CHEMICAL, BIOLOGICAL AND COMPARATIVE CLINICAL …
Transcript of CHEMICAL, BIOLOGICAL AND COMPARATIVE CLINICAL …
CHEMICAL, BIOLOGICAL AND COMPARATIVE CLINICAL
EVALUATION OF ENTOBAN TO DETERMINE SAFETY AND
EFFICACY FOR THE TREATMENT OF CHRONIC DIARRHEA
Thesis submitted in the partial fulfillment of degree of
Doctor of Philosophy (PhD) in
Pharmacy Practice
By:
Sadia Jamil, R.Ph.
B.Pharm, M.Pharm. (Karachi University), CRCP (DUHS)
Under the Supervision of
Supervisor: Prof.Dr.Usman Ghani Khan, M.Pharm, Ph.D.
Co-Supervisor: Dr.Somia Gul, Ph.D.
2016
Faculty of Pharmacy, Jinnah University for Women, Karachi
Dedicated
to my
Beloved Mother, Father, Husband
&
Son Mohammed Zohad Ali
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ACKNOWLEDGEMENTS
It is with all praise to almighty ALLAH, the creator of the universe, the most
beneficent and merciful and his Prophet Muhammad peace be upon him, the guide and
Rehmat-ul-Alamin from whom I take guidance to lead my life. It is ALLAH mercy that I
ventured to take up study in pharmacy and now availing the opportunity to write this
thesis for presentation.
There is no limit of learning knowledge, not withstanding of my present age only
my respected supervisor Prof. Dr. Usman Ghani Khan inculcate my chivalry with
rejuvenated zeal to perform this task by his in valuable guidance, deep interest, coaching,
sustained interest, orientation, advices, meticulous care in experimental work, pragmatic
suggestions, stimulating discussion, critism and he made me so to undertake the
strenuous research work like young student. I pay my gratitude to my supervisor, as
without his compassion, it was hard for me to accomplish the job. I am most thankful to
Dr. Somia Gul, Associate professor, Jinnah University for women and my co supervisor
for her kind cooperation and assistance for conducting the research.
My sincere thanks also go to the family of Jinnah University for women with
many good names who offered a helping hand in formulating necessary cooperation and
information regarding my thesis. My special thanks to Dr. Ghulam Server (Dean, Faculty
of Pharmacy), Dr. Safila Naveed and my colleagues both in Jinnah University for women
and Dow university of Health Sciences Karachi.
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I am most thankful to Mr. Nadeem Khalid, Mr. Zeeshan Ahmed Sheikh, Dr. Aqib
Zahoor and Dr. Saleha Suleman Khan of Herbion Pakistan (Pvt.) Ltd., for their kind
cooperation and assisting me to conduct some part of research in Herbion Pakistan (Pvt.)
Ltd., Karachi, Pakistan.
I am highly grateful to Dr. Hafiz Muhammad Asif (Department of Eastern Medicine &
Surgery, Faculty of Medical & Health Sciences, The University of Poonch, Rawalakot,
Azad Jammu & Kashmir Pakistan) , for his cooperation and directions in the clinical trial.
I may not forget my colleagues namely Dr. Najia Rahim, (Incharge Department of
pharmacy practice, Dow university of Health Sciences) and Wajiha Iffat whose blessings
have been a great source of my vitality in my profession all around.
At the outset I would like to pay gratitude to my parents particularly to beloved
mother who strongly stood with me, for my schooling in her own direct supervision. I
may not be doing justice if I missed the names of my family members specially my
father, husband and my son who offered support and care to concentrate on my research
work.
Sadia Jamil
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ABSTRACT:
Diarrhea is the third most frequent disease that affects people of all ages. In spite of the drop in
global mortality rate, diarrhea still accounts for more than 2 million deaths per annum. About
two-thirds of the total annual deaths in Pakistan of children under five are due to diarrhea.
Different drugs are prescribed to treat the symptoms of chronic diarrhea whereas an empirical
mode of treatment with antibiotics considered viable when the infection is elevated in the
community. However, the resistance of antibiotic is responsible as the main factor for treatment
failure. The adverse effects, inadequate accessibility of allopathic medicines and antibiotic
resistance have led to the resurgence of plant based drugs as an alternate treatment option.
Traditional herbal medicines have now been proven to be safe and effective and being utilized to
cure many disorders, including GI ailments. Herbal dosage forms have been shown to heal acute
as well as chronic diarrheal diseases.
In current study, standardized coded polyherbal mixture was formulated in hard gelatin capsule
and syrup. Various physicochemical parameters including physical appearance, weight
variation and disintegration time were calculated for the capsule. Average weight of 20
capsules was between 450 mg and 550 mg (with a mean of 506 mg ± 10%). The maximum time
for disintegration was 6 min. It was found that alkaloids and tanning agents in Entoban syrup
and capsules were within the specified limits. The different physicochemical parameters of
Entoban syrup were assessed. Entoban syrup revealed brown color, characteristic odor and
sweet taste. The pH of Entoban syrup was 3.7 and specific gravity was 1.324. Entoban capsules
and syrup were in agreement with the acceptable microbial limit. The prospective validation
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was executed to validate the manufacturing process of Entoban syrup and to make sure that it
fulfills the predetermined specifications. To execute prospective process validation, critical
process parameters were recognized, the protocol and reports were developed. Three
consecutive batches of Entoban formulations were analyzed to reassure reproducibility of the
results. It was found that the manufacturing process of syrup was reproducible for these batches
and every parameter analyzed was in accordance with the specifications and validated
according to the guiding principles stated in prospective process validation.
An antimicrobial activity was evaluated against five gram negative bacterial cultures namely
Salmonella enteric, Eschericia coli, Shigella dysenteriae, Pseudomonas aeruginosa, Vibrio
cholera and one gram positive bacterial culture Staphylococcus aureus by agar well diffusion
method. The prepared Entoban formulation inhibited the growth of these organisms. The
stability study on Entoban syrup demonstrated no changes in all the tested physicochemical
parameters during 24 hours, 48 hours and 72 hours.
Entoban syrup and capsules have outstanding antioxidant ability with 8.5 and 10.3 μg/ml IC50
values respectively. The reducing ability of Entoban syrup and capsules increased in a dose
dependent manner. It can be inferred that antioxidant activity could be helpful in slowing down
the development of a variety of diseases of gastro intestinal tract associated with oxidative stress.
Anti-inflammatory and anti-urease activities were determined on Entoban dosage form design to
overcome H. pylori-associated inflammation. Anti-H.pylori and cell cytotoxic activity of
Entoban syrup and capsule formulations was conducted by serial dilution method and cell
survival assay, respectively. Anti-adhesion activity of Entoban was then evaluated. Entoban did
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not demonstrate anti-adhesion outcome against the cell co-culture of H. pylori. Additionally,
Entoban syrup formulation suppressed H. pylori-induced IL-8 more as compared to capsule
formulation. Entoban syrup and capsules revealed antiurease activity increased in a dose
dependent way just like standard (Thiourea) using the indophenol method. The formulations
have an excellent antiurease potential that can be used in the cure of different problems occurring
due to urease enzymes. The Lipoxygenase inhibition activity of polyherbal formulation syrup
and capsules increased in a dose dependent manner and revealed that formulations under test
have good potential of lipoxygenase inhibition.
The quantization of biomarkers gallic acid and berberine was explored in polyherbal formulation
Entoban capsule and syrup. HPTLC was performed to evaluate the presence of gallic acid and
berberine applying toulene–ethyl acetate–formic acid–methanol in ratio of 12:9:4:0.5 v/v and
ethanol–water–formic acid in ratio of 90:9:1 v/v, as the mobile phase, respectively. The present
standardization provides specific and accurate tool to develop qualifications for identity,
transparency and reproducibility of biomarkers in Entoban formulations.
Entoban medicinal plant syrup was analyzed for As, Cd, Pb and Hg by flame atomic absorption
spectroscopy (FAAS). Contents of heavy metals in the examined samples were in the range: As
(0.074–10.0 ppm); Cd (0.020–0.3 ppm); Pb (0.00–10.0 ppm) and Hg (0.00–1.0 ppm). Results
were compared with permissible limit acceptability intake (AHPA). According to determined
amounts of heavy metals, the investigated Entoban syrup samples were validated and considered
safe for human consumption.
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In order to investigate the antidiarrheal activity albino mice were treated with Entoban at dosages
of 2.5, 5, 10 mg/kg. For evaluation of acute toxicity the animals were administered orally with 1
or 5 g/kg of the Entoban capsule aqueous extract, maintained under standard laboratory
conditions. Entoban was given in quantity of 50 mg/kg, 100 mg/kg and 200 mg/kg body weight
for a period of 28 days for determining sub chronic oral toxicity. The data collected were
summarized as mean ± SEM. Entoban showed significant inhibition of diarrhea in dose
dependent manner. Entoban was not found to be the reason of death in albino mice at the
specified doses of 1 g/kg or 5 g/kg. Toxicity indications including the loss of hair, mucus
membrane (nasal), loss in weight, lacrimation, drowsiness, gait and tremors were also not
observed. The study gave evidence of good tolerance of Entoban and the absence of detrimental
effects on functional state of the vital organs of experimental animals in acute and sub chronic
oral toxicity test.
For the evaluation of the clinical safety and efficacy of Entoban for treating patients of chronic
diarrhea, a controlled, randomized, multicenter clinical trial was conducted in Sharafi Goth
hospital Korangi Karachi, Nawaz Salik Hospital in Rawalpindi and Victoria Hospital in
Bahawalpur. The current trial enrolled 150 patients fulfilling the inclusion criteria, among them
95 were males and 55 were females. Among the total enrolled patients; 10 patients belonging to
the test group and 7 of the control group did not receive the allocated treatment due to unknown
reasons. Further 13 were dropped out during the treatment and 8 discontinued intervention due to
side effects in control group. In test group, 15 were dropped out during the treatment and 4
discontinued intervention due to side effects. Overall 47 and 46 in control and test group
completed the study. The trial was registered at http://www.ClinicalTrial.org, a service of the US
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National Institutes of Health (registry No. NCT02642250). A block-randomization procedure,
with a block size of 4, was adopted to assign participants either to treatment with allopathic
therapy or with a phytomedicine-based formulation. Metronidazole tablets (Flagyl) in strength of
400 mg manufactured by Sanofi-aventis Pakistan limited was used in a control group for 7-10
days. The test group received Entoban capsule 400mg tds, every 8 hours for five days. The stool
frequency was documented quantitatively, and semiquantitative factors including consistency of
stool, abdominal pain, distention and incomplete evacuation were noted. Stool DR was noted at
baseline and thereafter 2nd
and 4th
weeks of treatment. Adverse reactions were evaluated by
patient history and physical assessment on daily basis every 3 days until the completion of study.
The quantitative evaluation of daily bowel frequency was the primary outcome of the study and
evaluation of clinical symptoms including consistency of stool, distention, abdominal pain and
feeling of incomplete evacuation were the secondary outcome. Patients’ characteristic data was
demonstrated as the mean ± standard deviation (SD). A χ2 test using a 2 × 2 contingency table
was used to check for a statistically significant difference in the cure rate as well as in the
proportions of other categorical variables between 2 treatment groups. A Wilcoxon signed-rank
test was applied to analyze the intensity of symptoms at baseline (T0), after 2 weeks (T2) and 4
(T4) weeks of treatment, expressed through median values and interquartile ranges (IQRs) (p <
0.05 was considered significant). It has been found in current study that 39(84.78%) in test group
and 37(78.72%) in control group showed complete improvement who completed the study.
Participants in the test group exhibited a marked reduction in symptoms; the symptom score was
decreased from 3 (maximum) to 1 (minimum) or 0 (absent) in most of participants. Participants
in the test group with complete improvement exhibited significant decreases in overall GI
symptoms from baseline (T0)—with a median of 8 and an IQR of 6 to 10, to week 2 (T2)—with a
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median of 3 and an IQR of 2 to 5, and to 1 month after treatment (T4)—with a median of 4 and
an IQR of 3 to 6. There was a significant decrease in symptoms was observed for participants in
the test group with no improvement, also from T0—with a median of 9 and an IQR of 6 to 10, to
T2—with a median of 3 and an IQR of 2 to 5, and to T4—with a median of 4 and an IQR of 3 to
6. The intensity of individual symptoms in the test group was monitored and statistically
significant improvement was recorded after treatment. Participants in control group with
improvement exhibited a statistically significant reduction in the overall diarrheal symptom
score, from T0—with a median of 9 and an IQR of 6 to 10, to T2—with a median of 4 and an
IQR of 3 to 6, and toT4—with a median of 4 and an IQR of 3 to 7. No significant improvement
in symptoms was observed, however, for the participants with no recovery, showing scores from
T0—a median of 9 and an IQR of 6 to 10, to T2—a median of 6 and an IQR of 4 to 8, and to T4—
a median of 8.5 and an IQR of 5 to 10. Patients in control group reported more side effects as
compared to test (p value < 0.0001). Around 20% patient reported adverse effects in test group
however in control group 55.31% reported adverse effects. The major adverse effects reported in
control group were anorexia (14.89%), metallic taste (10.63%), dizziness (8.51%) and vomiting
(4.25%). Among test group the major adverse effects reported were metallic taste (6.52%),
anorexia and headache (4.34%).
Entoban possesses considerable therapeutic efficacy for the treatment of chronic diarrhea and it
is comparable with the standard conventional Metronidazole therapy. Entoban revealed high cure
rates of chronic diarrhea with little or no side effects as compared to Metronidazole. Furthermore
Entoban improves the well-being off over all sign and symptoms of diarrhea and has better
compliance.
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Entoban, also exhibits strong anti-inflammatory activity against inflammation in gastric
epithelial cells induced by H. pylori. Single poly herbal drug formulation with two modes of
action against H. pylori can act as a double bladed sword ensuring complete suppression of H.
pylori and its associated inflammation. Herbal drugs like Entoban are an excellent candidate for
future in vivo and clinical studies, which are required in order to establish its definitive role as
chemotherapeutic agent against H. pylori-induced gastric disease.
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1.1 CHRONIC DIARRHEA:
Chronic diarrhea can be defined as ―an intestinal disorder characterized by an abnormal
frequency and liquidity of fecal evacuations that lasts for more than 4 weeks is considered
persistent or chronic‖(1). Stool weight has been quoted often as a practical approach to define
diarrhea; however diarrhea not supposed to be defined simply in expressions of fecal weight.
Several persons have increased fecal weight although have normal consistency of stool and did
not complaint of diarrhea. Some have typical fecal weight and complaint of diarrhea for the
reason that their stools are thin or watery(2).
1.2 PATHOPHYSIOLOGY OF CHRONIC DIARRHEA:
Chronic diarrhea has several causes including motility, osmotic, secretory, iatrogenic, and
inflammatory. In common, no single root of chronic diarrhea is in fact unifactorial from a view
point of pathophysiology. During the past three decades, it was revealed that a number of ion-
transport systems may be affected in diarrheal disorders.
Diarrhea disorder is stimulation of the secretion of fluid and electrolytes in one or more
fragments of the small intestine or colon, or sometimes both. In secretory diarrhea, secretagogues
affect ion transport in the intestine along with chloride secretion by activating transmembrane
regulator and means to retain sodium and chloride absorption (Figure 1). In steatorrhea induction
of fluid and electrolyte secretion in the intestine through unabsorbed fatty acids causes diarrhea
(3). (Table 1)
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In developed part of the world, the most common diagnosis made in sufferers of chronic diarrhea
include malabsorption syndrome, irritable bowel syndrome (IBS), chronic infections, idiopathic
inflammatory bowel disease and idiopathic secretory diarrhea(4, 5). Whereas bacterial and
protozoal infections are the main commonly observed sources of persistent diarrhea in less
developed countries; however inflammatory bowel disease, functional disorders, and
malabsorption (due to diversity of unspecified causes) are also frequent in this setting(6).
Figure 1: Regulation of Absorptive and Secretory Processes in the Intestine.
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Table 1: Causes of Chronic Diarrhea According to Predominant Pathophysiological
Mechanism
INFLAMMATORY CAUSES
Idiopathic inflammatory bowel disease
Infections
Radiation injury
Gastrointestinal malignancies
Immune related mucosal disease (primary and secondary immunodeficiency,
food allergy)
STEATORRHEAL CAUSES
Intraluminal maldigestion (pancreatic exocrine insufficiency, bacterial
overgrowth)
Mucosal malabsorption (celiac sprue,Whipple's disease)
Post mucosal obstruction (lymphatic obstruction)
SECRETORY CAUSES
Laxative abuse
Chronic ethanol ingestion
Bowel resection, disease or fistula
Partial bowel obstruction
Diabetic autonomic neuropathy
Hormone producing tumors
Addison disease
OSMOTIC CAUSES
Osmotic laxatives
Lactase and other disaccharides deficiencies
DYSMOTILITY
Irritable bowel syndrome
Drugs (prokinetics)
Hyperthyroidism
1.3 ETIOLOGY OF DIARRHEA:
There are wide and varied causes of diarrhea; amongst them the deprived sanitary conditions and
low socio-economic statuses are of major concern. Infectious diarrhea, the most widespread form
of diarrhea globally may perhaps be caused as a result of viral, bacterial or protozoal
contamination. Rotavirus, EntericAdenovirus, Norovirus, Caliciviruses, Enteroviruses and
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Astroviruses are the exemplar of viruses that causes diarrhea. Infection due to Rotavirus
particularly in children is accountable for diarrhea, and may be the reason of 40% and 25% of
diarrheal cases in developed and developing countries respectively(7).
Figure 2: Rotavirus A
Figure 3: Norwalk virus
Infectivity due to bacterial causes for instance; 25% of diarrheal cases are due to enterotoxigenic
Escherichia coli (ETEC) and 18% due to Campylobacter jejuni in the developing countries (8).
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Further bacterial contributory agents are non-typhoidal species of Salmonella, Shigella species,
Vibrio cholerae and Salmonella typhi. Giardia lamblia, Cryptosporidium parvum and
Entamoeba histolytica are the protozoa that have also been documented as severe causes of
diarrhea in developing world.
Figure 4: Escherichia coli (ETEC)
Figure 5: Clostridium difficile
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Figure 6: Entamoeba Histolytica
Figure 7: Campylobacter jejuni
Figure 8: Giardia lamblia
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Figure 9: Vibrio cholera
Figure 10: Cryptosporidium parvum
A recent study from Ghana reported Giardia lamblia as the key source of childhood diarrhea
having a pervasiveness of 89.5% (9). Other researchers have accounted the role of Entamoeba
histolytica and Cryptosporidium parvum as most important sources of diarrhea (10).
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Figure 11: Shigella dysenteriae
Figure 12: Salmonella enteric
Different drugs, toxins and sometimes food allergens are accountable for non-infectious diarrhea.
Extended utilization of antibiotics ensuing in the disturbance of gut microflora may perhaps be a
source of diarrhea and sometimes pseudomembranous colitis ensuing from Clostridium difficile
infection. On the other hand, the incidence of diarrhea may sometimes also be investigative of
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clinical circumstances other than gastrointestinal tract. Antacids and other magnesium containing
drugs can affect the digestion or absorption process. Drug-related diarrhea is generally frequent
in the elderly. Diarrhea typically occur in case if the absorptive capability of the intestine exceed
and overall secretion is more than absorption leading to an imbalance among absorption and
secretion (11). Minimum variation in typical electrolyte balance and intestinal fluid may
consequence in diarrhea. This reaction is defensive for severe gut irritations however turn out to
be a problem when persistently present and not serve up as a physiological function. Greater
changes in thickness and volume of stool may owe to failure in ionic balance regulation,
dissimilarities in fluid assimilation and secretion. Unnecessary electrolytes loss, nutrients and
fluids loss owed to diarrhea may consequence in dehydration, malnutrition, acidosis and
haemolytic uremic syndrome. In geriatric population, imperfect physiological reserves and co-
morbidities raise the frequency and severity of complications associated with diarrhea including
electrolyte loss and dehydration, identified to be accountable for increase span of patient
hospitalizations and fatalities (11).
An osmotic pressure may be generated due to intake of inadequately absorbable aqueous solutes
of low molecular weight that pulls ions and water into the intestinal lumen thereby causes
recurrent fecal output. In case when persons with inherent lactase deficiency use diets rich in
lactose then it may cause this form of diarrhea. Sometimes loss / disturbance of epithelial cells
affects the intestinal epithelium‘s barrier function and hydrostatic pressure within lymphatics and
blood vessels causes water as well as electrolytes, protein and mucus to gather in the lumen that
leads to the formation of thin stools. It is known as exudative diarrhea and is frequent in bacterial
infection, predominantly Shigella (12). Furthermore infection due to Salmonella, Aeromonas,
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Yersinia, Campylobacter and Rotavirus may also cause exudative diarrhea. Destruction may
occur in the surface epithelium that causes inflammation by the invasion of these organisms.
General types of diarrhea as revealed by earlier studies are illustrated in Table 2.
Table 2: Common forms of diarrhea with etiologies, mechanisms and clinical features
Etiology Main site of action Primary mechanism Clinical features
ETEC Small intestine
Heat stable and heat labile toxins
produced by the organism induce
secretory diarrhea
Watery stools associated with fever,
abdominal cramps and vomiting
EPEC Proximal small
intestine
Attachment/effacement of enterocytes,
alteration of intracellular calcium and
cytoskeleton
Self-limiting watery diarrhea occasionally
accompanied with fever and vomiting.
EIEC Distal ileum and
colon
Tissue invasion and mucosal
destruction Watery occasionally bloody diarrhea
EHEC Colon Elaboration of potent shiga-like
cytotoxins l and ll
Bloody diarrhea in 90% of cases and
haemolytic uremic syndrome in 10%.
Vibrio cholerae
enterotoxin
Endocrine cells on
the villus surface of
the intestinal
epithelium
Enterotoxins cause an increase in cAMP
or cGMP inducing cAMP-mediated
alterations of ion transport.
Voluminous watery diarrhea without
abdominal cramps or fever; nausea and
vomiting.
Shigella
M-cells of the
colonic and rectal
epithelium
Bacteria invade the intestinal epithelium
damaging it and causing inflammation.
Diarrhea is due to epithelial damage and
inflammatory mediators.
Abdominal cramps and pain with initial
high volume watery stool that eventually
reduces in volume, becomes stained with
mucus and blood and associated with
urgency and painful defecation.
Salmonella
Peyers patches of
the
small intestine
Bacteria invade the intestinal epithelium
damaging it and causing inflammation.
Diarrhea is due to epithelial damage and
inflammatory mediators
Loose stools to profuse watery diarrhea,
nausea, vomiting and sometimes persistent
headache, especially in S. typhi infection.
Yersinia
enterocolitica
Intestinal
epithelium of the
terminal portion of
the ileum
Bacteria invade the intestinal epithelium
damaging it but inhibit host
inflammatory
responses
Abdominal pain and diarrhea
occasionally accompanied by fever,
nausea, vomiting and malaise.
Norovirus
Sub-mucosa of
proximal small
intestines
Continuous viral replication in the
submucosa of the proximal small
intestines is believed to interfere with
normal intestinal function
Stomach pain, fever, nausea, vomiting,
mild self-limiting and non bloody diarrhea
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Astroviruses
Epithelial cells of
the proximal small
intestines
Viral infection increases intestinal
barrier
permeability and causes sodium
malabsorption creating an osmotic
pressure which pulls water and ions into
the intestinal lumen.
Moderate to severe diarrhea characterized
by abdominal pain and vomiting.
Enteric
adenoviruses
Intestinal
epithelium,
peyers patches in the
ileum
Viral infection of the intestinal
epithelium
damages endothelial cells and interferes
with smooth functioning of the
intestines
Watery diarrhea accompanied by vomiting,
low grade fever and mild dehydration.
Cytomegalovirus
Entire
gastrointestinal tract
but frequently
involves the
oesophagus and
colon
Viral infection causes intestinal
inflammation, erosion and ulceration
with
inclusions in the stromal and endothelial
cells. Causes distal oesophageal
ulceration.
Acute watery diarrhea, stained with blood
and may be persistent
Cryptosporidium
parvum
Surface epithelial
cells lining the distal
jejunum and ileum.
Protozoan invades minimally the
intestinal mucosa causing self-limiting
diarrhea in immune competent
individuals
Mild to severe watery diarrhea
Giardia lamblia Small intestine
Colonization of the intestine is an
important step for diarrhea. Initially,
there is excystation followed by
attachment to the intestinal epithelium
and multiplication, then encystment.
This process disrupts and distorts the
microvilli of the intestine.
Asymptomatic, Stools are loose or
semiformed, mild abdominal discomfort
Entamoeba
histolytica Small intestines
Ingested cysts rupture in the small
intestine releasing trophozoites which
invade the mucin layer of the intestinal
mucosa. Protozoan has an ability to kill
and phagocytise host cells
Lumpy mucoid stools with blood stains,
diarrhea, cramping, abdominal pain,
flatulence, tenesmus rectal, headache and
vomiting
Balantidium coli Caecum and colon
Trophozoites produce proteolytic
enzymes that digest the mucus coating
of the colon facilitating tissue invasion,
abscess formation, ulceration and
perforation of the intestine.
Acute explosive watery diarrhea, stools
may be stained with blood. Cramping,
halitosis, abdominal pain. Tenesmus,
weight loss and intestinal perforations are
seen in severe cases.
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Figure 13: Symptoms of chronic diarrhea
1.4 DIAGNOSIS OF DIARRHEA:
An unusual frequency and nonconforming liquidity of fecal evacuations that lasts for more than
4 weeks is considered persistent or chronic diarrhea. Evaluation and culture of stool specimens
are usually carried out in laboratories for the diagnosis. Presence of yeast and bacteria flora may
be examined by Gram-stained slides. Stool specimens are inoculated into bacteriological media
and recognized by means of standard microbiological and biochemical techniques. Tissue
culture studies are often used to recognize viral agents. Polymerase chain reaction (PCR) has
been generally use for identification of diarrheic agents in the laboratory as it is rapid, sensitive
and specific. Yet, its appliance is restricted due to expensive equipment, be deficient in expertise
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and false positive results that often occurs due to contamination in stool as well as inadequately
processed apparatus(13).
1.5 PREVALENCE OF CHRONIC DIARRHEA:
Diarrhea is the major cause of childhood death worldwide as it kills more than 4 million children
under five in developing countries each year. Among the gastrointestinal tract infections,
diarrhea is the most familiar devastating infectious disease considered globally. Diarrhea is the
third most frequent syndrome seen in common practice that affects people of all ages. In spite of
the drop in global mortality rate, diarrhea still accounts for more than 2 million deaths per annum
(14).
Chronic diarrhea is mainly the common reason for referral to a gastroenterology clinic. WHO
reported the pervasiveness of chronic diarrhea in children globally varying from 3- 20%(15). In
1992 , CDC prepared the first national guidelines for the management of childhood diarrhea (16).
According to UNICEF (United Nations Children's Fund), diarrhea kills 1.5 million children
under five years every year (17). Although diarrhea can be represented as a simple symptom at
one end, it may be life threatening at the other. The proper approach to the analysis and
management of chronic infectious diarrhea is determined by the frequency and intensity of
disease (18).
The UNICEF and the WHO, in 2013 published the Global Action Plan for Pneumonia and
Diarrhea (GAPPD), which outlined a framework for eliminating preventable child deaths due to
both disease by 2025(19). The GAPPD aims to decrease diarrhea mortality to < 1 death per every
1000 births and to lessen the 2010 prevalence level of severe diarrhea as 75% by 2025 (20). In
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1990, 27 % of children under five deaths have been associated with this disease and for children
1-11 months , the association increased to 40 %(21). In 1995, 229,000 deaths of children under
five, constituting 30 percent of total deaths, were attributed to diarrheal disease(22).
Diarrhea is the number one killer of children in Pakistan; accounting for about 250,000 deaths.
Approximately 350,000 children pass away due to diarrhea each year earlier than reaching their
fifth birthday in five countries of the world; Pakistan is one of those countries. In profoundly
populated areas of Pakistan, parallel to other developing countries, the ecosystem contains an
elevated back-ground level of fecal pollution related with the transmission of enteric pathogens
all the way through water, food, humans, and animals. In the existence of these causes,
gastroenteritis remains one of the most important cause of disease in the paediatric population of
Pakistan(23). In Pakistan, infant mortality is high, and 40% of all deaths amongst children less
than five years of age are owed to diarrhea(24). Diarrhea caused 16% of child deaths in Pakistan.
About two-thirds of the total annual deaths in Pakistan are currently of children under five,
diarrhea being the major cause of these deaths (25).
1.6 EMPIRICAL THERAPY OF CHRONIC DIARRHEA:
The major treatment approaches for chronic diarrhea includes:
SUPPORTIVE THERAPY
The purpose of anti-diarrheal treatment is to restore the loss of fluid and electrolyte,
decrease frequency of stool and associated symptoms like abdominal pain, decrease fecal
losses and eventually decrease length and severity of disease. Hence, the management of
diarrhea with oral rehydration solutions to restore the loss of fluid and electrolyte is
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necessary for successful cure of disease. The basic components of such solutions are
water, electrolytes and glucose however various formulations of these solutions exist.
(26). Research has reported that the combination therapy of ORS and zinc improve
diarrheal symptoms and accelerate resurgence of numerous patients. This management is
being optimistic in view of the fact that it may be an approach to evade needless use of
antibiotics, particularly in children. Additionally, use of zinc during and after diarrhea has
been stated to reduce the reappearance of ailment in the subsequent 2–3 months(27).
ANTISECRETORY THERAPY AND ANTI-MOTILITY AGENTS
Numerous chemical compounds are used for the symptomatic treatment for undiagnosed
or poorly reactive chronic diarrhea (28). Natural and synthetic opioids for the most part
are commonly used, however further compounds, including bile acid–binders, bismuth
and medicinal fiber are use occasionally (29). Opium has been used for more than two
thousand years to manage diarrhea and still its a very effective therapy. The majority
cases react to adequately higher doses of opium or morphine. These drugs with the
exclusion of loperamide, falls into the category of controlled substances owing to the
potential for abuse. Potent narcotics can perhaps underuse in the management of severe
chronic diarrhea (30-32). Adsorbents like activated carbon, clays and binder resins ;
bismuth and medical fiber are commonly used as Intraluminal agents (32).
Cholestyramine and analogous binding resins decrease fecal weight in sufferers of
chronic idiopathic diarrhea who had an elevated rate of bile acid malabsorption (33-35).
Thriving treatment of acute travelers' diarrhea with bismuth subsalicylate has been
reported, however its competence in treating chronic diarrhea is not recognized(36). They
16
increases intestinal transit time thereby enhances the probability of fluids and electrolytes
re-absorption. Though, they are generally not suggested for young infants and children
owed to the probable adverse effects. Bismuth salicylate also possesses anti-
inflammatory and antibacterial characteristics other than its anti-secretory properties,
making it an excellent candidate for the management of diarrhea. Conversely, bismuth
salicylate is not an extraordinarily acceptable option for the reason that of its elevated
medication burden, deferred onset of action and the occurrence of obnoxious adverse
effects.
ANTIBIOTIC THERAPY
An antibiotic curtails the length of the disease, averts an increase in diarrheal problems
and decreases the severity of related symptoms for instance abdominal pain and fever.
Nevertheless, the utilization of antibiotics in the cure of diarrhea is being approached
with concern owed to probable harms of side-effects, drug-resistance and expense of
treatment. Antimicrobial therapy may deteriorate the condition of patients since their
consequence on gut microflora. In the majority of cases, antimicrobials are only
suggested in the management of acute bloody diarrhea in childhood. (Table 3)
Furthermore it has been commonly observed that there are several severe infections
which are due to bacteria had become resistant to frequently used antibiotics. It has been
revealed from the literature that bacteria have developed resistance nearly to all
antibiotics classes; the molecular mechanisms of developing resistance are diverse and
complex. The random and inapt use of antibiotics in patients is particularly the major
factor that leads to antibiotic resistance.
17
Table 3: Recommendations for therapy against specific pathogens
Pathogen Recommendations for therapy
Shigella
species
TMP-SMZ, 160 and 800 mg, respectively (pediatric dose, 5 and 25 mg/kg,
respectively) b.i.d. x 3 d (if susceptible) or fluoroquinolone(e.g., 300 mg ofloxacin,
400 mg norfloxacin , or 500 mg ciprofloxacin b.i.d. x 3 d) ; nalidixic acid, 55
mg/kg/d (pediatric) or 1 g/d (adults) x 5 d or ceftriaxone ; azithromycin
Non-typhi species of
Salmonella
TMP-SMZ (if susceptible) or fluoroquinolone as
above, b.i.d. x 5–7 d; ceftriaxone, 100 mg/kg/d in 1 or 2 divided doses
Campylobacter species Erythromycin, 500 mg b.i.d. x 5 d
Enterotoxigenic
TMP-SMZ, 160 and 800 mg, respectively, b.i.d., x 3 d (if susceptible), or
fluoroquinolone (e.g., 300 mg ofloxacin, 400 mg norfloxacin, or 500 mg
ciprofloxacin b.i.d. x 3 d)
Enteropathogenic As above
Enteroinvasive As above
Enteroaggregative Unknown
Enterohemorrhagic (STEC)
Avoid antimotility drugs ; role of antibiotics unclear, and administration should be
avoided
Aeromonas/Plesiomonas
TMP-SMZ, 160 and 800 mg, respectively, b.i.d. x 3 d (if susceptible),
fluoroquinolone (e.g., 300 mg ofloxacin, 400 mg norfloxacin, or 500 mg
ciprofloxacin b.i.d. x 3 d)
Yersinia species
Antibiotics are not usually required ; deferoxamine therapy should be withheld ; for
severe infections using combination therapy with doxycycline, aminoglycoside,
TMP-SMZ, or fluoroquinolone
Vibrio cholerae
Doxycycline, 300-mg single dose; or tetracycline, 500 mg q.i.d. x 3 d; or TMP-SMZ,
160 and 800 mg, respectively, b.i.d. x 3 d; or single-dose fluoroquinolone
Toxigenic Clostridium difficile Metronidazole, 250 mg q.i.d. to 500 mg t.i.d. x 10 d
Giardia Metronidazole, 250–750 mg t.i.d. x 7–10 d
Cryptosporidium species If severe, consider paromomycin, 500 mg t.i.d. x 7 d
Isospora species TMP-SMZ, 160 and 800 mg, respectively, b.i.d. x 7–10 d
Cyclospora species TMP/SMZ, 160 and 800 mg, respectively, b.i.d. x 7 d
Entamoeba histolytica
Metronidazole, 750 mg t.i.d. x 5–10 d, plus either diiodohydroxyquin, 650 mg t.i.d. x
20 d, or paromomycin, 500 mg t.i.d. x 7 d
18
In current time, the innovation in antimicrobial discovery research and development has
been curtailed and the integer of novel antibiotics licensed for human use has been lower
as compared to the recent past. Pharmaceutical industries are not investing the obligatory
resources to manufacture the next generation of novel harmless and effectual
antimicrobial drugs (37).
1.7 RESURGENCE OF HERBAL MEDICINES:
Although there are advancements in modern medicine, yet traditional medicine has always been
accomplished for treating gastrointestinal infections. The traditional medicine has become an
alternate resource in health care, particularly in rural and urban areas of the country(38).
According to the WHO, the use of herbal remedies exceeds to that of the conventional drugs by
two to three folds all over the world(39). Herbal remedies are astonishingly successful in curing
chronic diarrhea and acute diarrheal diseases. Therapeutic medicines obtained from aboriginal
plants are the most commonly available and reasonable treatment for the management of
diarrhea in several rural communities. The available text is loaded with data on the anti-diarrheal
actions of plants and several of them have been analytically validated, by means of isolated
active components. The anti-diarrheal activities of many plants owed to the saponins, alkaloids,
steroids, tannin and flavonoids present in them. Yet, only some of these have ultimately been
used in pharmacy as anti-diarrheal agents following numerous years of evaluation. Among the
350,000 plant species originated, approximately 20–30% has been investigated systematically
and only 5–10% is at present recognized to be used in conventional medication. In hospitals one
forth of medicines prescribed are derivative obtained from plants, most of them revealed by the
utilization of aboriginal remedial plants(40).
19
It is obligatory to set up logical evidences for rational utilization of such traditional medicinal
products. Herbal medicine is still the support of about 75 - 80% of the globe population,
primarily in the budding countries (39, 41) owing to the universal faith that plant based drugs are
devoid of side effects moreover being economical and easily accessible (42-45). The resurgence
of herbal medicines has increased the international trade enormously. Herbal medical
information point towards that herbal medicine sells in Asia and Japan had reached $2.3 and
2.1billion, respectively(46). Pharmaceutical companies have established renewed concern in
exploring plants as a major source for new lead structures and for the expansion of standardized
phytotherapeutic drugs with potential of safety, efficacy and quality(47)-(48).
Traditional systems of medication have been in inclination round the globe for centuries.
According to an approximation, 80 % of the global populace relies on plant based medicines to
accomplish their basic healthcare desires. The deleterious adverse effects and inadequate
accessibility of modern medicines for various ailments have led to the resurgence of the plant
based drugs, with evidence based treatments for various diseases. Hence, the utilization of herbs
as an alternate medicine is increasing all over the globe(49), particularly for cough,
gynecological disorders, arthritis, gastrointestinal disorders and many other ailments (50, 51).
Plants with diverse phytochemical mixtures are found effective in treating diseases, with an
additional benefit of being devoid of adverse drug reactions and side effects. WHO supports to
make use of plant-based medicine, in particular for the developing and developed countries, to
trim down the fiscal burden on the relevant government in health sector. The current antibiotic
renaissance has given way for herbal drugs for its amplification in the requirement to treat
disease. Stipulation for herbal products for the preventive and curative mode of treatment has
been growing at the rate of 7 % per year. In 2000, the international market of herbal medicinal
20
products was estimated at about US$ 60 billion and in 2015 US$ 110 billion, which is
predictable to accomplish US$ 5 trillion by 2050. In view of the rising demand for herbal
products herbal products now, these in turn are verified for their effectiveness and safety as have
been pioneered in the preceding two decades.
1.8 STANDARDIZATION OF HERBAL DRUGS:
Plants produce complex combination of wide-ranging chemicals, exhibiting a challenge in
standardization and quality control. This fact is accountable for imparting herbal drugs with the
attribute of being therapeutically effectual with the advantage of synergistic and additive effects
and simultaneously being having fewer side effects. An escalation of the global marketplace for
the herbal products have drawn the attention of public health establishment to ensure the safety
and quality of these products (52). The considerable disparity in the quality control impacts the
public health, since the presence of contaminants may represent avertable risks for customers
(53).
Traditional herbal products have a complex character and they are a heterogeneous combination
of phytoconstituents. The majority of herbal products on the market place nowadays have not
been subjected to regulatory consent process to reveal their safety. A number of these products
include mercury, lead, arsenic, corticosteroids and poisonous organic substances in detrimental
quantity (54). Owed to poor quality control, it is not probable for most of the herbal drugs to
establish their efficacy in clinical practice. Furthermore, the issues related with the preparation,
storage, unpleasant taste and odor acts as an obstacle and interferes with the pharmacological
activity of these traditional dosage forms. Considering the clinical use of these drugs, the focus
21
has been shifted to the ease of medication rather than the traditional dosage form, which reduces
the patient compliance(55).
In spite of the assurance that plant-based medicine emphasis should rely on using herbal drugs
has been its reproducibility of the activity. Revival of significance and the emergent market of
herbal medicinal products demands strong commitment by the stake holders to protect the
consumer. Various side effects, hypersensitivities, consequences due to adulterants, and
interaction with other plant derived drugs have been brought into notice, which have drawn the
consideration of many regulatory authorities for the standardization of plant based drugs. WHO
has developed specific guiding principles to support the associated countries to instigate
nationalized policies on plant based drugs and to study their prospective safety, efficacy and
quality, as a qualification for universal synchronization(56). Advancement of standards for
herbal drugs is an exigent task and it needs pioneering and inventive approaches, unlike the
practicing technique to develop herbal dosage form design (57) (58). The need of the hour is to
develop an organized approach and to evolve ingenious methodologies for the standardization of
herbal formulations(59). In National Policy on the Indian Systems of Medicine(60), precedence
is being given to research on clinical trials, standardization, toxicology and pharmacology of
plant-based drugs. WHO emphasized on the significance of qualitative and quantitative methods
for distinguishing the samples, quantification of the biomarkers and the fingerprint profiles. The
recognition of valid drug, apart from the adulterants and in sustaining the excellence and
reliability of the drug (61, 62).
1.9 AIMS AND OBJECTIVES OF STUDY:
The current study was conducted with the aim to develop an herbal formulation Entoban and
22
evaluate its safety and efficacy for the treatment of diarrhea. Outstanding combinations of herbs
used to eliminate microbes and worms from GIT have been incorporated in the formulation
including Helicteres isora, Berberis aristata, Holarrhena antidysenterica, Querecus infectoria
and Symplocos racemosa. The developed formulation underwent different chemical, biological,
preclinical and clinical evaluation parameters to ensure standardization for the quality that could
be reproducible as well as validation for its effectiveness for the indications.
CHEMICAL EVALUATION:
For the chemical assessment of Entoban; following parameters were evaluated:
Elemental analysis
Biomarkers quantification
BIOLOGICAL EVALUATION:
The following parameters were evaluated
In vitro Antioxidant ability
In vitro Reducing ability
In vitro Enzyme inhibition investigations (urease and lipoxygenase enzymes inhibition)
In vitro Antimicrobial properties
In vitro anti-Helicobacter pylori activity
Acute and subchronic toxicity
CLINICAL EVALUATION:
The clinical study was conducted by making coded herbal formulation (Entoban) and
compared with Metronidazole for treatment of osmotic and secretory diarrhea and anti H
pylori activity for the improvement of the community health.
23
2.1 HOLARRHENA ANTIDYSENTERICA:
Holarrhena antidysenterica Wall. belonged to the class Apocynaceae. Research has shown that
different parts of H. antidysenterica executed antibacterial activity. It is reported that bark of the
plant showed anti diarrheal and astringent activity(56). In China the extract of the dried bark of
H. antidysenterica was given orally in adult for treating diarrhea and dysentery. It has also been
cited from India that H. antidysenterica bark infusion was administered orally in human adult to
cure diarrhea. The water extract of the bark of H. antidysenterica was given orally for dysentery
(63). Warm water extract of the dried stem bark of Holarrhena antidysenterica was given orally
and was found active against dysentery in Thailand (64). Decoction in the combination of dried
bark, flower and leaf of H. antidysenterica was administered orally to treat adult man suffering
from dysentery, India (65). In Middle East countries decoction from seeds of Holarrhena
antidysenterica was administered orally to adult man for treating dysentery. In children seeds
paste of Holarrhena antidysenterica is used with Cow‘s Milk to treat dysentery(66).
Figure 14: Holarrhena antidysenterica
About 30 steroidal alkaloids have been isolated from Holarrhena antidysenterica, generally from
the stem bark. These include kurchinine, kurchinine, kurchinidine, holarrifine, holadiene,
24
regholarrhenines, pubescimine, pubescine, norholadiene, kurchamide, kurcholessine,
kurchessine, kurchilidine,conessimine, conessine and isoconessimine. Stem bark of the plant
possess antidiarrheal properties(67).
Figure 15: Chemical structure of Betulinaldehyde
Figure 16: Chemical structure of Betulinic acid
2.2 BERBERIS ARISTATA:
Berberis aristata belonging to the family of Berberidaceae has profound antibacterial activity
and used in the treatment of diarrhea(68). Berberis aristata have protoberberine and bis
isoquinoline form of alkaloid.
25
Figure 17: Berberis aristata
B. aristata contains alkaloid which are Berberine, oxycanthine, berbamine, palmatine,
dehydrocaroline, epiberberine, jatrorhizine and columbamine, dihyrokarachine,
taximaline,oxyberberine, karachine, aromoline. The main alkaloid originated from B. aristata is
Berberine have yield of 2.23% followed by palamatine(69). Berberine has been known as an
antidiarrheal since ancient times (70, 71). The extract have antibacterial activity restricted against
S. typhimurium, E. coli, S. dysenteriae type 1 and V. cholera, the greatest activity being in
opposition to V. cholera; important pathogens accountable for diarrhea and dysentery(72).
Figure 18: Chemical structure of Berberine
26
2.3 SYMPLOCOS RACEMOSA:
Symplocos racemosa exhibited significant antimicrobial properties (73). This weed possesses a
broad range of ethnomedicinal uses that include cure for bowel complaints, inflammations,
vaginal discharges, dysentery, abortion and miscarriages, snake bites. A wide range of bioactive
compounds including flavonoids, tannins, loturine, loturidine, colloturine, linoleic acid,
salireposide, symplocososide, betasito-glycoside, symploveroside, benzoylsalireposide,
salireposide etc. have been isolated from this plant. The antidiarrheal activity of the drug
Symplocos racemosa was performed in-vivo and was found that the crude extract on isolated
tissue of rabbit intestine was decreased in the tone of smooth muscle(74). The bark of Symplocos
racemosa contains Locoracemosides A, B and C which are active against α-chymotrypsin.
Traditionally S. racemosa is very effective and have antidiarrhoeal, anti-inflammatory and
analgesic activities(74).
Figure 19: Symplocos racemosa
27
Figure 20: Chemical structure of Loturine
Figure 21: Chemical structure of Symposide
Figure 22: Chemical structure of Ellagic acid
28
Figure 23: Chemical structure of Salireposide
Figure 24: Chemical structure of Oleanolic acid
Figure 25: Chemical structure of Betulinic acid
29
2.4 QUERCUS INFECTORIA:
Quercus infectoria Olivier (Fagaceae) is an inhabitant of Greece, Iran and Asia (75). Research
has shown that the galls of Q. infectoria have been recognized to have antidiabetic, astringent,
Figure 26: Querecus infectoria
local anaesthetic, antimicrobial, larvicidal and anti-inflammatory activities. Tannin and small
quantity of ellagic acid and gallic acid are the major components originated from the galls of Q.
infectoria (76). The nutgalls of dyer‘s oak are traditionally used for abdominal pain and as
antidiarrheal and antidysentery agent(77)
Figure 27: Chemical Structure of gallic acid
30
Figure 28: Structure of ellagic acid
2.5 HELICTERES ISORA LINN.
Helicteres isora Linn. belonged to the family Sterculiaceae prescribed for different intestinal in
the Indian traditional systems of medicine (78). Research has shown that the plant possess
antioxidant, hypolipidaemic, antibacterial, cardiac antioxidant, anti-diarrheal activity,
hepatoprotective activity and wormicidal activity. It is broadly prescribed in healing colic pain,
complaint of bowels and flatulence. The fruit contains tannins, sterols, friedelin, triterpenes, a
and b amyrin, lupeol, cardiac glycosides, taraxerone and b-sitosterol. The pods are fried and
administered in children to eradicate intestinal worms. Acetone fruit extract of H. isora exhibited
96.44% potent antioxidant action in comparison of hexane, and IPA. (79).
Figure 29: Helicteres isora
31
Figure 30: Structure of Rosmarinic acid
2.6 MYRTUS COMMUNIS L:
Myrtus communis L. (Myrtaceae) has been for therapeutic, food and spices reasons. Flavonoids,
tannins and volatile oils are found in the leaves (80). A furocoumarin marmalosin mainly
accountable for its therapeutic characteristics is present in the fruits whereas the bark contains
umbelliferone and other coumarins.
Figure 31: Myrtus communis L
Ripe fruits were used in earlier period, as food integrators for the reason that it contains high
vitamin contents; it is a simple therapy for dyspepsia as well. One of the major constituents of
32
myrtle essential oil is 1,8-cineole (80). The leaves oil of M. communis budding in Turkey
contains myrtenyl acetate 1,8-cineole, linalool and myrtenol as main constituents (81).
Figure 32: Chemical structure of myrtenyl acetate
Figure 33: Chemical structure of myrtenol
Zaidi et al., reported that berries can be used for the cure of peptic ulcers. It decreases the gastric
juice volume and overall acidity; rising the gastric pH and gastric wall mucus component(82). In
Ethiopia, to treat dysentery, leaves of Myrtus communis were crushed and boiled in water, the
water extract was administered orally to adult human early in the morning. Extract of dried leaf
of Myrtus communis was used to treat dysentery in Tunisia. Whereas hot water extract of dried
33
entire plant of Myrtus communis in the concentration of 50mg/person found to be effective
against Entamoeba, India(83). A mixture of Myrtus communis, Quercus infectoria, Coptis teeta
and Hyoscyamus niger was used against dysentery for 1-7 days, India. The unripe dried fruit is
found to be astringent, digestive and stomachic. The fruits are used in treating chronic diarrhea
and dysentery. Sweet drink prepared from the pulp of fruits creates a relaxing effect on the
patients improved from bacillary dysentery. The unripe fruits recover appetite and digestion.
(84).
2.7 ZINGIBER OFFICINALE:
Ginger (family Zingiberacae) is commonly used around the globe for the cure of different
diseases(85). The powdered rhizome of ginger has extensively been used for improving the
complaints of GIT. Ernst and Pittler (2000) reviewed the worth of ginger against nausea and
vomiting from different scientific data(86). O‘Mahony et al. evaluated the bactericidal action of
ginger and claimed that ginger was very successful in killing H. pylori. (87). Siddaraju and
Dharmesh,(2007) reported that ginger was strong inhibitor of gastric cell proton potassium
ATPase mechanism and H.pylori growth (88).
Figure 34: Zingiber officinale
34
Figure 35: Chemical structure of zingeberene
Figure 36: Chemical structure of zingiberol
Figure 37: Chemical structure of zingerol
35
2.8 BUTEA FRONDOSA:
Butea frondosa Koen. Ex Roxb (Papilionaceae) has been conventionally used as an astringent,
in colic, for worms and in piles. Butea frondosa have flavanoids, glucosides and lectins. In
traditional remedy, Butea frondosa is being used as an antidiarrheal as it is revealed that the
extract of stem bark of Butea frondosa have antidiarrhoeal activity.
The leaf extracts of Butea frondosa project the similar action on these enteric neurotransmitters
ensuing in decreased propulsion of intestinal contents. This could consequently serve as an
additional contributor to its anti-diarrhoeal action(89).
Figure 38: Butea frondosa
Figure 39: Chemical structure of Stigmasterol
36
Figure 40: Chemical structure of isobutrin
Figure 41: Chemical structure of butrin
2.9 AEGLE MARMELOS:
Aegle marmelos Linn. belonged to the family Rutaceae. Half ripe fruit have slight astringent
activity and used to treat gastrointestinal problems including dysentery, diarrhea and dyspepsia.
Methyl Hydroxide and water extracts of Aegale marmelos was administered by intragastric route
to male mouse and found successful treatment against diarrhea. In traditional medicine, the
young fruit of A. marmelos possess anti-diarrheal activity. Infectious diarrheal diseases due to V.
cholera and S. flexneri can be controlled by the decoction of A. marmelos. Though, it may
37
perhaps not successful against diarrhea due to ETEC. The major chemical components found in
Aegle marmelos Linn include skimmin, beta-sitosterol, alloimperatorin, marmelide, marmin,
umbelliferone, isoimperatorin, tannic acid, isopimpinellin, marmesin, marmelosin,marmesinin,
and fatty acids. It has been reported that A. marmelos exhibited considerable antirotaviral and
antigiardial activity however it did not demonstrated any considerable activity against bacteria.
Adherence and invasive assays which are responsible for the pathogenic organisms‘ colonization
to the epithelium of intestine, point toward that A. marmelos does not allow the establishment of
pathogens. The pathogen adherence to the gut epithelium is the primary phase of the infection
progression; if the adherence is inhibited it could be a significant aspect in the antidiarrheal
action of the plant (90).
Figure 42: Aegle marmelos
38
Figure 43: Chemical structure of marmelosin
Figure 44: Chemical structure of marmin
Figure 45: Chemical structure of marmesin
Figure 46: Chemical structure of skimmin
39
PART I: DEVELOPMENT AND QUALITY CONTROL EVALUATION OF ENTOBAN:
3.1 COLLECTION OF HERBS:
The herbs utilized in Entoban syrup and capsules were procured from the market place of local
vicinity and evaluated for their prescribed part, microscopic and macroscopic descriptions and
compared morphologically with the samples available at Quality Control department of Herbion
Pakistan Pvt. Ltd. All the herbs were also verified and authenticated by Prof. Dr. Iqbal Azhar,
Dean, Faculty of Pharmacy, University of Karachi. The herbs were dried and coarsely powdered
in electronic mixer, sieved through mesh no. 40 and they were then stored in air tight, well
closed container till further use.
3.2 PREFORMULATION PARAMETERS:
3.2.1 Bulk density and tap density and Carr’s index (91):
A weighed quantity (15g) of powdered material was taken in a 50ml measuring cylinder. Initial
volume (vo) was recorded. The contents were tapped and powdered volumes was recorded after
50 taps(v50).
Fluff density = w/vο g/cc
Tapped density = w/vο50 g/cc
Carr‘s index = Tapped density- Fluff density/ Tapped density * 100
Value for Carr‘s index below 15 indicate excellent flowing material and value over 20-30
suggested poor flowing material.
40
3.2.2 Angle of repose:
A paper was placed under the funnel on the table. The powdered drug was passed gradually
through the funnel until it forms a pile. The radius of the pile was noted down. An angle of
repose of the different powders used in manufacturing of herbal formulation was determined as
follows:
tanθ = h/r θ = tan (h/r)
where , h = height of the pile, r = radius.
3.2.3 Hausner’s ratio (92):
The common procedure was used to determine the apparent volume (V0) and the final tapped
volume (Vf), of the powdered material.
Hausner‘s ratio = V0 / Vf
The Hausner‘s ratio between 1.00 and 1.11 shows excellent flow and value more than 1.60
shows very, very poor flow.
3.3 EXTRACT PREPARATION:
The herbs used in the preparation were sieved through mesh #60. Each grinded herb was taken
into extractor and water was added as solvent in the ratio of 1:10 with herb: solvent. The
decoction was obtained by heating the extractors with steam for 2 - 3 hours. Filtration was done
and the filtered decoction was shifted to evaporators to eradicate the additional solvent.
3.4 COMPOSITION OF ENTOBAN CAPSULE:
Each 500mg capsule contains:
Holarrhena antidysenterica (Kura Chaal) 40 mg
41
Myrtus communis (Hab-ul-aas) 40 mg
Symplocos racemosa( Lodh Pathani) 20 mg
Aluminum silicate (Gil – e – Armani) 20 mg
Quercus infectoria( Mazu) 10 mg
Zingiber officinalis (Soanth) 10 mg
Helicteres isora( Maroor Phali) 10 mg
Berberis aristata (Zarishk ) 10 mg
Butea frondosa (Kamarkas) 10 mg
Aegle marmelos (Belgiri) 10 mg
Acacia Arabica (Acacia) 10 mg
3.5 PREPARATION OF FORMULATION BY WET GRANULATION METHOD:
All herbs were finely powdered (# 40), and taken for preparation of capsules by wet granulation
technique. Starch (20%) solution was used as binder. Then it was passed through sieve # 30 to
obtain granules which were dried at 45οC in tray dryer. Diluents and preservatives were added
and filled in capsules colored green–size ‗00‘ in capsule filling machine. The capsules were
evaluated for different quality control parameters.
3.6 QUALITY CONTROL PARAMETERS OF CAPSULE:
3.6.1 Description:
Size, shape, colour were evaluated
3.6.2 Weight uniformity:
Randomly selected 20 capsules were weighed (individually and together) using electronic
42
balance (Mettler Toledo B204-S, Switzerland)(93).
3.6.3 Determination of moisture content:
The test was executed by means of using Karl Fischer instrument(94).
3.6.4 Disintegration test:
Disintegration test was executed by means of the digital microprocessor based disintegration test
apparatus (Erweka ZT-2, Heuesnstanm, Germany). For the test, a 1000 ml beaker was filled with
distilled water (approx. 900ml), equilibrated to 37±0.5ºC. Six capsules were subjected to the test.
Time required for the last capsule to disintegrate was recorded (95).
3.6.5 Qualitative Identification Reactions:
3.6.5.1 Test for polysaccharides:
Preparation under test in quantity of 5 ml was taken into a 50 ml flat-bottomed flask. Then 96%
spirit in amount of 20 ml was added into it and blended.
3.6.5.2 Test for tanning agents:
Preparation under test was filtered cautiously by means of filter paper after leaving it for 1 hour
for the separation of layer (Solution А). A solution of Ferric chloride (3 drops) was added to
solution A. Subsequent to shaking tanning agents of greenish-yellow color were observed.
3.6.6 Microbial Analysis(96):
3.6.6.1 Pre Treatment of the Sample:
Sample (5 g) was dissolved in 50 ml of buffered NaCl - peptone solution of pH-7.0 that has no
43
antimicrobial action under the test condition and in Lactose Broth that have a pH of 7.2 ± 0.2.
Then they were subsequently incubated for a period of 2 - 4 hours at 37°C.
3.6.6.2 Primary Treatment:
0.1 of the sample was pipetted out from buffered NaCl - peptone solution and spreaded onto
Soya bean Casein Digest Agar plates (SCDA) and for Total Fungal Count, 0.1 ml onto
Sabouraud‘s Dextrose Agar (SDA) that have a pH range of 5.6 ± 0.2. SCDA plates were then
upturned and incubated for 24 hours at 37°C after which the numeral of colonies were counted
up, it was then multiplied by the dilution factor and were presented in the unit of cfu/g/ml.
3.7 QUANTITATIVE EVALUATION OF GALLIC ACID AND BERBERINE IN
ENTOBAN CAPSULE BY HPTLC-DENSITOMETRY:
3.7.1 Materials and Methods:
Chloroform, formic acid, ethyl acetate, toulene (Merck, Pakistan). Methanol and ethanol of
analytical reagent grade (Merck, Darmstadt, Germany). Gallic acid and berberine reference
standard (Sigma-Aldrich GmbH, Germany).
3.7.2 Apparatus:
100 μL syringe (Hamilton, Bonaduz, Switzerland), Linomat V Automatic Sample Spotter
(CAMAG, Muttenz, Switzerland), glass twin trough chamber (20 cm × 10 cm × 4 cm)
(CAMAG), TLC Scanner 3 linked to Win Cats software (CAMAG), 0.2 mm thickness pre-
coated with silica gel 60 F254 (Merck) were used in this study. The experiment was carried out
under the conditions with (25±2) °C temperature and 40% relative humidity.
44
3.7.3 Standard Preparation of Gallic Acid:
The standard solution was prepared containing known concentration of 0.4 mg/ml. 4 mg
gallic acid (standard) was dissolve in methanol (10 ml).
3.7.4 Sample Preparation of Gallic Acid:
The sample (2.5 gm) was weighed accurately in to conical flask in which 30 ml of methanol was
added. It was heated to boil on water bath for 15 minutes and then cooled at room temperature.
Methanol soluble part was filtered. This step was repeated four more time. The filtrate was
concentrated up to 5 ml, and then transferred the concentrated syrup in to volumetric flask.
Methanol was used to make up the volume (washing of conical flask).
3.7.5 Standard Preparation of Berberine:
The standard solution was prepared of 0.1 mg/ml concentration using 1mg standard of
berberine hydrochloride in 10 ml of MeOH.
3.7.6 Sample Preparation of Berberine:
1.0 g of capsule powder was weighed accurately in to volumetric flask; 10 ml MeOH was added.
For 5 min it was sonicated, shaked (wrist- action shaker 10 min).
3.7.7 Procedure:
TLC Development for Gallic acid:
a. The plate was developed by dipping sample HPTLC plate into glass chamber containing
the toulene- ethyl acetate – formic acid –methanol in ratio of 12:9:4:0.5 (v/v/v/v).
b. The plate was allowed to dry in fume cupboard till ten minutes.
45
c. The brown spot present in the chromatogram refer gallic acid under 273 nm.
TLC Scanning for Gallic acid:
The plate was scanned in the densitometer by linear scanning at 273 nm for gallic acid by
using a TLC Scanner III CAMAG with a D2 source, and integrate the area of the spots
corresponding to Gallic acid standard.
Development of TLC for Berberine:
a. TLC plate was developed by dipping sample HPTLC plate into glass chamber containing
the solvent system ethanol: water: formic acid in ratio of 90:9:1 (v/v/v),
b. The plate was allowed to dry in fume cupboard till ten minutes.
TLC Scanning for Berberine:
The plate was scanned in the densitometer by linear scanning at 366 nm for berberine by
using a TLC Scanner III CAMAG with a mercury source, and integrate the area of the
spots corresponding to berberine hydrochloride standard
The quantity of gallic acid and berberine in Entoban capsules was calculated by:
ASMP x WSTD x f x Dilution of Smp x application vol. of sample × P x WCAP
ASTD x Dilution of Std x WSMP x application of vol. standard
ASMP = Average area of Sample
ASTD = Average area of Standard
WSTD = Weight of Standard in mg
46
WSMP = Weight of Sample in g
Dilution of Smp = Dilution of Sample in ml
Dilution of Std = Dilution of Standard in ml
P = Percent Purity of Standard
f = conversion factor
WCAP = Average capsule weight in g
3.8 COMPOSITION OF ENTOBAN SYRUP:
Each 10 ml contains:
Aegle marmelos Syrup; Oral; 100 mg
Berberis aristata Extract 30 mg
Butea frondosa Dry extract 20mg
Holarrhena antidysenterica Dry Extract 50mg
Myrtus communis Dry Extract 200 mg
Quecrus infectoria Dry Extract 50 mg
3.9 MANUFACTURING OF SYRUP:
The herbs used in the preparation were sieved through mesh #60. Each grinded herb was taken
into extractor and water was added as solvent in the ratio of 1:10 with herb: solvent. The
decoction was obtained by heating the extractors with steam for 2 - 3 hours. Filtration was done
47
and the filtered decoction was shifted to evaporators to eradicate the additional solvent. To
formulate syrup, 666.7 gm of sucrose was dissolved in purified water by heating with alternating
stirring. Enough hot water was put in to make up the volume 1000 ml. For the preparation of
finished herbal syrup, 1 part of decoction was blended with 5 parts of plain syrup. Then
propylparaben, citric acid, propylene glycol, methyl paraben and glycerol were added to the
mixture.
3.10 PROSPECTIVE PROCESS VALIDATION FOR POLYHERBAL ORAL LIQUID
PREPARATION:
For executing prospective process validation, the protocol and report were developed and critical
process parameters were recognized. Three batches were analyzed to reassure reproducibility of
the results.
3.10.1 Critical quality attributes:
Following parameters were recognized as critical quality attributes that effect quality of final
product.
• Quality of distilled water used in developing formulation.
• The velocity of stirrer during the procedure.
• Final product‘s viscosity, density and pH.
• Organoleptic properties of the finished product.
• The filled volume of finished product.
• Sealing excellence of the filled bottle.
48
Above mentioned parameters were analyzed for three batches and report and validation protocol
were produced. Brookfield Viscometer was used to measure viscosity. Conductivity meter was
used to measured conductivity of the purified water.
3.10.2 Sampling for process validation:
At first, purified water sample (30 ml) was taken and evaluated for its appearance, conductivity
and pH. Syrup sample (30 ml) was taken finally which was stored after filtration in storage
vessel. It was then evaluated for critical process parameters stated in protocol. The samples were
collect in amber colored bottles.
3.11 QUALITY CONTROL ESTIMATION OF HERBAL SYRUP:
3.11.1 Physicochemical properties (97-99):-
The prepared syrup was assessed for diverse physicochemical properties including colour, odour,
specific gravity, taste and pH.
3.11.2 Qualitative Identification Reactions:
Preparation under test in quantity of 5 ml was taken into a 50 ml flat-bottomed flask. Then 96%
spirit (20 ml) was added into it and blended. Preparation under test was filtered cautiously by
means of filter paper after leaving it for 1 hour for the separation of layer (Solution А). A
solution of Ferric chloride (3 drops) was added to 3 ml solution A.
3.11.3 MICROBIAL ANALYSIS (96, 100):
3.11.3.1 Pre Treatment of the Sample:
Sample (5 ml) was dissolved in 50 ml of buffered NaCl - peptone solution of pH-7.0 that has no
antimicrobial action under the test condition and in Lactose Broth that have a pH of 7.2 ± 0.2.
49
Then they were subsequently incubated for a period of 2 - 4 hours at 37°C.
3.11.3.2 Primary Treatment:
To determine the Total Bacterial Count, 0.1 of the sample was pipetted out from buffered NaCl -
peptone solution and spreaded onto Soya bean Casein Digest Agar plates (SCDA) and for Total
Fungal Count, 0.1 ml onto Sabouraud‘s Dextrose Agar (SDA) that have a pH range of 5.6 ± 0.2.
SCDA plates were then upturned and incubated for 24 hours at 37°C after which the numeral of
colonies were counted up, it was then multiplied by the dilution factor and were presented in the
unit of cfu/g/ml.
3.11.4 STABILITY TESTING (101):
The stability of Entoban syrup was conducted by keeping the samples at accelerated
temperatures. The samples were evaluated for different physicochemical parameters, turbidity
and homogeneity at 40C, Room temperature and 47
0C during the period of 24, 48 and 72 hours to
examine any change.
3.11.5 ANTIMICROBIAL ACTIVITY:
3.11.5.1 Apparatus:
Glass petri dishes (pre sterilized), volumetric flask, metallic borer, Pyrex A (Germany), Sanyo
lab autoclave, MLS-3780, S.NO-2Y0301, Made in Japan, Streamline Horizontal laminar flow
cabinet, ESCO. HEPA filters, ISO 14644.1 Class 4. Digital constant temperature tank (China).
3.11.5.2 Test Microorganism:
Five gram negative bacterial cultures namely Salmonella enteric, Eschericia coli, Shigella
dysenteriae, Pseudomonas aeruginosa , Vibrio cholera and one gram positive bacterial culture
50
Staphylococcus aureus were used in this investigation. All the cultures were obtained from Dr
Essa laboratories, Karachi, Pakistan.
3.11.5.3 Preparation of McFarland (0.5) Index:
0.5 ml of 1.175%w/v BaCl2 solution was added to 99.5ml of 1.0% H2SO4 solution and blended
cautiously to prepare McFarland (0.5) index. The index was identical to estimated bacterial cell
density of 1.5×108 CFU/ml. Absorbance of the index was 0.136 as noted down by
spectrophotometer. The solution was stored at room temperature in dark in screw capped test
tubes and absorbance was checked after storage.
3.11.5.4 Preparation of Tryptic Soy Agar:
Tryptic Soy Agar (TSA) medium gave the essential nutrients to carry out the growth of the
microorganisms tested and an appropriate medium to execute susceptibility testing. Tryptic Soy
Agar (TSA) was prepared according to the procedure mentioned by producer (OXOID, USA).
TSA powder was dissolved in distilled water and subsequently it was autoclaved.
3.11.5.5 Culture preparation:
Cultures were kept for 24 hours at 36ºC ± 1ºC all night and the clarity of cultures was evaluated
later than 8 hours of incubation. Dilution of bacterial suspension (inoculum) was made with
sterile physiological solution, to 108 CFU/mL after 24 hours of incubation,.
3.11.5.6 Preparation of inoculum and standardization:
Result interpretation of sensitivity test can be affected by the turbidity of inoculum related to
bacterial cell density in TSA. McFarland Index (0.5) was used for standardization of inoculated
51
TSA. Absorbance by means of spectrophotometer was noted to make adjustment in the turbidity
of inoculated broth.
3.11.5.7 Antimicrobial Activity Assay:
The antimicrobial assay was carried out by agar well diffusion method (102, 103). According to
the method, 0.1 ml of diluted inoculums (106 CFU/ml) of the test organism was carefully mixed
with 20 ml of molten sterile TSA and poured in pre sterilized petri dishes in sterile condition.
Every plate was left to set for 30-40 minutes at room temperature. A well of 6mm diameter was
made in the centre of each seeded plates by means of sterile cork borer. Holes were subsequently
filled aseptically with 0.1 ml of Entoban syrup. Ciprofloxacin was used in contrast as a positive
control. 1mg of ciprofloxacin was dissolved in 1ml of triple distilled water. Antibacterial plates
were incubated at 37±10C for 24 hours. The zone of growth inhibition around the well was
measured by vernier caliper. All investigation was repeated quadruplicate to reduce the error and
the mean values are presented.
3.12 IN VITRO ANTIOXIDANT ABILITY AND REDUCING ABILITY:
3.12.1 Chemicals and reagents:
Butylated hydroxyanisole, solvents and other chemicals used were of HPLC grade and got from
Merck, Pakistan.
3.12.2 DPPH Radical Scavenging Activity:
The antioxidant capacity of Entoban syrup and capsules was evaluated by measuring the
scavenging capability of the syrup and capsules on free radical 2,2‘-diphenyl-1-picryl hydrazyl
(DPPH; C18H12N5O6) at 517 nm (104).
52
DPPH radical scavenging effect (%) =Ac – As x 100
Ac
Where,
Ac = Absorbance of Control
As = Absorbance of Test compound
3.12.3 Determination of the reducing power:
Ferric was converted into ferrous state by antioxidant compounds to observe the reducing ability
(105).Percent reduction ability was calculated as:
Percent Reduction Activity = At x 100
As
Where,
At = Absorbance of test
As= Absorbance of standard capability.
3.12.4 Superoxide Scavenging Activity By alkaline DMSO method:
Absorbance was measured at 560 nm against control and the percentage of super oxide radical
scavenging by the test compounds were calculated by
53
3.13 INHIBITION OF HELICOBACTER PYLORI-INDUCED INFLAMMATION:
3.13.1 Chemicals and reagents:
Sodium nitroprusside and urease (Jack beans, EC 3.5.1.5) were obtained from Sigma
(St. Louis, MO, USA). Potassium phosphate buffer (100 mM), pH 8.2 was prepared in distilled
water.
3.13.2 Bacterial strains and culture conditions:
For this study, H. pylori strain 193C(106) was isolated from a patient of gastric cancer,
and strain NCTC(107) was isolated from a patient of gastric ulcer. Brucella broth (BB) medium
complemented with 10% fetal bovine serum (FBS) was used to cultured these H. pylori strains.
The formula absorbance of 0.1 = 108 bacteria/ml was used to estimate the concentration of
bacteria in each culture.
3.13.3 Determination of non-bactericidal concentration:
Entoban was evaluated for non-bactericidal concentration by adopting the method
formerly illustrated with slight variation (108). Serial dilution of bacteria was done and then they
were inoculated onto commercial selective Pylori agar plates (Kyokuto; Tokyo, Japan) under
microaerophilic condition. The colony forming units (CFUs) were calculated after the incubation
of 2 – 3 days. Data was articulated as percentage of bacterial survival. The results represent three
independent experiments.
3.13.4 Cell survival study:
CCK-8 assay was used to measure number of viable cells over a period of time after
treatment with Entoban(109). Briefly, 100µl of 5000 cells/well were dispensed in 96-well plate.
54
After 24 hours of incubation, cells were washed with PBS for 3 times and fresh medium was
added. The cells were then treated for 6 hours with various concentration of Entoban both
capsule and syrup formulation. Results were analyzed after correction with OD from blank and
untreated wells. The OD was calculated as absorbance unit (AU). Data is expressed as
percentage of viable cells.
3.13.5 Experimental design for co-culture experiments:
Human gastric epithelial cell line (AGS) were grown-up in RPMI 1640 containing 2
mmol/L l-glutamine complemented with antibiotics and 10% FBS at 37oC in 5% CO2. Cells
were regularly passage every three days. The medium RPMI 1640, without antibiotics and FBS,
was added. The AGS cells treated with or without Entoban for 60 minutes, were then incubated
in the presence of H. pylori at a bacterium/cell ratio of 50:1 for anti-adhesion and anti-IL8
assays.
3.13.6 Anti-adhesion activity assay:
AGS cells were used for the investigation of H. pylori adhesion (110). Cells were
preincubated with or without Entoban in 96-well plates at 37oC, 5% CO2 for sixty minutes and
H. pylori was added in the treated and untreated wells and incubated for 4 hours. The cells were
then fixed at 4oC for 60 min and followed by washing, 100 µL of rabbit anti-H. pylori polyclonal
antibody (Dako Cytomation, Glostrup, Denmark) was added to every well, and the plates were
incubated for 2 hours at 37oC. Subsequent to washing, 100 µL of peroxidase-conjugated goat
anti-rabbit Igs (Wako) diluted 1 : 1000 in PBS was added to all wells and incubated for two
hours at 37oC. Following final washing, 100 µL of substrate reagent pack (DY999; R&D System
Inc., Minneapolis, MN, USA) was added for 15 min then of 100 µL of 1 mol/L H2SO4 was
55
added to conclude the reaction. The optical density (OD) of the reaction was calculated at 490
nm with a microplate reader. The OD corresponds to the quantity of H. pylori adhering to target
cells. Data is expressed as percentage of adherent bacteria.
3.13.7 Enzyme Linked Immunosorbent assay (ELISA) for effect on IL-8 secretion:
AGS cells were co-cultured with H. pylori at multiplicity of infection (MOI) of 50 : 1
for a period of 4 hours in the presence or absence of Entoban. IL-8 secretion in the supernatant
from the treated cells was analyzed by using ELISA (R & D System). The supernatant medium
was collected after 4 hours of culture, and IL-8 contents were determined as per the instructions
of manufacturer. A standard curve of recombinant IL-8 (R & D) was used to find out IL-8
concentrations in pg/ml.
3.14 ANTI-UREASE ACTIVITY:
3.14.1 Chemicals and reagents:
Urease (EC 3.5.1.5) and sodium nitroprusside (sodium pentacyanonitrosyloferrate III) were
purchased from Sigma (St. Louis, MO, USA). The potassium phosphate buffer pH 8.2 was
prepared in distilled water. Analytical reagent grade chemicals obtained from Merck were used.
3.14.2 Procedure:
Urease activity of Entoban was evaluated by calculating production of ammonia by means of the
indophenol method (111). Thiourea was used as the standard inhibitor of urease.
56
3.15 LIPOXYGENASE INHIBITION ACTIVITY:
3.15.1 Chemicals and reagents:
Linoleic acid and lipoxygenase were procured from Sigma (St. Louis, MO, USA). All other
chemicals were of analytical reagent grade from Merck. Cadmium chloride (CdCl2), Dalbeco
Eagle‘s Minimum Essential Medium (D-MEM) , Dimethyl sulphoxide (DMSO) and Ethylene
diamine tetra acetic acid (EDTA) from Wako Pure Chemical Industries Ltd. and Trypsin (0.25%)
from Gibco,Canada.
3.15.2 Procedure:
1. Lipoxygenase enzyme solution was prepared in sodium phosphate buffer with such
concentration to give 130 U per well.
2. Sodium phosphate buffer (pH 8.0: 160µl:100 mM) was taken in each well of plate
labelled as Blank (Bsubstrate and Benzyme), Control and Test.
3. Test compound solution in methanol (10-1000 M: 10 l) was added in each well labeled
as test.
4. Lipoxygenase solution (LOX: 20l) was added in each well including B enzyme, Control
and Test except Bsubstrate and the mixture was incubated at 25 C for ten minutes.
5. Substrate solution was prepared by adding linoleic acid (155 µl:0.5 mM) into 0.12 % w/v
tween 20 (257 µl). The mixture was mixed and 0.6 ml NaOH (1N) was added to remove
turbidity and volume was making up to 20 ml with deionized water. This mixture was
flushed with nitrogen gas to avoid autoxidation before adding to each well.
6. The reaction was initiated by the addition of 10 l substrate in each well except B
(enzyme) and the absorbance was measured after five minutes at 234 nm.
57
3.16 CELL VIABILITY ASSAY:
3.16.1 Cell line and culture:
HepG2 cells were purchased from Riken, Japan. The cells were grown in10 cm culture plate
containing D-MEM supplemented with 10% FBS, L-glutamine and phenol red at 37˚C in a CO2
incubator (SANYO, Japan) in an atmosphere of humidified 5% CO2 in 95% air. The cells were
then trypsinized to seed in the 96 well-plate for the experiment.
3.16.2 Cell Viability Assay:
Cell viabilities in response to polyherbal formulations on the HepG2 cells were measured to
check any cytoprotective response and antioxidant activity of the drugs by using cell counting kit
8 (CCK-8) (Dojindo, Japan). Cells were seeded at density of 5000 cells per well in 96-well plates
in a triplicate and pre-incubated overnight for adherence at physiological conditions of 5% CO2
and 37˚C, in a humidified atmosphere. The cells were then pre-treated at concentrations of
0µg/ml (NT), 100µg/ml, 200µg/ml, 300µg/ml, 400µg/ml, 500µg/ml from both formulations;
capsule and syrup of Entoban separately in two groups; one without cadmium and one with
cadmium. In this study cadmium was used to induce oxidation in the HepG2 cells and one
sample with only cadmium as a positive control. After 12 hours incubation, add 25µM cadmium
and incubated again for 24 hours. After incubation, 10µl of CCK-8 was added to each well
including blank sample and incubated at 37˚C for 3 hours; trailed by measuring the absorbance
using an automated micro-plate reader ELx 800 (BioTek, UK) at 450 nm.
58
3.17 QUANTITATIVE ESTIMATION OF BIOMARKERS IN ENTOBAN SYRUP
3.17.1 Materials and Methods:
Chloroform, formic acid, ethyl acetate, toulene (Merck, Pakistan). Methanol and ethanol of
analytical reagent grade (Merck, Darmstadt, Germany). Gallic acid and berberine reference
standard (Sigma-Aldrich GmbH, Germany).
3.17.2 Apparatus:
100 μL syringe (Hamilton, Bonaduz, Switzerland), Linomat V Automatic Sample Spotter
(CAMAG, Muttenz, Switzerland), glass twin trough chamber (20 cm × 10 cm × 4 cm)
(CAMAG), TLC Scanner 3 linked to Win Cats software (CAMAG), 0.2 mm thickness pre-
coated with silica gel 60 F254 (Merck) were used in this study. The experiment was carried out
under the conditions with (25±2) °C temperature and 40% relative humidity.
3.17.3 Standard Preparation of Gallic acid:
The standard solution was prepared containing known concentration of 0.4 mg/ml. 4 mg
gallic acid (standard) was dissolve in methanol (10 ml).
3.17.4 Sample Preparation of Gallic acid:
A total of 12.0 g of syrup was weighed accurately in 100 mL conical flask then, 30 mL of water
was added and mixed thoroughly. The solution was transferred carefully in 250 mL separating
funnel and 50 mL of ethyl acetate was added in the funnel and was shaked carefully for 3 min.
After complete separation of layers, upper ethyl acetate layer was filtered by using the paper
filter with anhydrous sodium sulphate (about 10 g) in 500 mL round bottom flask. Extraction
was repeated four times more and ethyl acetate fraction was collected into the same RB flask.
59
The organic fraction was evaporated under vacuum. 5 ml methanol was used to dissolve the dry
residue and transferred quantitatively into volumetric flask. Methanol was used to make up the
volume.
3.17.5 Standard Preparation of Berberine:
The standard solution was prepared of 0.1 mg/ml concentration using 1mg standard of
berberine hydrochloride in 10 ml of MeOH.
3.17.6 Sample Preparation of Berberine:
About 40.0 g of syrup was weighed accurately in 100 mL conical flask then, thirty mL of water
was added and mixed carefully. The resulting solution was transferred in 250 mL separating
funnel. The solution was extracted by adding 50 mL of chloroform in the separating funnel and
shaked carefully for 3 min. The layers were allowed to separate, after full division, lower
chloroformic layer was filtered by means of the filter paper with anhydrous sodium sulphate in
250 mL conical flask. The top water layer was further extracted with chloroform (50 mL). The
extraction was repeated using 50 mL portions of chloroform (5 times). The extract was
evaporated to dryness under vacuum. 5 mL methanol was used to dissolve the dry residue and it
was then transferred quantitatively into 10 mL volumetric flask. Volume was brought up to the
mark.
3.17.7 Procedure:
TLC Preparation:
Analysis was performed on 10 x 10 cm HPTLC silica gel G60F254 plates with fluorescent
indicator. Before starting the analysis, HPTLC plate were cleaned by predevelopment
with methanol by ascending method. (HPTLC plate was immersed in a CAMAG glass
60
chamber (20 x 10 cm), containing 30 ml methanol (HPLC grade) as solvent system. The
chamber was covered with glass lid and left till development of the plate to the top with
methanol. After complete development, the plate was removed from TLC glass chamber
and dried in an oven at 105ο C for 5 min).
Application Procedure:
3 spots of 10 μl of standard were applied along with 3 spots of 10 μl of sample on the similar
plate by means of a CAMAG Linomat 5.
TLC Development for Gallic acid:
a. The plate was developed by dipping sample HPTLC plate into glass chamber containing
the toulene- ethyl acetate – formic acid –methanol in ratio of 12:9:4:0.5 (v/v/v/v).
b. The plate was allowed to dry in fume cupboard till ten minutes.
c. The brown spot present in the chromatogram refer gallic acid under 273 nm.
TLC Scanning for Gallic acid:
The plate was scanned in the densitometer by linear scanning at 273 nm for gallic acid by
using a TLC Scanner III CAMAG with a D2 source, and the area of the spots was
integrated corresponding to Gallic acid standard.
Development of TLC for Berberine:
a. TLC plate was developed by dipping sample HPTLC plate into glass chamber containing
the solvent system ethanol: water: formic acid in ratio of 90:9:1 (v/v/v),
b. The plate was allowed to dry in fume cupboard till ten minutes and then kept in hot air
oven at 105 °C for five minutes.
61
TLC Scanning for Berberine:
The plate was scanned in the densitometer by linear scanning at 366 nm for berberine by using a
TLC Scanner III CAMAG with a mercury source, and the area of the spots was integrated
corresponding to berberine hydrochloride standard
Amount of gallic acid and berberine in Entoban syrup was calculated as:
ASMP x WSTD x f x Dilution of Smp x application vol. of sample × P x D x 10
ASTD x Dilution of Std x WSMP x application of vol. standard x 100
Where
ASMP is average area of sample; ASTD is average area of standard;
WSTD is weight of standard in mg;
WSMP is weight of sample in g;
Dilution of Smp is dilution of sample in mL;
Dilution of Std is dilution of standard in mL;
P is percent purity of standard; f is conversion factor;
D is density of syrup, mg/mL.
3.18 DETERMINATION OF HEAVY METAL CONTENTS:
3.18.1 Chemicals and equipment:
All reagents were analytical reagent (AR) grade. Reagents and standards were prepared and
diluted by using distilled and deionized water whose purity was equivalent to specification
62
ASTM Type II reagent water (Eaton, 1995). All reference standards were prepared from BDH
SpectrosoL A standard. Determination of trace elements was executed by atomic absorption
spectrometry flame (FAAS) by means of standard addition techniques. Analysis for each sample
was performed three times to obtain representative results. The test was conducted at the
Pakistan Council of Scientific and Industrial Research (PCSIR) Laboratory at Karachi, Pakistan,
the certification of which is recognized by the regulatory authority.
3.18.2 Sample preparation:
Syrup samples were dried in a rotary evaporator. Known amount of dry sample was incinerated
wet ashed with 5 to 10 ml (1: 1) HNO3 - HClO4 mixture and heated close to dryness in a
platinum dish. The remains was treated with 10 ml of concentrated HCl, and after boiling for 30
minutes, 20 ml of distilled water was added and the solution heated for another 15 minutes, then
the solution was filtered and made up to 50 ml. Entoban syrup does not contain additional
vitamins, minerals and amino acids, except excipients.
PART II PRE CLINICAL STUDY OF ENTOBAN
3.19 ANTIDIARRHEAL ACTIVITY OF ENTOBAN:
3.19.1 Animal handling:
In order to investigate the antidiarrheal activity, acute and sub chronic oral toxicity of Entoban, 3
to 4 weeks aged NMRI albino mice of both sex, of around 25 to 35g weight, were collected from
the animal house facility of Herbion Pak. Pvt. Ltd. They were kept in temperature of 25 ± 1 C
63
and 12 h dark / light cycle dark for the entire study time. Food and water were obtainable ad
libitum.
3.19.2 Castor oil induced diarrhea:
All procedures for testing were complying the Guide for the Care and Use of Laboratory
Animals(112) and OECD guideline for acute toxicity(113, 114). The animals used were divided
in to control, test and positive groups. Each group contains six animals. 0.2 ml castor oil was
used orally to induce diarrhea to mice(115, 116). The animals in control group received only
distilled water (10 ml/kg) per oral (p.o); loperamide (2 mg/kg, p.o.) was given to the animals in
positive control group; test group received Entoban at doses of 2.5, 5, 10 mg/kg, p.o., body
weight thirty minutes earlier to the administration of castor oil. The parameters observed
throughout an examination phase of 4 h, were: onset of diarrhea, total weight of stool output,
total weight of wet stools, total number of stool output, and number of wet stools(115). Percent
inhibition of diarrhea was calculated as follows:
% inhibition of diarrhea = Mean Number of wet defecation (control – test) x 100
Mean wet defecation of control
3.19.3 Magnesium sulphate induced diarrhea
Analogous procedure as intended for castor oil induced diarrhea was followed with the exception
that magnesium 2 g/kg, p.o was administered (115).
64
3.20 ACUTE TOXICITY STUDIES:
Healthy NMRI albino Mice of either sex (n = 15/sex) weighing between 25 – 35 g were treated
orally with doses (1 or 5 g/kg) of the Entoban capsule aqueous extract, in standard laboratory
setting. Animals were evaluated singly as a minimum for once for the duration of first 30 min
after dosing. Daily observations on the changes in skin and fur, eyes and mucus membrane
(nasal), respiratory rate, heart rate, blood pressure, autonomic effects (salivation, perspiration,
piloerection, lacrimation, urinary incontinence and defecation) and central nervous system
(ptosis, lethargy, gait, tremors and seizure) were noted for 1 week.
3.20.1 Sub chronic toxicity:
Healthy NMRI albino mice of either sex (n=40) were divided into 4 groups. Each group contains
10 animals of either sex. Group I was given water and standard diet. Group II was given
standard diet, water and 50 mg/kg body weight of Entoban, group III was given standard feed,
water and 100 mg/kg body weight of Entoban while group IV was given standard feed, water
and 200 mg/kg body weight of Entoban. The treated animals were weighed up at 0, 7, 14 and 28
day of the doses.
3.20.2 Statistical analysis
The data collected were summarized as mean ± SEM. Student‘s t- test was used to determine
significant differences.
65
3.21 CLINICAL TRIAL OF ENTOBAN
3.21.1 Hypothesis, Objective and Method
Objective of the Study
Clinical evaluation of chronic diarrhea and its treatment was undertaken to evaluate the
therapeutic efficacy of herbal coded medicine Entoban along with a comparative study of an
allopathic medicine Metronidazole.
Null Hypothesis (H0)
The herbal coded formulation Entoban is of the same value as allopathic medicine Metronidazole
and there is no difference between these medicines and is evenly effectual for the treatment of
chronic diarrhea.
ALTERNATE HYPOTHESIS
Research Hypothesis (H1)
Entoban (H1) is of great value and will show differences with control medicine Metronidazole in
reducing the symptoms of chronic diarrhea.
Research Hypothesis (H2)
Metronidazole is effective and having difference in with other group of medicines.
Research Hypothesis (H3)
The Efficacy of Entoban shows good result with reduction in symptoms of chronic diarrhea.
66
Point of Error or Level of Significance
P stands for probability and expressed as the level of significance in the study. The smaller the p-
value more significant is the results and it means less is the chance of making alpha error. The
alpha error is the error, which occurs if null hypothesis is being rejected but when indeed the null
is true then the type of wrong decision is Type 1 or Alpha error.
The statement p = 0.05 means that there is less than a 5 % risk that Entoban is misrepresentative
sample if the null hypothesis is true. This would be reasonable evidence for concluding that the
null hypothesis is false.
VARIABLES
To evaluate the hypothesis, statistical analysis was made by applying independent variable (IV)
to dependant variable (DV) and confounding variables. The variables are;
Independent / Predictor variable: Entoban and Metronidazole
Dependent / Outcome variable: Symptoms of chronic diarrhea or levels of improvement after
the treatment.
Bias
Any systematic error that resulted in an incorrect estimation of the association between the
controlled and experimental group was considered bias.
Blinding
To alleviate the possibility of experimental bias blinding was used, and in this experimental
study single blinding was used in which two investigators were involved in the collection of data.
67
The principal investigator performed assessments including identification of chronic diarrhea,
reduction symptoms of chronic diarrhea. An independent observer to see if full improvement had
occurred assessed clinical inspection of side effects. This obtained the maximum level of test
reliability and intra-observer reliability.
Table 4: Work Plan of clinical trial
Activity Outline Description
No. of Drugs for
comparison
2 Entoban Syrup/Capsules
Metronidazole Syrup/Tablets (400 mg)
No. of Patients Approx. 100 50/group
No. of Test Before,Mid and After Treatment Stool D/R
Time Period Five Days
Entoban syrup:
1-2 teaspoons, every 4 hours
Entoban Capsule:
1 capsule, every 8 hours
Total Duration 2 Years January 2014 to December 2015
Presentation of
report After every 3 months Follow up period
3.21.2 Study Design and setting
The study was conducted in Sharafi Goth hospital Korangi Karachi, Victoria Hospital in
Bahawalpur, and at Nawaz Salik Hospital in Rawalpindi during the period from January 2014 to
December 2015. Patients which were clinically diagnosed based on clinical history; clinical
68
presentation and stool DR of patients were enrolled. The data including demographic
information and vitals of subject, number of stool per day, stool with or without mucus or mixed
with blood along with abdominal cramps, dehydration, nausea, vomiting and related clinical
information‘s were carefully recorded.
3.21.3 Inclusion Criteria
Patients between age group of 05-60 years.
Either sex (male or female)
Every socio-economical class include lower, middle and upper.
Those who were willing to participate and provide written consent.
Suffering of chronic diarrhea
3.21.4 Exclusion Criteria
Patient with uncontrolled hypertension and diabetes.
Patient having hyper pyrexia (103 0 F or more).
Patient having Amoebic Liver Abscess.
Patient with hepatic or renal impairment.
Severe dehydration.
Spasmodic condition.
Pregnant and lactating women.
Patients treated with a medication against diarrhea in the five days prior to the study
3.21.5 Ethical approval
The study was carried out after taking approval from Ethical Committee of Faculty of Pharmacy,
Jinnah University for Women. The study design and synopsis were presented to the Board of
69
Advanced Studies and Research for their clearance and permission before the start of clinical
trial. The trial was registered at http://www.ClinicalTrial.org, a service of the US National
Institutes of Health (registry No. NCT02642250).
3.21.6 Randomization and Study Protocol
A block-randomization procedure, with a block size of 4, was adopted to assign participants
either to treatment with allopathic therapy or with a phytomedicine-based formulation. The study
was unblinded because the number of drugs and the dosing regimens differed between the 2
treatment groups. However, the statistician was blinded while performing the comparative
analysis of data. Metronidazole tablets (Flagyl) in strength of 400 mg manufactured by Sanofi-
aventis Pakistan limited was used in a control group for 7-10 days. The test group received
Entoban capsule 400mg tds, every 8 hours for five days. Stool was examined to confirm the
diagnosis. A comprehensive proforma, soliciting required demographic characteristics,
presenting complaints, general examination, stool consistency, frequency, patient‘s weight and
treatment option was filled for every patient by skilled healthcare personnel.
3.21.7 Clinical Diagnosis of Chronic Diarrhea:
Fecal evacuations that lasts for more than 4 weeks
Blood or mucus can appear in the stools with some infections.
Crampy pains and tenderness in abdomen
A high temperature (fever), headache and aching limbs sometimes occur
Nausea and vomiting
70
Clinical evaluation
Patient history
_ Frequency, type and volume of stool
_ Presence of blood in stool
_ Vomiting
_ Medical history
_ Underlying circumstances
_ Epidemiological signs
Physical examination
_ Body weight / height
_ Temperature
_ Pulse rate
_ Respiratory rate
_ Blood pressure
Biochemical and microscopic investigation of stool.
3.21.8 Criteria for Assessment of Therapeutic Evaluation
Complete improvement: Where there is a complete relief from all the clinically and biochemical
and pathological signs/symptoms within a period of 2 week in chronic diarrhea without
recurrence.
71
Slight improvement: Where there is trivial response is observed from the above signs/symptoms
clinically and biochemically within a period of 2 weeks in chronic diarrhea.
No improvement: No noticeable response of the drugs is observed clinically and biochemically
from the above sufferings within a period of 2 weeks in chronic diarrhea.
Other words utilize for the criteria of assessment in the four categories as indicated in different
tables further specify as follows.
Completely Improve / Significant Improvement, Little Improvement No Improvement.
Good Response, Mild Weakness, Feeling Better, No Weakness.
Still Feeling, Not Feeling.
Subsides.
Occurred, Not Occurred.
3.21.9 Primary and Secondary Outcomes
The quantitative evaluation of daily bowel frequency was the primary outcome of the study and
evaluation of clinical symptoms including consistency of stool, distention, abdominal pain and
feeling of incomplete evacuation were the secondary outcome. The details of relevant diarrheal
symptoms (e.g, abdominal pain, anorexia, flatulence, nausea, vomiting, rectal urgency,
incontinence and bloating) were obtained for each patient, by a special scoring system (absent, 0;
mild, 1; moderate, 2; and severe, 3). Scores for each of the symptoms could range from 0, no
symptoms, to a maximum of 3, severe symptoms. The follow up information about improvement
of the symptoms and the appearance of any side effects was recorded in the relevant file of each
72
patient. The stool DR was performed at baseline, after 2 weeks and 4 weeks of treatment. Adverse
reactions were evaluated by patient history and physical assessment on daily basis every 3 days until the
completion of study.
3.21.10 Data analysis:
The filled questionnaires were entered into Statistical Package for Social Sciences (SPSS 20.0)
for analysis to compare the effect of two drugs. Patients‘ characteristic data was articulated as the
mean ± standard deviation (SD). A χ2 test using a 2 × 2 contingency table was used to check for
a statistically significant difference in the cure rate as well as in the proportions of other
categorical variables between 2 treatment groups, such as age, gender, occupation, and marital
status. A Wilcoxon signed-rank test was applied to analyze the intensity of symptoms at baseline
(T0), after 2 weeks (T2) and 4 weeks (T4) of treatment, expressed through median values and
interquartile ranges (IQRs) (p < 0.05 was considered as a significant value).
73
Diarrhea is the third most frequent disease that affects people of all ages. In spite of the drop in
global mortality rate, diarrhea still accounts for more than 2 million deaths per annum.
Approximately two-thirds of the total annual deaths in Pakistan of children under five are due to
diarrhea. Diarrhea is the number one killer of children accounting for around 250,000 deaths in
Pakistan. Approximately death of 350,000 children occurs due to diarrhea every year earlier than
reaching their fifth birthday in five countries, Pakistan is one of them. In Pakistan, infant
mortality is high, and 40% of all deaths amongst children less than five years of age are owed to
diarrhea(24). Diarrhea caused 16% of child deaths in Pakistan (25). In profoundly populated
areas of Pakistan, parallel to other developing countries, the ecosystem contains an elevated
back-ground level of fecal pollution related with the transmission of enteric pathogens all the
way through water, food, humans, and animals. In the existence of these causes, gastroenteritis
remains one of the most important cause of disease in the paediatric population of Pakistan(23).
Different drugs are prescribed to treat the symptoms of chronic diarrhea whereas an empirical
mode of treatment with antibiotics considered viable when the infection is elevated in the
community. However, the resistance of antibiotic is responsible as the main factor for treatment
failure. Antimicrobial therapy may deteriorate the condition of patients since their consequence
on gut microflora. In the majority of cases, antimicrobials are only suggested in the management
of acute bloody diarrhea in childhood. There are several severe infections which are due to the
fact that bacteria had become resistant to frequently used antibiotics. It has been revealed from
the literature that bacteria have developed antibiotics resistance; the molecular mechanisms of
developing resistance are diverse and complex. The random and inapt utilization of antibiotics in
patients is chiefly the major factor that leads to antibiotic resistance. The innovation in
74
antimicrobial discovery research and development has been curtailed and the integer of novel
antibiotics licensed for human use has been lower as compared to the recent past.
The adverse effects, inadequate accessibility of allopathic medicines and antibiotic resistance
have led to the resurgence of plant based drugs as an alternate treatment option. Traditional
herbal medicines have now been proven to be safe and effective and being utilized to cure many
disorders, including GI ailments. Herbal dosage forms have been shown to heal acute as well as
chronic diarrheal diseases. The present study compare the clinical therapeutic activity of Entoban
with Metronidazole for the treatment of chronic diarrhea. The different quality control
parameters of both Entoban syrup and capsules were evaluated to ensure the reproducibility and
effectiveness of developed formulation Entoban.
4.1 PREFORMULATION PARAMETERS OF CAPSULES:
In the present study, standardized polyherbal mixture was formulated in hard gelatin capsule to
replace the traditional liquid dosage form. Before converting the blended powder extract into
dosage form it was passed through different procedures to estimate the flow ability of the powder
extract that was necessary for getting pharmaceutically equivalent dosage. The flow property of
powdered extract was evaluated and it was observed that they showed good flow property. Table
5 depicts the report of various preformulation parameters.
Table 5: Preformulation studies
Characterization Parameters Data obtained
Bulk density 0.65 ± 0.01
Tap density 0.78 ± 0.02
Compressibility index 19.31 ± 2.63
Hausner‘s ratio 1.24 ± 0.08
Angle of repose 29.82 ± 0.75
75
4.2 PHYSICOCHEMICAL PARAMETERS OF CAPSULES:
To enhance the acceptability of the herbal medicine by consumers, many of the products have
been formulated into conventional dosage forms such as tablets, capsules, suspensions, and
powders. The present formulation is the combination of Holarrhena antidysenterica (Kura
Chaal), Myrtus communis (Hab-ul-aas), Symplocos racemosa (Lodh Pathani) , Aluminum silicate
(Gil – e – Armani), Quercus infectoria (Mazu), Zingiber officinalis (Soanth), Helicteres isora
(Maroor Phali), Berberis aristata (Zarishk), Butea frondosa (Kamarkas) , Aegle marmelos
(Belgiri) and Acacia arabica (Acacia). Proper and complete identification is one of the most
important parameter incase of herbal medicine because the formulation cannot produce desired
effects, if the herbs are not properly identified. Various physicochemical parameters including
physical appearance, weight variation and disintegration time were calculated for the polyherbal
formulation. (Table 6)
Table 6: Physicochemical parameters
Characterization Parameters
(n=20) Data obtained
Physical appearance
Green capsules filled with brown
color powder
Average weight 500mg ± 10%
Moisture contents 2.05% ± 0.5
Disintegration Time 06 minutes
Average weight of 20 capsules was between 450 mg and 550 mg with a mean of 506 mg ± 10%.
The rate of absorption and bioavailability are dependent upon how fast the drug dissolves in GI
fluid. This means that drugs administered orally in solid dosage forms (tablets, capsules, etc)
than dissolve in the GI fluid before absorption (117). Hence the rate of absorption and
76
availability may be improved by improving the disintegration and the rate of dissolution of drug.
In present study, six capsules were taken to determine the rate at which the active drug substance
dissolved in the fluid of gastrointestinal tract. The maximum time for disintegration was 6 min.
Table 7 illustrates the determination of different phytochemical components in capsules. An
enormous presence of bacteria has been commonly observed in soil or derivative of fertilizers.
(118)(119). The developed formulation was found in agreement of the allowable microbial
limits.
Table 7: Determination of different components
Quantitative determination Specified limit
Quantity
present
Total alkaloids as Berberine
Hydrochloride
Total alkaloids as Berberine hydrochloride
should not be less than 0.100 %. 0.311 %
Total tanning agent as gallic
acid
Total tanning agent as gallic acid should be not
be less than 1.5 % 2.780 %
Table 8: Admissible contents for 1g of preparation
Microbial Analysis Limit CFU/g Observation
Total aerobic viable count not more than 104 CFU/g Comply
Salmonella Absent Absent
Escherichia coli Absent Absent
Staphylococcus aureus Absent Absent
P. aeruginosa Absent Absent
Total fungal count not more than 102 CFU/g Comply
77
4.3 PROCESS VALIDATION OF SYRUP:
Manufacturing process of liquid syrup involves many steps production operations starting from
raw material procurement then its analytical testing for purity and strength to packaging of
finished product and it‘s testing for assays (120). FDA necessitate that the drug product should
be evaluated in terms of its purity, quality and stability (121). Hence, pharmaceutical validation
and process controls are imperative tool considered in developing a quality product(122, 123).
(124, 125). In current study, samples from Batch # 1, 2 and 3 were tested for critical process
parameters. Table 5 depicts the results for individual batches. The description for purified water
given in the USP was used as control variable(126) and the quality of water was evaluated to
make sure that it comply with USP specifications. The conductivity was measured by means of
conductivity meter at 250C(127). All the specifications of the other parameters were decided
depending upon the pilot study in the R&D department of Herbion Private Limited. Filled
volume was analyzed to make sure that accurate quantity of syrup has been filled in the bottle by
means of filling machine (Table 10).
Table 9: Details of critical process parameters for Batch # 1, 2 & 3
TEST PARAMETERS SPECIFICATIONS Batch # 1 Batch # 2 Batch # 3
Quality of purified water
Description It should be clear colorless
liquid, odorless and tasteless. Comply Comply Comply
pH 5.00-7.00 6.75 6.53 6.61
Conductivity (250C): 1.0-1.5 μs/cm 1.4 μs/cm 1.3 μs/cm 1.1 μs/cm
Temperature Should not exceed 80° C 65 C 63 C 65 C
Stirrer speed 2000-3000 rpm 2000rpm 3000rpm 2500rpm
78
Final mixing Time 50-60 mins. 50 mins. 60 mins. 54 mins.
Clarity of final batch Must be clear Clear Liquid Clear Liquid Clear Liquid
Quality of syrup
Description Brown color syrup Comply Comply Comply
Density From 1.25 to 1.35 g/ml 1.297 g/ml 1.328 g/ml 1.282 g/ml
pH From 3.0 to 6.0 3.7 3.7 3.7
Viscosity 100-200poise 161poise 170poise 175poise
Taste Characteristic sweet taste Comply Comply Comply
Odor Characteristic Comply Comply Comply
Table 10: Details of filling volume and sealing quality
Batch # 1 Batch # 2 Batch # 3
Fill
Volume
Sealing
Quantity
Fill
Volume
Sealing
Quantity
Fill
Volume
Sealing
Quantity
90 ml Comply 90 ml Comply 90 ml Comply
90 ml Comply 90 ml Comply 90 ml Comply
90 ml Comply 90 ml Comply 90 ml Comply
90 ml Comply 90ml Comply 90 ml Comply
90 ml Comply 90 ml Comply 90 ml Comply
4.4 PHYSICOCHEMICAL PARAMETERS OF SYRUP:
Entoban exhibited brown color, characteristic odor and sweet taste and. An imperative practice
of taste masking is used to put off unpleasant drugs from coming in contact with the taste buds
(128). The pH of developed polyherbal syrup was 3.7 and specific gravity of 1.324 (Table 11).
pH determination and other physico-chemical estimation like appearance, plays a noteworthy
79
role in the quality assessment(100). Table 12 depicts various phytochemical components and
preservatives used in syrup.
The stability of a product ensures that the product remains in specified criteria of identity,
strength, quality and purity. Temperature, light, air and humidity can have an effect on stability
(129). The results of stability study of the syrup (Table-13) revealed that no variation was
observed in all the evaluated parameters during 24 hrs, 48 hrs and 72 hrs.
Table 11: Physicochemical parameters of poly herbal syrup.
Physicochemical
parameters Observed Values
Color Brown color syrup
Odor characteristic
Taste sweet
Pourability Good
pH 3.7
Wt/ml at 250C 1.254 g/ml
Table 12: Determination of different components and preservatives in syrup.
Quantitative determination Specified limit
Quantity
present
Determination of total
alkaloids
Total alkaloids in the preparation as Berberine
hydrochloride should not be less than 0.03 %.
0.06%
Determination of tanning
agents
Total tanning agents as gallic acid in the
preparation should not be less than 0.2 %.
0.47%
Methyl paraben Should be within 80% - 120% of the claimed
amount of preservatives
107.04%
Propyl paraben Should be within 80% - 120% of the claimed
amount of preservatives
99.43%
80
Table 13: Stability studies through Physicochemical parameters of poly herbal Syrup.
Sample
Code
Time
Duration
(in hour)
Temperature
Physicochemical parameters
Color Odor Taste pH
Wt/ml
at 250C
Specific
gravity
Turbidity/
Homogeneity
Entb1a
24 hr
40C NC NC NC 3.7 1.254 1.324 NC
Entb1b Room temp NC NC NC 3.7 1.254 1.324 NC
Entb1c 470C NC NC NC 3.7 1.255 1.325 NC
Entb2a
48 hr
40C NC NC NC 3.7 1.254 1.324 NC
Entb2b Room temp NC NC NC 3.7 1.254 1.324 NC
Entb2c 470C NC NC NC 3.7 1.255 1.325 NC
Entb3a
72 hr
40C NC NC NC 3.7 1.254 1.324 NC
Entb3b Room temp NC NC NC 3.7 1.254 1.324 NC
Entb3c 470C NC NC NC 3.7 1.255 1.325 NC
NC = No change
Table 14: Bioburden Analysis of Polyherbal Syrup
Microbial Analysis Limit
CFU/ml Observation
Total aerobic viable
count not more than 104
cfu/ml Comply
Salmonella Absent Absent
Escherichia coli Absent Absent
Staphylococcus aureus Absent Absent
P. aeruginosa Absent Absent
Total fungal count not more than 102 cfu/ml Comply
81
4.5 IN VIVO ANTIMICROBIAL ACTIVITY:
The global dilemma of antimicrobial resistance is predominantly pressing in developing countries,
where the infectious disease burden is elevated and cost constraints put off the appliance of
expensive agents. Traditionally, plants based drugs have proven to be an excellent source of
inspiration for novel drug compounds and extremely successful in the fight against microbial
infections. Polyherbal formulation Entoban syrup was prepared and its in vivo antimicrobial
activity was observed. Screening of anti-microbial activity was carried out by agar well dilution
method. An antimicrobial activity was evaluated against five gram negative bacterial cultures
namely Salmonella enteric, Eschericia coli, Shigella dysenteriae, Pseudomonas aeruginosa ,
Vibrio cholera and one gram positive bacterial culture Staphylococcus aureus. (Table 15) The
prepared syrup inhibited the growth of these organisms. Zone of inhibition of the developed
formulation was comparable with the positive control. (Figure 47)
Table 15: Zone of Inhibition for formulation
Test Organisms Diameter of zone of inhibition (in mm)
1 2 3 4
Mean ± S.D
(n= 4)
Positive
control
Gram positive bacteria
Staphylococcus aureus 19 17 18 19 18.25±0.957 23
Gram negative bacteria
Salmonella enterica 24 21 21 22 22 ± 1.438 26
Eschericia coli 19 19 20 18 19 ± 0.816 23
Shigella dysenteriae 17 20 19 18 18.5 ± 1.290 19
Pseudomonas aeruginosa 20 17 17 19 18.25 ± 1.5 24
Vibrio cholerae 20 19 19 18 19 ± 0.816 25
82
Figure 47: Comparative zone of inhibition of Entoban and ciprofloxacin
4.6 ANTIOXIDANT ACTIVITY OF POLYHERBAL FORMULATION:
DPPH radical scavenging activity increased in a dose dependent manner while evaluating the
formulations of syrup and capsules at different concentrations (Table 16). It was observed that
Entoban syrup and capsules have excellent antioxidant and reducing capability (Figure 48 &49).
The reducing ability of syrup and capsules is illustrated in Table 17. Both syrup and capsules
have reasonable activity to scavenge superoxide radicals at different concentrations (Table 18,
Figure 50).
Table 16: Antioxidant Activity of Syrup, capsules and Standard
Concentration
tested
Percent Activity
(%)(syrup) ±
SEM
Percent
Activity ± SEM
(%)(capsule)
Percent
Activity ± SEM
(%)(standard)
1 10 μg/ml 64.6 ± 0.21 51.5 ± 0.43 71.2± 0.33
2 50 μg/ml 75.2 ± 0.32 76.8 ± 0.54 87.9± 0.41
3 100 μg/ml 84.8 ± 0.65 83.4 ± 0.89 96.8± 0.40
83
Table 17: Reducing Ability of Syrup, capsules and Standard
Concentration
tested
Percent Activity
(%)(syrup) ±
SEM
Percent Activity
(%)(capsules) )
± SEM
Percent Activity
(%)(standard) )
± SEM
1 10 μg/ml 32.5 ± 0.32 43.3 ± 0.12 43.7 ± 0.55
2 50 μg/ml 43.4 ±0.21 45.6 ± 0.65 65.8 ± 0.66
3 100 μg/ml 68.9 ± 0.42 71.2 ± 0.31 87.4 ± 0.92
Table 18: Superoxide scavenging activity of Syrup, capsules and Standard
Concentration
tested
Percent Activity
(%)(syrup) ± SEM
Percent
Activity
(%)(capsule)
Percent Activity
(%)(standard)
1 10 μg/ml 39.6 ± 0.54 21.4 ±0.43 34.2 ± 0.11
2 50 μg/ml 45.8 ± 0.76 32.8 ± 0.55 59.7 ±0.27
3 100 μg/ml 62.3 ±0.91 50.2 ± 0.42 75.2 ±0.29
0
20
40
60
80
100
Syrup Capsules Standard
10 μg/ml
50 μg/ml
100 μg/ml
Figure 48: Comparison of Antioxidant Potential of Formulations with Standard
84
Figure 49: Comparison of reducing ability of Formulations with Standard
Figure 50: Comparison of reducing ability of Formulations with Standard
85
Certain diseases and ageing can be controlled by antioxidant supplementation (130)(131).
Antioxidants originated from natural sources protect the human body from harmful free radicals
(132). Entoban syrup and capsules integrates herbs whose antioxidant activity have already
reported in the literature (133), (134), (135) (136). DPPH radicals is frequently used as screening
technique for evaluating the antiradical activity of compounds (137). DPPH is a stable free
radical that have a distinctive absorption maximum between 515 and 517 nm(138). Certain plant
extracts have direct relationship among antioxidant capability and reducing potential (139) (140).
There is an association between antioxidant and antidiarrheal activities (141). Rahman and
Wilcock revealed that the plants having antidiarrheal potential owed to their reducing potential
(142). World Health Organization has given specifics value to the application of conventional
medicines in the management and treatment of diarrhea, by virtue of its economic viability,
availability and experience of our ancestors.
4.7 INHIBITION OF HELICOBACTER PYLORI-INDUCED INFLAMMATION:
Providentially, H.pylori eradication with antibiotics can consequence in healing ulcer and stop
peptic ulcer (143)(144). A number of drug regimens have been approved for the removal of
Helicobacter pylori with varied combinations of therapeutic agents including bismuth
subsalicylate, antibiotics, H2-blockers and proton pump inhibitors. Nevertheless, resistance to
these antibiotics, particularly clarithromycin and metronidazole restricted their utilization in the
management of infections. Research has shown that about 20% of the patients using antibiotics
treatment would experience therapeutic letdown (145).
86
4.7.1 Non-bactericidal concentration and effects of Entoban on human gastric cells
The viability of H. pylori in the presence of Entoban-S and Entoban-C at different
concentrations was analyzed to determine non-bactericidal concentration and the effect of
Entoban on H. pylori. The results demonstrated no significant effect of both the Entoban
formulation on H. pylori viability at the concentrations of ≤ 500 µg/ml. However, Entoban at
1000 µg/ml showed slight bactericidal effect (Figure 51A and 51B).
AGS cell viability was calculated in a concentration dependent manner at 6 hours of
incubation with Entoban. The results showed non-significant consequence on cell viability at
concentrations of both Entoban formulations at concentration of ≤ 1000 µg/ml (Figure 52) when
evaluated with untreated cells. Therefore, a lower concentration of Entoban (≤ 500 µg/ml was
used for later experiments.
4.7.2 Anti-adhesion activity of Entoban against H. pylori binding to gastric epithelial cells
To examine anti-adhesion effect of Entoban different non-bactericidal concentrations of
Entoban (≤ 500 µg/ml) were evaluated by ELISA on H. pylori-infected gastric cancer cell line
AGS. The results showed that adhesion of H. pylori to gastric epithelial cells was not inhibited
by Entoban pretreatment (Figure 53A and 53B). We consider that any anti-inflammatory effect
of Entoban (≤ 500 µg/ml) on gastric epithelial cells is not an outcome of bacterial viability
alteration or not by inhibition of bacterial adhesion.
87
Figure 51(A &B): Effect of Entoban syrup (Entoban-S) and capsule (Entoban-C) formulation on viability
of two clinical strains of Helicobacter pylori (193C and NCTC). Bacteria were left untreated or treated
for 1 hour, then serially diluted and plated for 2 - 3 days at specified condition. At concentration ≤ 500
µg/ml viability of both strains of H. pylori, (A) H. pylori 193C and (B) H. pylori NCTC, is not affected by
any of the Entoban formulations Result represent percentage survival CFU‘s. *p < .05 (compared to
control). (n = 3)
88
Figure 52: Effect of Entoban syrup (Entoban-S) and capsule (Entoban-C) formulation on gastric
epithelial cells. At ≤ 500 µg/ml Entoban had mild but non-significant cytotoxic effect on AGS cells. *p <
.05 (as compared to control). (n = 3)
89
Figure 53(A&B): Anti-adhesion activity of Entoban syrup (Entoban-S) and capsule (Entoban-C) formulation
against Helicobacter pylori (193C and NCTC) binding to AGS cells. Cells were treated with various concentrations
of Entoban (125 – 500 µg/ml) for 60 minutes. Even at high concentration (500µg/ml) Entoban had no significant
anti-adhesion activity against of both strains of H. pylori, (A) H. pylori 193C and (B) H. pylori NCTC. Each value
represents the mean ± SD (n = 3).
90
4.7.3 Effect of Entoban on H. pylori-induced IL-8 secretion
IL-8 is a critical chemokine accountable in mediating H. pylori-induced inflammatory
response in gastric mucosa (146). Earlier it was explored the pharmacological basis of
Cinnamomum cassia by investigating the effect of its component active compound
cinnamaldehyde (CM) on IL-8 secretion in H. pylori-infected gastric epithelial cells. At a
concentration of 250 µM, CM revealed strongest inhibitory activity against IL-8 secretion in H.
pylori-infected gastric cells(106).
In current study anti-inflammatory effect of two formulations of Entoban (Syrup and
Capsule) was estimated. To find out anti-inflammatory activity of Entoban, low non-bactericidal
concentrations (≤ 500 µg/ml) of Entoban was used. There was a strong increase in IL-8 content
in H. pylori-infected cells when there was no drug as compared to the uninfected cells which was
suppressed by Entoban in a concentration-dependent manner (Figure 54). At 500 µg/ml of
Entoban-S, the maximum suppression of IL-8 secretion was observed (p < 0.01) whereas only
mild suppression was observed at 500 µg/ml of Entoban-C (p < 0.05).
Zaidi et al. reported bactericidal activity of 50 native Pakistani therapeutic plants
including two constituents of Entoban (i.e., B. aristata and M. communis) against H. pylori
which are usually prescribed in Unani system of medicine to treat various gastrointestinal (GI)
disorders (82). However, several medicinal plants including B. aristata and M. communis
exhibited less anti-H. pylori activity. In this study also, B. aristata and M. communis along with
other constituents of Entoban showed very weak anti-H. pylori activity against two H. pylori
clinical strains not tested in previous study.
91
Figure 54: Dose-dependent inhibitory effect of Entoban syrup (ES) and capsule (EC) formulation on IL-8
secretion from Helicobacter pylori-infected gastric epithelial cells. Cells were pretreated with
concentrations of 125-500 µg/ml and supernatants from H. pylori-co-cultured cells were analyzed for IL-
8 content. At 500µg/ml of ES showed significant suppression of IL-8 secretion, while mild suppression
was seen at 500 µg/ml of EC. Each value represents the mean ± SD (n = 3). **p < .01, *p < .05
(compared to untreated H. pylori-infected cells).
As, these plants are broadly prescribed for GI disorders; it was assumed that Entoban might
possess anti-inflammatory activity against H. pylori-induced gastric inflammation. Therefore in
the current study it was examined whether Entoban could modulate IL-8 secretion from the H.
pylori-infected gastric cells and urease inhibition. We showed that Entoban dose-dependantly
and significantly inhibits IL-8 secretion from H. pylori-infected gastric epithelial cell.
92
Previously, one group also reported cytoprotective activities and anti-inflammatory of twenty-
five selected medicinal plants originated from Pakistan including B. aristata and M.
communis(147). M. communis inhibited IL-8 secretion and also suppressed reactive oxygen
species generation in H. pylori-infected gastric epithelial cells. This data supports and justify the
medicinal use of Entoban in inflammatory GI diseases.
HPLC data showed that both formulations of Entoban consisted gallic acid and berberine
in abundant quantitiy. Gallic acid is a non-flavonoid polyphenol found abundantly in many fruits
and berries. A study by Díaz-Gómez et al. reported significant decrease in H. pylori viability
after 30 min of incubation with 1mg/ml of gallic acid (148). Berberine was supposedly used in
traditional Chinese medicine as a broad-spectrum anti-microbial medicine. In vitro study
reported minimum inhibitory concentration of berberine to be as low as 200µg/ml against the
clinical strains of H. pylori (149). We also found that both gallic acid and berberine possess
stong anti-H. pylori activity at high concentrations. Furthermore, pretreatment of gastric cell with
low concentrations (<200µg/ml) of gallic acid and berberine did not showed any anti-
inflammatory activity against H. pylori induced IL8 secretion (data not shown). This suggested
that anti-inflammatory activity of Entoban is due to some other compound besides gallic acid or
berberine.
4.8 ANTIUREASE ACTIVITY:
Plant based drugs are an unexploited affluence for the innovation of compounds to treat diverse
diseases (150-152). Antiurease activity of Berberis aristata, Querecus infectoria and Helicteres
isora has already been reported in the literature(82, 153). Entoban syrup and capsules possess
antiurease activity like standard Thiourea. (Table 19) Entoban syrup and capsules have excellent
93
antiurease potential. (Figure 55) It is obvious from Table 13 that the capsules are having more
potential of antiurease activity as compared to syrup. Urease inhibitors can act by binding in a
substrate or active-site directed mode or by binding in a non-substrate like or mechanism-based
directed mode. Thiourea and hydroxyurea are the main examples of the substrate-like urease
inhibitors. Most of the early inhibitors of urease contained strongly basic groups such as mimics
of the amide bond of its substrate molecule i.e. urea. Hydroxamic acid derivatives and
phosphazenes are also substrate like inhibitors(154) (155).
Table 19: Antiurease Activity of Syrup, capsules and Standard
Concentration
tested
Percent Activity
(%) of syrup
Percent Activity
(%) of capsules
Percent Activity (%)
of Standard
1 10 μg/ml 33.9 55.6 64.5
2 50 μg/ml 64.9 71.6 76.5
3 100 μg/ml 75.9 83.5 89.9
Figure 55: Assessment of urease inhibitory activity of the Entoban syrup (Entoban-S) and capsule
(Entoban-C) formulations. Anti-urease activity increased in a dose dependent manner for both
formulations. Thiourea is used a positive control for this experiment.
94
Urease inhibitors have attracted a great deal of attention in recent times as potential innovative
anti-ulcer drugs (156). It is a significant factor for gastric ulcers as it counteracts the gastric acid
locally by the producing ammonia, consequently providing a suitable microenvironment for the
continuation of H. pylori (157)(158). Owed to the diversity in enzyme functions, the inhibition of
it by compelling and explicit compounds could direct the management of infections due to
urease-producing bacteria(159).
Urease is an important antigen of the H. Pylori and act as an influential immunogen for the
organism. H. pylori is acid sensitive and only imitates at pH of 7-8, it continues to exist in the
stomach in extremely acidic conditions. Urease action in bacteria is supposed to be fundamental
for the colonization of and survival of H. pylori at very acidic pH(160). Thus virulence of H.
pylori could be controlled by means of chemicals that restrain urease activity(161), (154)(162).
Hence, scientists are persistently incisive for compounds that can inhibit urease enzyme (163).
Consequently, looking for innovative and effectual urease inhibitors with excellent
bioavailability and minimum toxicity are of great significance particularly in low income
countries with escalated infection rate of H. pylori is desirable.
4.9 LIPOXYGENASE INHIBITION ACTIVITY:
Table 20 depicted lipoxygenase inhibition activity for both formulations. However the Entoban
syrup revealed better lipoxygenase inhibition activity as compared to Entoban capsules. At 10
µg/ml syrup possess 31.2% whereas capsules possess 12.3% inhibition activity. At 50 µg/ml
syrup exhibit 45.6% inhibition activity whereas capsules 32.4%. At 100 µg/ml syrup possess
67.3% inhibition activity whereas capsules possess 45.6% inhibition activity. The standard
95
bacilein revealed 61.3%, 73.4% and 85.3% lipoxygenase inhibition activity at concentrations of
10, 50 and 100 µg/ml respectively. Entoban syrup and capsules have good lipoxygenase
inhibition potential when compared to bacilein. (Figure 56) LTs are deemed as compelling
mediators of hypersensitivity and inflammatory reactions (164). Numerous studies have revealed
that LTs may participate significantly in the development of pathological conditions including
kidney stones, pyelonephritis, peptic ulcer and other inflammatory diseases of digestive tract
(165). Concerning their pro-inflammatory possessions the inhibition of 5-lipoxygenase pathway
is believed to be remarkable in the management of inflammatory diseases (166). Owing to the
increase production of LTs concerned in several inflammatory diseases, there has been
substantial interest in the generation of 5-LO inhibitors intended for therapeutic purpose. The
compounds recognized as 5-LO inhibitors can be divided into antioxidants, substrate-analogous,
and miscellaneous grouping of inhibitors(167).
Though exercise of using numerous anti-inflammatory drugs is in vogue, the persistent use of
these for an extended period of time can have unfavorable side effects. Hence, there is need to
explore substitution approaches to decrease the production of inflammatory mediators with
natural dietary products (150-152). Many phenolic/flavonoid compounds originated from
vegetables source are revealed to modulate 5-LO and prostaglandin H synthase pathways of
arachidonic acid (167).
It was found that Entoban capsule can rescue the cell protection at the dose of 300µg/ml around
78-80% in the HepG2 cells (Figure 57). The activity of 1-ethyl brachiose-3‘-acetate and
triacontyl palmitate present in Symplocos racemosa displayed the inhibitory potential against
96
lipoxygenase enzyme in a dose dependent manner(168). Research has shown that owing to the
active constituents of galls of Querecus infectoria comprises a large amount of tannins, the
activity of the Querecus infectoria the aphthous powder and aphthous gel could inhibit the
synthesis of the inflammatory mediators such as IL-6 and PGE2 (169).The present research
confirms the similar findings that both formulations of Entoban executing in vitro liopxygenase
inhibition due to the presence of herbs depicting such activities. Enzyme inhibition is a
noteworthy part of pharmaceutical research leading to the innovations of drugs having
remarkable performance in diverse physiological conditions(170). Entoban syrup and capsules
revealed good anti lipoxygenase potential at various concentrations. However the Entoban syrup
revealed better lipoxygenase inhibition activity as compared to Entoban capsules.
It was also found that Entoban capsule at the dose 300µg/ml concentration has cytoprotective
activity (approx. 76%), to rescue the cell viability after induction of apoptosis by using strong
oxidant agent, cadmium. Previous studies conducted in vivo and in vitro also indicated that
similar plants used in the formulation of capsule and syrup, shown anti-oxidant and
hepatoprotective effects (170, 171).
Table 20: Lipoxygenase Inhibition Activity of Entoban Syrup, capsules and standard
Concentration tested Percent Activity (%)
syrup
Percent Activity (%)
capsules
Percent Activity (%)
standard
10 μg/ml 31.2 12.3 61.3
50 μg/ml 45.6 32.4 73.4
100 μg/ml 67.3 45.6 85.3
97
Figure 56: Comparison of Lipoxygenase inhibition potential of Formulations with
Standard Error
Figure 57: Anti-oxidant effect of Entoban capsule and syrup on the HepG2 cell line by cck-8
cell viability assay. NT showed non-treated cells and Cd was used as a positive control and the
drugs were examined in 3 different concentrations.
98
4.10 QUANTITATIVE ESTIMATION OF BIOMARKERS IN ENTOBAN CAPSULES:
Quantitative analysis of gallic acid was conducted by TLC, using silica gel 60 F254 coated plates
as a stationary phase to augment the identification and determination of gallic acid components.
Thin layer chromatographic analysis of gallic acid was conducted by using toulene- ethyl acetate
– formic acid –methanol in the ratio of 12:9:4:0.5 (v/v/v/v) as a solvent system. After developing
and drying, the plates were observed under UV light for the presence of gallic acid, which was
detected by prominent dark brown color spot (Figure 58). The Rf value (0.58) of gallic acid in
both sample (Figure 59) and reference standard (Figure 60) was found comparable under UV
light at 273 nm. HPTLC was executed to confirm the quantitative presence of berberine
employing ethanol: water: formic acid in the ratio of 90:9:1 (v/v/v) as a solvent system at a
wavelength of 366 nm. After developing and drying, the plates were observed under UV light for
the presence of berberine. The Rf value (0.76) of berberine in both sample (Figure 62) and
reference standard (Figure 63) was found comparable under UV light at 366 nm. Specific
biologically active gallic acid and berberine components were identified in the poly herbal
formulation while quantitative assay measures the level of biomarker in the capsules thereby
establishing the standard of those particular compounds for validation.
K. Dhalwal developed a simple TLC method for the concurrent quantification of catechin,
bergenin and gallic acid from different parts of Berberis ciliata and B. ligulata using HPTLC. He
proposed that method was found to be accurate, precise and specific(172). In the current study
HPTLC densitometric analysis of gallic acid was conducted by using toulene- ethyl acetate –
formic acid –methanol in the ratio of 12:9:4:0.5 (v/v/v/v) as a mobile phase. Sample preparation
and development of appropriate mobile phase are two imperative stages in analytical procedures,
which becomes more considerable for plant based medicines owing to their complexity of the
99
chemical compounds and their affinity towards different solvent systems (173). The Rf value
(0.58) of gallic acid in both sample and reference standard was found comparable under UV light
at 273 nm. HPTLC was also performed to confirm the quantitative presence of berberine (Rf
value 0.76) employing ethanol: water: formic acid 90:9:1 (v/v/v) as a solvent system at a
wavelength of 366 nm.
TLC densitometry methods were also developed by Shah for the quantification of two marker
compounds berberine and gallic acid for standardization of the polyherbal formulation,
Punarnavashtak kwath. He reported that results of estimation berberine and gallic acid were
found to be 0.08 and 4.9%, respectively (174). Solvent systems were optimized to achieve finest
resolution of the marker compounds from the sample extracts (175). A quantitative HPTLC
method for analysis of gallic acid and tannin in extracts of Arctostaphylos uva-ursi (L.) Sprengel,
bearberry leaves (Ericaceae), and validation of the method, were also described by Renata
Slaveska. He reported HPTLC method to be simple, reliable, and convenient for routine analysis
(176).
HPTLC technique is generally applied in the pharmaceutical industry in the development process
, identification and detection of adulteration of herbal products and helps in identifying content
of pesticides, mycotoxins and quality control of herbs and healthy food (177). Sachin U Rakesh
proposed that HPTLC method enables a high-quality resolution of gallic acid from other
components present in hydroalcoholic extract of N. stellata and can be used for quantization of
gallic acid (98.33%) (178). HPTLC technique was reported for simultaneous evaluation of Rutin,
gallic acid, quercetin in Terminalia chebula (179).
100
Figure 58: TLC image of Gallic Acid in Entoban Capsule
Figure 59: Peak response of Gallic acid in Entoban capsules
101
Figure 60: Peak response of Gallic acid Standard
Figure 61: TLC image of Berberine in Entoban Capsule
102
Figure 62: Peak response of Berberine in Entoban capsules
Figure 63: Peak response of Berberine Standard
103
4.11 QUANTITATIVE ESTIMATION OF BIOMARKERS IN ENTOBAN SYRUP:
In the current study quantitative estimation of gallic acid and berberine components were
conducted in the poly herbal formulation using HPTLC. Among the different solvent systems
tried the solvent system containing toulene- ethyl acetate – formic acid –methanol in the ratio of
12:9:4:0.5 (v/v/v/v) resulted in good separation of the gallic acid. TLC plate was observed under
UV light for the presence of gallic acid, which was detected by prominent dark brown spots. The
spots developed were dense, compact and typical peaks of gallic acid were obtained. The Rf
value (0.58) for gallic acid in both sample (Figure 64) and reference standard (Figure 65) was
found comparable under UV light at 273 nm.
Different solvent systems were used for the detection of berberine of which; the solvent system
containing ethanol: water: formic acid in the ratio of 90:9:1 (v/v/v) resulted in good resolution of
berberine in the presence of other compounds in formulation. TLC plate was observed under UV
light for the presence of berberine, detected by prominent violet color spot. The Rf value (0.76)
for berberine in both sample (Figure 66) and reference standard (Figure 67) was found analogous
under UV light at 366 nm. The method employed in current study resulted in good peak shape of
berberine and gallic acid. Specific biologically active gallic acid and berberine components were
identified in the poly herbal formulation thereby establishing the standard of those particular
compounds for validation.
Herbal medicines are usually obtainable as a mixture of more than one plant constituent and its
therapeutic activity depends on its phytochemical constituents (180). Accurate identification and
quality reassurance is an indispensable requirement to make sure reproducible quality of herbal
medicines (177). Phytochemical assessment signifies the quality measurement, including
104
preliminary phytochemical analysis, chemoprofiling, and marker compound analysis employing
innovative investigative techniques. HPTLC is a significant tool for the quantitative
phytochemical analysis of the naturally occurring drugs (181).
In current study the Rf value (0.58) of gallic acid in both sample and reference standard was
found comparable under UV light at 273 nm. The gallic acid inhibits different forms of
microbiological organisms so it is useful in acute gastroentitis. It is already reported in the
literature that Berberis aristata contain biomarker berberine, a quaternary alkaloid which has
antibacterial, antiamoebic, antifungal, antihelminthic, leishmanicidal and tuberculostatic
properties(182). HPTLC was performed to confirm the quantitative presence of berberine (Rf
value 0.76) employing ethanol: water: formic acid 90:9:1 (v/v/v) as a solvent system at a
wavelength of 366 nm. Sample preparation and development of appropriate mobile phase are
two imperative stages in analytical procedures, which becomes more considerable for plant
based medicines owing to their complexity of the chemical compounds and their affinity towards
different solvent systems(173). Therefore in present study the development of mobile phases for
biomarkers were optimized by using the appropriate mixture of solvents.
Standardization promise constant composition of all herbals including analytical operations for
identification, markers and assay of active principles(183). Different researchers have proposed
that HPTLC method enables high-quality resolution and can be used for quantization of
biomarkers. HPTLC method was found to be simple, reliable, and convenient for routine analysis
(178, 179), (176). The present work confirms such findings. It has advantages that include its
simplicity, accuracy and selectivity (184-186).
105
Figure 64: Peak response of gallic acid in Entoban syrup
Figure 65: Peak response of gallic acid standard
106
Figure 66: Peak response of berberine in Entoban syrup
Figure 67: Peak response of berberine standard
107
4.12 HEAVY METAL CONTENTS OF ENTOBAN HERBAL MEDICINAL PRODUCT
The element group that has been shown to be toxic when consumed by humans and these are
specified as Arsenic (As) Cadmium (Cd), mercury (Hg) and lead (Pb). The elements specific
quantity cause different types of diseases and may influence with the absorption of useful metals
such as calcium and zinc. The average concentration of As, Cd, Pb and Hg were analyzed in
three different batches of syrup samples are given in Table 21. The range of different heavy
metals detected in these plants varies in case of arsenic (AS) 0042 to 0.0884 ppm, cadmium (Cd)
0.015 to 0.020 ppm, Lead (Pb) 0.023 to 0.074 ppm and mercury 0.016 to 0.020 ppm. All these
are quite below the permissible limit of As 10 ppm, Cd 0.3 ppm, Pb 10 ppm and Hg 1 ppm.as
prescribed in all the samples. Metal content in all these samples was found to be within these
limits. American Herbal Product Association guidelines on maximum quantities limit for orally
consumed herbal supplements have cited the limit mg/day for As 10 ppm, Cd as 4.1 ppm, Pb as
10 ppm and methyl mercury as 2.0 ppm(187). All these herbal ingredients are also a reputed
medicinal herb of Pakistan which herbal ingredients cultivated and has been in use to
combat diarrhea, as astringent and infection. In some ingredients of Entoban some of the
elements were not detected which clearly shows the plant part components utilized was even
devoid of metals in these experiments.
108
Table 21: Heavy metal contents (ppm, Mean +SEM ) of plants Component of Entoban syrup
product
Plant Specification Heavy metals
Aegle marmelos Arsenic: Not more than 10 ppm
Cadmium: Not more than 0.3 ppm
Lead: Not more than 10 ppm
Mercury: Not more than 1 ppm
0.084 ppm
Not detected
Not detected
Not detected
Berberis aristata Arsenic: Not more than 10 ppm
Cadmium: Not more than 0.3 ppm
Lead: Not more than 10 ppm
Mercury: Not more than 1 ppm
0.074 ppm
0.020 ppm
Not detected
Not detected
Butea frondosa Arsenic: Not more than 10 ppm
Cadmium: Not more than 0.3 ppm
Lead: Not more than 10 ppm
Mercury: Not more than 1 ppm
Not detected
Not detected
0.051 ppm
0.020 ppm
Holarrhena
antidysenterica
Arsenic: Not more than 10 ppm
Cadmium: Not more than 0.3 ppm
Lead: Not more than 10 ppm
Mercury: Not more than 1 ppm
0.042 ppm
0.016 ppm
0.030 ppm
Not detected
Myrtus cmmunis Arsenic: Not more than 10 ppm
Cadmium: Not more than 0.3 ppm
Lead: Not more than 10 ppm
Mercury: Not more than 1 ppm
Not detected
Not detected
0.023 ppm
0.016 ppm
Quercus infectoria Arsenic: Not more than 10 ppm
Cadmium: Not more than 0.3 ppm
Lead: Not more than 10 ppm
Mercury: Not more than 1 ppm
Not detected
0.015 ppm
0.074 ppm
Not detected
Figure 68: Heavy metal contents in Plants of Entoban syrup
109
The safety and quality of these herbal medicines has become incrementally important for health
authorities, academia and the public alike. Dzomba et al conducted study to verify the amount of
heavy metals in raw materials selected in traditional medicines. Heavy metal content in these
herbal medicinal herbs has been reported that, in the range of 0.23 to 19.01 for Pb, 0.12 to 0.39
for Cu, 0.25 to 1.30 for Zn, 0.01 to 0 14 for Ni, 1.41 to 30.84 for Fe and 0.01 to 0.46 mg Kg -1
for AS. Concentrations of heavy metals found above the permissible values were: 19.01 for Pb
in the Uapaca. kirkiana bark and roots, Uapaca. kirkiana cortex (12 25 ± 0.01), roots (12,11 ± 0.
00) and Ocimum americanum leaves (33.61. ± 0.07) and roots (30.84 ± 0.02) for iron. That most
drugs were found toxic and unsafe for human consumption primarily due to elevated
concentrations of heavy metals, Cu, Fe, As and Pb (188).
Naithani and co-workers have examined the presence of lead, cadmium, chromium, nickel,
arsenic and mercury in Azadirachta indica and Curcuma longa contents of a polyherbal product
Ampucare. All metals were within these limits, therefore Ampucare was found to be safe
(189)(190).
Baye et al worked for Ni, Co, Cu, Cr and As to evaluate the content of eight medicinal plants in
Ethiopia. Out of 26 samples studied, three (11.5%) Ni (10.42 ± 0.21 to 11.25 ± 0.01 mg / kg) and
nine (~ 34.6%) to Cr (± 2.77 0.06 to 13.24 ± 0.21 mg / kg) containing the 10 and 2 mg / kg upper
limits. None of the samples concentrations were found in cobalt, copper and arsenic contained in
the foregoing WHO limit for safe human consumption (25, 40 and 5 mg / kg) (191).
Singh and colleagues cited quantitative analyzes the level of six heavy metals such as arsenic
(As), lead (Pb), cadmium (Cd), mercury (Hg), chromium (Cr) and nickel (Ni) in ten Indian
medicinal plants. Vegetable powders were subjected to microwave assisted wet fermentation and
110
samples were analyzed by atomic absorption spectrophotometry. The results showed that all six
heavy metals were below the permissible limits in all the ten medicinal plants have been studied
(192). Ziarati conducted study to determine the level of Cd, Pb, Ni, Cu, Hg determine in natural
medicine and medicinal plants products in Iran. The 6 different herbs and 6 herbal plants
permissible limits (PL) were determined according to the acceptable Daily Intake (ADI) and the
provisional maximum tolerable (PMTDI) daily intake as set forth by the WHO (193). Saper and
colleagues studied the concentration of heavy metals and shown that 5 Ayurvedic HMPs
includes potentially harmful levels of lead, mercury and / or arsenic (194).
The growth of medicinal plants not only need nutrients for normal plant growth, but the selective
absorption of the possibility of toxic heavy metals can transmitted in human by herbal medicines
grown in the polluted affected areas is the concern for undesirable activity spam. From the
above discussion, though some studies have shown that elements not within the limits as
required clearly show that medicinal plants are used for human consumption or for the
production of herbal medicines must be obtained from a pristine natural habitat. It is advisable
that the large-scale cultivation of medicinal herbs can be promoted and used in the manufacture
of design dosage form. As desired to obtain a therapeutic benefit, the quality of these vegetable
raw sources in terms of heavy metals must ensure determinants.
4.13 ANTIDIARRHEAL ACTIVITY OF ENTOBAN:
4.13.1 Effect of Entoban on castor oil induced diarrhea:
During 4 hours, every mouse produced copious diarrhea in control group. Different doses of test
product (Entoban) caused decrease in the rate of purging and weight of wet stools which was
dose dependent. Entoban showed 59 %, 79.51 %, 91.3 % inhibition of diarrhea at doses of 2.5
111
mg/kg, 5 mg/kg and 10 mg/kg while loperamide at dose of 2 mg/kg showed 93 % inhibition of
diarrhea as shown in Table 22.
4.13.2 Effect of Entoban on magnesium sulphate induced diarrhea:
Every mice in control group produced diarrhea subsequent to the administration of magnesium
sulphate. Entoban showed 45.9 %, 73.07 %, 85.42 % inhibition of diarrhea at doses of 2.5
mg/kg, 5 mg/kg and 10 mg/kg whereas loperamide at dose of 2 mg/kg showed 89.96 %
inhibition of diarrhea (Table 23).
4.14 ACUTE TOXICITY STUDIES:
No mortality in NMRI albino mice was reported following the administration of the Entoban at
the given doses of 1 or 5 g/kg. Other signs of toxicity like loss of hair, reduction in weight,
mucus membrane (nasal), lacrimation, drowsiness, gait and tremors were also not observed. The
test product (Entoban) appeared to be safe on doses 1g/kg or 5g/kg.
4.14.1 Sub chronic toxicity:
The tested product (Entoban) was given for 28 days. The body weights of the treated animals
were observed at 0, 7, 14 and 28 day of the doses. The weight of animals in control group
slightly increases. There was not any noticeable change in the weights of tested animals.
World Health Organization has given specifics value to traditional medicines in the management
and treatment of diarrhea, by virtue of its economic viability, availability and experience of our
ancestors(195). Diarrhea occurs when the intestine secrete more electrolytes and
water(196)(197).
112
Table 22: Effects of Entoban on Castor oil induced diarrhea in mice
Group Dose
(/Kg)
Onset of
diarrhea
(min)
Total weight
of
stools (g)
Weight of
wet
stools (g)
Total
number
of stools
Number of
wet
stools
% Inhibition
Control
52 ± 2.21 0.362 ± 0.010 0.325 ± 0.020 13.43 ± 0.23 12.00 ± 0.56
Entoban 2.5 mg 85 ± 2.06 0.157 ± 0.005 0.14 ± 0.006 6.15 ± 0.31 4.92 ± 0.210 59*
Entoban 5 mg 112 ± 4.57 0.121 ± 0.048 0.081 ± 0.036 3.46 ± 0.24 2.46 ± 0.011 79.51**
Entoban 10 mg 172 ± 4.28 0.050 ± 0.012 0.035 ± 0.002 1.14 ± 0.26 1.04 ± 0.15 91.3***
Loperamide 2 mg 213 ± 5.12 0.036 ± 0.003 0.030 ± 0.003 1.04 ±0.23 0.84 ± 0.14 93 ***
Results were analyzed by Student‘t-test. * p < 0.05; ** p < 0.01; *** p < 0.001 vs control.
Table 23: Effects of Entoban on Magnesium sulphate induced diarrhea in mice
Group Dose(/Kg)
Onset of
diarrhea
(min)
Total weight
of
stools (g)
Weight of
wet
stools (g)
Total
number
of stools
Number of
wet
stools
%Inhibi
tion
Control
43 ± 1.20 0.392 ± 0.060 0.285 ± 0.060 11.14 ± 0.53 9.47 ± 0.32
Entoban 2.5 mg 87 ± 3.01 0.251 ± 0.010 0.14 ± 0.003 6.15 ± 0.43 5.12 ± 0.120 45.9*
Entoban 5 mg 116 ± 1.45 0.136 ± 0.043 0.085 ± 0.054 3.46 ± 0.36 2.55 ± 0.310 73.07**
Entoban 10 mg 192 ± 2.12 0.045 ± 0.014 0.035 ± 0.024 2.23 ± 0.17 1.38 ± 0.09 85.42***
Loperamide 2 mg 232 ± 3.01 0.036 ± 0.023 0.020 ± 0.013 1.85 ±0.23 0.95 ± 0.34 89.96***
Results were analyzed by Student‘t-test. * p < 0.05; ** p < 0.01; *** p < 0.001 vs control.
Research has shown that different parts of H. antidysenterica executed various medicinal
properties(63) (198). D Kavitha reported that alkaloids of H. antidysenterica reduced diarrhea in
castor oil induced rats(63). Shamkuwar revealed that aqueous extract of Berberis aristata treated
mice, considerably reduced the stimulation time of diarrhea (199).
113
Entoban showed 59 %, 79.51 %, 91.3 % inhibition in castor oil induced diarrhea and 45.9 %, 73.07
%, 85.42 % inhibition in magnesium sulphate induced diarrhea at the doses of 2.5 mg/kg, 5 mg/kg
and 10 mg/kg. No mortality in NMRI albino mice was reported following the administration of the
Entoban at the given doses of 1 or 5 g/kg. Other signs of toxicity like loss of hair, reduction in
weight, mucus membrane (nasal), lacrimation, drowsiness, gait and tremors were also not
observed. The test product (Entoban) appeared to be safe on doses 1g/kg or 5g/kg. Entoban
possesses antimotility and antisecretory activity. Results of the present study gave evidence of
good tolerance of Entoban and the absence of detrimental effects on the functional state of the vital
organs of the experimental animals in acute and sub chronic oral toxicity test.
4.15 CLINICAL EVALUATION OF ENTOBAN:
A current study enrolled 150 patients but 10 in the test group and 7 in the control group did not
receive the allocated treatment due to unknown reasons. Further 13 were dropped out during the
treatment and 8 discontinued intervention due to side effects in control group. In test group, 15
were dropped out during the treatment and 4 discontinued intervention due to side effects. Overall
47 and 46 in control and test group completed the study (Figure 69).
Both treatment options receiving Entoban and Metronidazole were evaluated for diarrheal
symptoms and no significant difference was observed between the two with respect to age,
duration of disease, and symptom scoring, in addition to daily bowel frequency (3.89±1.05 in test
group, 3.41±1.35 in control group; p=0.54). Mean and standard deviation of the ages of
participants for test and control groups were 25 ± 11.86 years and 23 ± 13.76 years, respectively.
Mean height of group one and two was 152 ±7.54 cm and 149 ± 12.76 cm, respectively. Mean
114
weight of all the participants was 56 ± 9.7 kg (Table 24). The skewness and kurtosis of the data
were 0.67 and 0.16, respectively.
Figure 69: Flow diagram of randomization, allocation, follow-up, and analysis.
115
Table 24: Baseline characteristics of patients who completed the study
Characteristics Treatment
Group 1
Treatment
Group 2 Test of significance p value
Herbal
(n=46)
Allopathic
(n=47)
Age(years) 25 ±11.86 23 ±13.76 0.368 Student t test 0.7
Weight (kg) 56 ±9.7 53 ±10.5 1.32 Student t test 0.342
Height (cm) 152 ±7.54 149 ±12.76 0.041 Student t test 0.654
Gender
Male 28(60.86%) 26(55.31%) 0.132 (Chi Square) 0.719
Female 18(39.13%) 21(44.68%)
Marital status
Married 26(56.52%) 23(48.93%) 1.107 (Chi Square) 0.293
Unmarried 20(43.47%) 24(51.06%)
The standard error of mean was 0.95 and inconsistent age was normally distributed. At the 2nd
week of treatment, it was observed that the mean bowel frequency was significantly lower in the
test group than in the control group (1.88±1.24 vs 2.64±1.12, p<0.05). The variation was
confirmed at the 4th
week (1.39±0.92 in the test group vs 2.19±1.05 in the control group; p<0.05)
(Figure 70). The study revealed that 39(84.78%) in test group and 37(78.72%) in control group
showed complete improvement (Table 25).
116
Figure 70: Mean bowel frequency of participants
Table 25: Improvement of symptoms in herbal and allopathic group
Treatment Total Complete
Improvement
Slight
improvement
No
improvement
Chi
square
value
p value
Herbal 46 39(84.78%) 4(8.69%) 3(6.52%)
0.35 0.62
Allopathic 47 37(78.72%) 6(12.76%) 4(8.51%)
Watery Stool is most common factor in diarrhea. Wherein, there were 21 patients recorded for
complains watery stool treated with Entoban. After the treatment with herbal drug formulation
Entoban, out of 21 recorded cases the complete improvement showed by 16 cases, and 5 cases
recorded in slight improvement, while there was no any patient who did not respond to this drug
and therefore no case recorded for no improvement and these 21 patients on second follow-up did
not complain for the watery stool. Watery stool is usually a form of diarrhea.
117
With the Entoban oral administration 5 patients were registered for Feeling of incomplete
Evacuation in which 3 patients out of them after the treatment reported complete improvement and
2 patients revealed slight improvement and these patients on repeated dose did not complain for the
Feeling of Incomplete Evacuation
Stool with Mucus is another complain of diarrhea and was commonly observed during clinical
trials. There were 25 patients registered with this complain. As the result indicated the
improvement levels in stool with mucus, so it was observed that 17 patients recorded as completely
improved out of 25 patients, slight improvement was recorded in 6 patients and there was no
failure complaint. The appearance of mucus in stool was determined as an important symptom, as
it determines whether actually the mucus was present in the stool or blood, fat or pus was also
present in stool.
Participants in the test group with complete improvement exhibited significant decreases in overall
GI symptoms from baseline (T0)—with a median of 8 and an IQR of 6 to 10, to week 2 (T2)—with
a median of 3 and an IQR of 2 to 5, and to 1 month after treatment (T4)—with a median of 4 and an
IQR of 3 to 6 (Figure 71). A significant decrease in symptoms was observed for participants in the
test group with no improvement, also from T0—with a median of 9 and an IQR of 6 to 10, to T2—
with a median of 3 and an IQR of 2 to 5, and to T4—with a median of 4 and an IQR of 3 to 6
(Figure 72). The intensity of individual symptoms in the test group was monitored and statistically
significant improvement was recorded after treatment (Table 26). Participants in control group
with improvement exhibited a statistically significant reduction in the overall diarrheal symptom
score, from T0—with a median of 9 and an IQR of 6 to 10, to T2—with a median of 4 and an IQR
of 3 to 6, and toT4—with a median of 4 and an IQR of 3 to 7 (Figure 71). No significant
improvement in symptoms was observed, however, for the participants with no recovery, showing
118
scores from T0—a median of 9 and an IQR of 6 to 10, to T2—a median of 6 and an IQR of 4 to 8,
and to T4—a median of 8.5 and an IQR of 5 to 10 (Figure 72). In control group, the intensity of
individual symptoms was recorded in the course of treatment and is given in Table 27.
Figure 71: Overall severities of symptoms at baseline (T0), two weeks after treatment (T2) and one
month after treatment (T4) by herbal and allopathic therapy in patients who show complete
improvement. Horizontal bar: median; box: 25–75th interquartile range; vertical lines: range of
values. a p < .001. b p < .0001.
119
Figure 72: Overall severity of symptoms at baseline (T0), two weeks after treatment (T2) and one
month after treatment (T4) by herbal and allopathic therapy in patients with no improvement.
Horizontal bar: median; box: 25–75th interquartile range; vertical lines: range of values.
a p < 0.01. b p <0 .001 c p < 0.0001.
Table 26: Overall Improvement of symptoms for patients in herbal group
Symptom
To T2
p Value
T4
p Value
Median
Interquartile
range Median
Interquartile
range Median
Interquartile
range
Abdominal pain 2.5 2-3 1 0-2 <0.0001 1 1-2 <0.0001
Anorexia 2 1-3 1 1-2 <0.001 0.5 0-1 <0.0001
Nausea/vomiting 2 1-3 1 0-2 <0.001 1 1-2 <0.001
Flatulence 2 2-3 1 1-2 <0.001 1 1-3 <0.001
Rectal urgency/
Incontinence 2.5 2-3
1.5
1-2 <0.001 0.5 0-2 <0.0001
Bloating 2 2-3 1 1-2 <0.001 1 1-2 <0.001
T0, baseline; T2, 2 wks after start of treatment; T4, 4wks after start of treatment.
120
Table 27: Overall Improvement of symptoms for patients in allopathic group
Symptom
To T2
p Value
T4
p Value
Median
Interquartile
range Median
Interquartile
range Median
Interquartile
range
Abdominal pain 2.5 2-3 1.5 1-2 <0.001 1 1-2 <0.0001
Anorexia 2 2-3 1 1-2 <0.001 1 0-1 <0.001
Nausea/vomiting 2.5 1-3 1 1-2 <0.001 1 1-2 <0.001
Flatulence 2.5 2-3 1.5 1-2 <0.001 1 1-3 <0.0001
Rectal urgency/
Incontinence 2 2-3
1
1-2 <0.001 1 0-2 <0.001
Bloating 2 2-3 1 1-2 <0.001 1 1-2 <0.001
T0, baseline; T2, 2 wks after start of treatment; T4, 4wks after start of treatment.
Table 28: Distribution of side effects by treatment option
Side effects
reported
Treatment option Total
Herbal Allopathic
Yes 9(19.56%) 26(55.31%) 35(37.63%)
No 37(80.43%) 21(44.68%) 58(62.36%)
Pearson chi square value 26.04 and p value < 0.0001
There was a significant difference as regards the side effects between two treatment groups (p
value < 0.0001). Patients in control group reported more side effects as compared to test (Table
28). The number of patients receiving allopathic and herbal medicine reported side effects
including anorexia, metallic taste, headache, vomiting dizziness color of urine, mouth and tongue
irritation and there percentage ratio is delineated in table 29.
121
Table 29: Types of side effects by treatment option
Types of side effects Treatment option
Herbal Allopathic
Anorexia 2(4.34%) 7(14.89 %)
Metallic taste 3(6.52%) 5(10.63%)
Headache 2(4.34%) 3(6.38%)
Vomiting 1(2.17%) 2(4.25%)
Dizziness 0 4(8.51%)
Dark or reddish-brown urine 1(2.17%) 0
Mouth or tongue irritation 0 2(4.25%)
Any other 0 3(6.38%)
Around 20% patient reported adverse effects in test group however in control group 55.31%
reported adverse effects. The major adverse effects reported in control group were anorexia
(14.89%), metallic taste (10.63%), dizziness (8.51%) and vomiting (4.25%). Among test group the
major adverse effects reported were metallic taste (6.52%), anorexia and headache (4.34%).
Evidence based herbal remedies are successful in curing chronic diarrhea and acute diarrheal
diseases. It is obligatory and regulatory requirement to set up logical evidences for rational
utilization of such traditional medicinal products. The present study evaluated the safety and
efficacy of herbal formulation Entoban used for the treatment of gastrointestinal infections(200).
Holarrhena antidysenterica Wall has shown a pronounced antibacterial activity and its bark is
utilized for anti-diarrheal and astringent activity(201). The antidiarrheal effect of the alkaloids
from H. antidysenterica is due to the inhibition of production of watery stools or fluid(63).
Berberis aristata (BA) has profound antibacterial activity and used in the treatment of
diarrhea(68)(199). Qualitative data analysis of nineteen clinical trials indicated that berberine
122
(major biomarker found in BA) has potentially valuable antisecretory effects against diarrhea
caused by Vibrio cholerae and enterotoxigenic Escherichia coli(202). Extracts of the galls of Q.
infectoria have high potential as an antibacterial agent(203). Symplocos racemosa possess
antimicrobial activity(204)(205). Thus Entoban possesses antimotility and antisecretory activity
due to the presence of different phytochemicals (206). In this study Metronidazole has been used
as a control drug to treat diarrhea and Entoban has been compared with respect to the healing rate
and side effects of the two therapies. Fred reported that treatment with Metronidazole among the
patients with severe diarrhea resulted in 76% clinical cure(207). C. Wenisch revealed that
treatment resulted in clinical cure for 94% of the patients treated with Metronidazole(208).
In this study, participants showing complete and no improvement in the test group exhibited a
marked reduction in the symptoms; the symptom score was decreased from 3 (maximum) to 1
(minimum) or 0 (absent) in most of the participants. Research has shown that Metronidazole
produces side effects including nausea, diarrhea, headache, loss in weight, dizziness, abdominal
pain, vomiting, and metallic taste in the mouth(209). Similar adverse effects were reported by the
participants in current study. A significant difference was observed concerning the side effects
between two treatment groups (p value < 0.0001). Patients in control group reported more side
effects as compared to test. Plants produce complex combination of wide-ranging chemicals,
accountable for imparting herbal drugs with the attribute of being therapeutically effectual with the
advantage of synergistic and additive effects and simultaneously being having fewer side effects.
It is revealed from the literature that herbal preparations have fewer side-effects since they are a
balance of naturally occurring ingredients(210). Tomoo Kuge conducted a clinical trial to evaluate
the safety and tolerability of Seirogan, an herbal medicine used to treat diarrhea and reported that
27% subjects receiving Seirogan reported adverse events. The recurrent adverse actions were
123
altered taste and somnolence(211). This was in compliance with our study the major adverse
effects reported by Entoban was metallic taste (6.52%). Entoban produce high cure rates of chronic
diarrhea as compared to Metronidazole. Furthermore Entoban improves the well-being off over all
sign and symptoms of diarrhea and has better compliance(212).
124
CONCLUSION:
The manufacturing process of polyherbal Entoban syrup was found to be reproducible for
three batches and all parameters were complying with the specifications and validated as
per the guidelines mentioned in Prospective Process Validation.
In-vitro antioxidant analysis revealed excellent antioxidant potential and reducing
capability which might be supportive in preventing a variety of oxidative stress-related
diseases associated with gastro intestinal tract.
Entoban syrup formulation suppressed H. pylori-induced IL-8 more compared to capsule
formulation. The formulations have an excellent antiurease potential as well as good
potential of lipoxygenase inhibition and prospective to be used in the management of
different complications due to urease enzymes, such as gastric ulcers.
The quantitative estimation of biomarkers gallic acid and berberine was explored in
polyherbal formulation Entoban capsule and syrup. The standardization provides specific
and accurate tool to develop qualifications for identity, transparency and reproducibility of
biomarkers in Entoban formulations. According to determined amounts of heavy metals,
Entoban syrup samples were validated and considered safe for human consumption.
Entoban showed significant inhibition of diarrhea in dose dependent manner. The study
gave evidence of good tolerance of Entoban and the absence of detrimental effects on the
functional state of the vital organs of the experimental animals in acute and sub chronic oral
toxicity test.
125
Entoban possesses considerable therapeutic significance for the treatment of chronic
diarrhea and its associated symptoms that are comparable with those of the standard,
conventional therapy. Entoban is a better tolerated drug as there were considerably more
side effects accrue due to Metronidazole by comparison to Entoban.
This study showed that poly herbal drug, Entoban, exhibits strong anti-inflammatory
activity in gastric epithelial cells against inflammation induced by H. pylori. Entoban also
possess urease inhibitory activity. Single poly herbal drug formulation with two modes of
action against H. pylori can act as a double bladed sword ensuring complete suppression of
H. pylori and its associated inflammation. Herbal drugs like Entoban are an excellent
candidate for future in vivo and clinical studies, which are required in order to establish its
definitive role as chemotherapeutic agent against H. pylori-induced gastric disease.
126
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LIST OF PUBLISHED PAPERS
1. Sadia Shakeel, et al. Development and Quality Assessment of Polyherbal Entoban
Capsules Int J Pharm 2015; 5(4): 1068-1072
2. Sadia Shakeel, et al. Development and evaluation of polyherbal Entoban syrup Spatula
DD.2015;5(2)97-102
3. Sadia Shakeel, et al. A novel HPTLC method for quantitative estimation of biomarkers
in polyherbal formulation. Asian Pac J Trop Biomed 2015; 5(11): 930-934
4. Shakeel et al. In Vitro Evaluation of Antimicrobial Activity of Entoban Syrup; A
Polyherbal Formulation. World Journal of Pharmaceutical Research.Volume 4, Issue 5,
504-511.
5. Shakeel et al. Prospective Process Validation for Polyherbal Oral Liquid Preparation
World Journal of Pharmaceutical Research.Volume 4, Issue 6, 89-95.
6. Shakeel et al. Determination of the heavy metal content of Entoban herbal medicinal
product. J. Chin. Pharm. Sci. 2015, 24 (11), 764–769
7. Shakeel et al. Evaluation of in vitro antioxidant capacity and reducing potential of
polyherbal drug Entoban. Afr. J. Pharm. Pharmacol. 2015;9(40),982-987
8. Shakeel et al. Standardization of Biomarkers Gallic Acid and Berberine in
Polyherbal Formulation Entoban Capsules by High- Performance Thin-Layer
Chromatography–Densitometry. Journal of Planar Chromatography 28 (2015) 5, 386–
390
9. Shakeel et al. (2015) Evaluation of Acute and Sub-Chronic Oral Toxicity of Entoban:
A Polyherbal Drug on Experimental Mice. J Med Diagn Meth 4:1000187. doi:
10.4172/2168-9784.1000.187
145
10. Shakeel et al. Evaluation of in vitro lipoxygenase Inhibition and Antioxidant Activity
of Polyherbal Formulation Entoban. RADS Journal of Pharmacy and Pharmaceutical
Sciences 2015; 3(2):82-88
11. Evaluation of in vitro urease inhibition activity of polyherbal drug Entoban submitted
12. Clinical evaluation of herbal medicine Entoban for the treatment of chronic diarrhea:
A randomized control trial submitted to World Journal of Gastroenterology