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

ii

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.

iii

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

i

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

ii

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

iii

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

v

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.

1

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)

2

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.

3

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

4

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).

5

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

6

Figure 6: Entamoeba Histolytica

Figure 7: Campylobacter jejuni

Figure 8: Giardia lamblia

7

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).

8

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

9

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,

10

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.

12

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

13

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

15

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).

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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.

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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