence along with prokinetic drugs,
suppression of gastric acid is the current
pharmacotherapeutic approach for its treatment [3].
There are many drugs used for the treatment of
acid peptic disorder, such as antacids, proton pump
inhibitors or antihistaminic agents and gastroprokinetic
agent such as mosapride or metoclopramide, but most
of these drugs produce several adverse reactions. Thus,
there is a need of more effective and less toxic agents
for this ailment. Plants are some of the most attractive
sources, and have been shown to produce promising
results for the treatment of various GIT disorders. Even
though single herbal is effective in the treatment of human
ailments, but drugs with multiple mechanisms of
protective action may be one way forward in minimizing
tissue injury in human disease [4].
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Journal of Pharmacy Research Vol.1.Issue 1. July-September 2008
PHF used in this study consists of seven medicinal
plants namely Aegle marmelos, Elettaria cardamomum,
Glycyrrhiza glabra, Citrus aurantifolia , Rosa
damascena, Cissus quadrangularis and Saccharum
officinarum. The indigenous plants used in the formulation
have been recognized to treat various gastrointestinal
illnesses. Warrier et al mentioned the uses of plants listed
in PHF in various gastrointestinal ailments, which was
given in Table I. Based on previous reports, the present
study was aimed to investigate the effect of PHF on
experimentally induced reflux oesopagitis as well as
gastrointestinal motility in rats[5].
MATERIALS AND METHODS
Plant material
The plants Aegle marmelos, Elettaria
cardamomum, Citrus aurantifolia, Rosa damascena,
Cissus quadrangularis and Saccharum officinarum
were collected locally during the month of December
and Glycyrrhiza glabra, obtained from the Horticulture
Research Station, Kodaikanal, Tamilnadu. Prof. R.
Duraisamy, Botanist., authenticated the botanical identity
of the plants and voucher specimen (NCP/Phcog/2008/
0201) has been retained, for future reference in the
herbarium of Pharmacognosy department, Nandha
College of Pharmacy, Erode, India.
Formulation development
The collected plant materials were shade dried
separately and pulverized to get a fine powder. The fine
powders were passed through the sieve number 100.
The graded powders were weighed individually and
mixed to give the specified composition. This herbal
formulation was stored in air tight container and used
for pharmacological studies. The PHF was administered
orally to the animals by suspending in 1 % carboxy methyl
cellulose solution.
Composition
Each gram of PHF contains powders of Aegle
marmelos Corr. (Rutaceae; fruit, 150 mg), Elettaria
cardamomum Maton. (Zingeberaceae; seeds, 125 mg),
Glycyrrhiza glabra L. (Papilionaceae; root, 150 mg),
Citrus aurantifolia Swingle. (Rutaceae; Fruits, 150
mg), Rosa damascena Mill. (Rosaceae; flower petals,
150 mg), Cissus quadrangularis Linn. ( Vitaceae;
Whole Plant, 150 mg) and Saccharum officinarum
Linn (Poaceae; root, 125 mg).
Animals
Male Swiss albino mice weighing between 20 –
25 gm and male Wistar rats weighing between 150 –
220 gm were used for this study. The animals were
obtained from animal house, IRT Perundurai Medical
College, Erode, Tamilnadu, India. On arrival, the animals
were placed at random and allocated to treatment groups
in polypropylene cages with paddy husk as bedding.
Animals were housed at a temperature of 24±2oC and
relative humidity of 30 – 70 %. A 12:12 light: day cycle
was followed. All animals were allowed to free access
to water and fed with standard commercial pelleted rat
chaw (M/s. Hindustan Lever Ltd, Mumbai). All the
experimental procedures and protocols used in this study
were reviewed by the Institutional Animal Ethics
Committee (688/2/C-CPCSEA) and were in
accordance with the Institutional animal ethical guidelines.
Acute toxicity study
Acute toxicity studies were performed
according to OECD-423 guidelines [6]. Male Swiss
albino mice, selected by random sampling technique,
were employed in this study. The animals were fasted
for 4h with free access to water only. PHF was
administered orally at a dose of 5 mg/kg initially and
mortality if any was observed for 3 days. If mortality
observed in two out of three animals, then the dose
administered was considered as toxic dose. However,
if the mortality observed in only one animal out of three
animals then the same dose was repeated again to
confirm the toxic effect. If no mortality was observed,
then higher (50, 300, 2000 mg/kg) doses of PHF were
employed for further toxicity studies. During the study,
the animals were closely observed for the first 12 hours
for any toxic symptoms and 72 hours for mortality rate.
Evaluation of reflux esophagitis
The animals were divided into five groups each
consisting of six rats. Group 1, represented control group
of animals, received suspension of 1% carboxy methyl
cellulose (CMC) in distilled water. Group 2 received
Omeprazole (10 mg/kg).  Groups 3–5 received PHF in
doses of 100, 200and 400 mg/kg. The drugs were
administered for three days, orally by suspending in 1%
CMC solution. On day 3, after the last dose, the rats
were kept for 12 hour fasting and care was taken to
avoid coprophagy. Under ether anesthesia, the abdomen
Singaravel et al : The Effect Of Polyherbal Formulation - PHF On Experimentally Induced Reflux Esophagitis In Rats
12
Journal of Pharmacy Research Vol.1.Issue 1. July-September 2008
was incised along the midline and both the pylorus and
limiting ridge (transitional region between the forestomach
and corpus) were then ligated simultaneously [7]. An
approximately 1 cm long longitudinal cardiomyotomy
across the gastroesophageal junction was performed to
enhance reflux. Six hours later, the rats were sacrificed
by a cervical dislocation, and the esophagus was
harvested. The total area of the lesion that had developed
in the esophagus was determined using a magnifying glass
and the diameter of the lesion was measured using a
vernier caliper.  Lesion index was determined by
following the scoring method of Suzuki et al [8].
Score 1: maximal diameter of 1 mm.
Score 2: maximal diameter of 1–2 mm.
Score 3: maximal diameter of 2–3 mm.
Score 4: maximal diameter of 3–4 mm.
Score 5: maximal diameter of 4–5 mm.
Score 10: an ulcer over 5mm in diameter.
Score 25: a perforated lesion.
Charcoal meal gastrointestinal transit test
Charcoal meal test was used to evaluate the
gastrointestinal motility of PHF [9]. Mice were divided
into five groups of six mice each and fasted for 24 h
before the experiment.  Group 1 served as control with
suspension of 1% CMC in distilled water (10 ml/kg),
group 2 was administered carbachol (1 mg/kg), a
standard cholinergic agent, as the positive control.
Groups 3 - 5 were then treated orally with three
increasing doses of the PHF 100, 200and 400 mg/kg.
The drugs were administered by suspending in 1%
Carboxy methyl cellulose solution. After 15 min, the
animals were given with 0.3 ml of freshly prepared
charcoal meal (distilled water suspension containing 10%
gum acacia, 10% vegetable charcoal and 20% starch).
Following 30 min of charcoal administration, the mice
were sacrificed by cervical dislocation and the abdomens
immediately opened to excise the whole small intestine
(pylorus region to cecum). The length of the small intestine
and the distance between the pylorus region and the
front of the charcoal meal was measured for obtaining
the charcoal transport ratio or percentage.
Statistical analysis
 The values were expressed as mean ± SEM.
The statistical analysis was carried out by one way
analysis of variance (ANOVA) followed by Dunnet’s
‘t’ - test. P values <0.05 were considered significant.
RESULT
All the doses (5, 50, 300, 2000 mg/kg) of PHF
employed for acute oral toxicity studies were found to
be non-toxic. PHF did not produce any mortality even
at the highest dose (2000 mg/kg) employed. Three sub
maximal doses (100, 200 and 400 mg/kg) which was
found to be safe were employed for further
pharmacological investigations.
Reflux esophagitis
The effect of PHF on surgically induced
13
Singaravel et al : The Effect Of Polyherbal Formulation - PHF On Experimentally Induced Reflux Esophagitis In Rats
Table-I. Shows the Ethnobotanicol uses of plants present in Polyherbal formulation
Plant name and Family Ethnobotanicol Uses
Aegle marmelos Corr. Dyspepsia, Stomachalgia, Gastric irritability, Digestive,
Rutaceae Laxative
Elettaria cardamomum Maton. Carminative, Digestive, Stomachic, Dyspepsia, Gastropathy,
Zingeberaceae Hyperdipsia
Glycyrrhiza glabra  L. Laxative, Hyperdipsia,   Gastralgia, Gastric ulcers
 Papilionaceae
Citrus aurantifolia Swingle. Laxative, Appetiser, Stomachic, Digestive, Anthelmintic,
Rutaceae Dyspepsia, Flatulance, Helmenthiasis,
Saccharum officinarum  Linn. Laxative,  Dipsia, Gastropathy,
Poaceae
Cissus Quadrangularis Linn. Laxative,  Anthelmintic, Carminative, Digestive,
Vitaceae Stomachic,  Helminthiasis, Anorexia, Dyspepsia,
Flatulance, Chronic ulcers, Haemorrhoids,
Rosa damascena Mill. Laxative
Rosaceae
Journal of Pharmacy Research Vol.1.Issue 1. July-September 2008
Figure I. Shows the effect of PHF the charcoal meal
transit in mice.
***P<0.001 Vs control; **P<0.01 Vs control; *P<0.05
Vs control
0
10
20
30
40
50
60
70
80
90
100
1
%
 In
te
st
in
al
 T
ra
ns
it
 Normal Control Positive Control PHF 100mg/kg PHF 200mg/kg PHF 400mg/kg
***
***
***
esophagitis was studied in rats. The effect of PHF on
lesion index and % protection against surgically induced
esophagitis is shown in table II. The PHF showed dose
dependent decrease in lesion index. The PHF showed
significant protective effect at the doses of 100, 200
and 400 mg/kg body weight. The percentage protection
of lesion was 33.29 % (P< 0.01), 78.47% and 82.35%
(P< 0.001) by PHF at the doses of 100, 200 and 400
mg/kg respectively; the percentage protection of lesion
by Omeprazole, the standard antiulcer agent was
Table-II: Protective effect of poly herbal formulation on
experimentally induced Reflux esophagitis in rats
Values are presented as mean ± SEM (n = 6)
***P<0.001 Vs control; **P<0.01 Vs control; *P<0.05 Vs control
84.35% (P< 0.001) at the dose of 10mg/kg.
Charcoal meal gastrointestinal transit test
Figure I. Shows the activity of the PHF to
influence gastrointestinal transit of the charcoal meal in
mice. It was determined by its effect on the traverse of
charcoal meal through the length of the small gut of mice.
PHF dose-dependently propelled the charcoal meal
travel through the small intestines. The distance traveled
by the solvent control was 14.93±1.89 %. The PHF at
the dose of 100 mg/kg moved the charcoal meal to
20.97±2.99 %,   while 50.76±4.56% and 78.95±1.66%
(P< 0.001) was caused with the next higher doses (200
mg/kg and 400 mg/kg respectively). Carbachol (1 mg/
kg) was used as positive control which moved the meal
to 83.85±2.66% (P< 0.001).
DISCUSSION
Gastro-esophageal reflux disease is a common
condition with a complex pathophysiology. Despite the
wide range of abnormalities, since gastric acid plays a
key role in mucosal damage, the suppression of gastric
acid secretion is the basis of many treatments [10].
Additionally, promotility agents increase lower
esophageal sphincter pressure and enhance lower
esophageal peristalsis, both actions are specifically target
the pathology of GERD. PHF, a Polyherbal formulation,
consist of plants that are mentioned in Indian system of
medicine (Ayurveda) for their curative properties. The
anti-ulcer activity of PHF has been proved against
14
Singaravel et al : The Effect Of Polyherbal Formulation - PHF On Experimentally Induced Reflux Esophagitis In Rats
Drug treatment Ulcer Index % Protection
Control
(1% Carboxy 8.50 ± 0.43 —
methyl cellulose)
Omeprazole
(10 mg /kg) 1.33 ± 0.21*** 84.35
PHF 100 5.67 ± 0.56*** 33.29
PHF 200 1.83 ± 0.31*** 78.47
PHF 400 1.50 ± 0.22*** 82.35
Journal of Pharmacy Research Vol.1.Issue 1. July-September 2008
different experimental gastric and duodenal ulcers [11].
It appears to strengthen the gastric mucosal resistance
and scavenging the free radical generation. The results
showed that the protective effect of PHF against
surgically induced reflux esophagitis in rats may be due
to the inhibition of gastric secretion and the prevention
of oxidative stress.
Gastrointestinal prokinetics promote the
coordination of the gut wall contractions leading to
enhancement of propulsive motility and consequently
caudal displacement of luminal contents. Currently, they
are considered as drugs of choice for the treatment of
upper gastrointestinal tract functional and motor disorders
such as those associated with gastro oesophageal reflux
disease, chronic dyspepsia and acute or chronic
idiopathic intestinal pseudo obstruction [12]. Charcoal
meal gastrointestinal transit test was adopted for
prokinetics activity of PHF in rats. Gastrointestinal transit
was enhanced by PHF at all the three doses employed.
The enhanced gastrointestinal transit by the PHF may
be mediated through increased of prostaglandin
synthesis. Prostaglandin was shown to contract the
intestinal longitudinal muscle and relax the circular muscle
in vitro, acting seemingly at pre and postganglionic sites
in the intramural plexus [13]. The increase production
of prostaglandin by PHF was already described by its
anti-ulcer activity against indomethacin induced ulcer in
rats [11].
The study indicates that the PHF has protective effect
against surgically induced reflux esophagitis. It may be
due to its gastro protective, anti-oxidant and prokinetics
activity. However, further study is necessary to pinpoint
the mechanism of  reflux esophagitis of PHF and to
identify the active constituents responsible for its activity.
ACKNOWLEDGEMENTS
Authors are thankful to Shri.V.Shanmugan,
Chairman, Nandha College of pharmacy, Erode, India
for providing infrastructural facilities to carry out this
project.
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Singaravel et al : The Effect Of Polyherbal Formulation - PHF On Experimentally Induced Reflux Esophagitis In Rats


The Effect Of Polyherbal Formulation -PHF On Experimentally Induced Reflux Esophagitis In Rats

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The Effect Of Polyherbal Formulation -PHF On Experimentally Induced Reflux Esophagitis In Rats

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