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CHAPTER 1: INTRODUCTION

In recent time there is an increase in global utilization of herbal medicine in the

treatment of various decease affecting human. The highly safety profile and low cost of

herbal medicines have been reported as the major factors responsible for the increased

upsurge in herbal medication. The subject of the phytochemical analysis, phytochemistry

or plant chemistry has developed in recent years as a distinct discipline, somewhere in

between natural product organic chemistry and plant biochemistry and it’s closely related

to both. It is concerned with the enormous variety of organic substances that are

elaborated and accumulated by plants and deals with the chemical structures of these

substances, their biosynthesis, turnover and metabolism, their natural distribution and

their biological function.

Herbal medicine is still the mainstay of about 75–80% of the world population, mainly in

the developing countries, for primary health care because of better cultural acceptability,

better compatibility with the human body and lesser side effects. However, the last few

years have seen a major increase in their use in the developed world.

The human being exploited to alleviate his suffering from injuries of deceases utilizing

plant growing around him. The plant kingdom still hold many species of plant containing

substance of medicinal value which have yet to be discovered and the large no. of plant

are constantly being screened for their possible pharmacological value in addition to

already exploited plants. As the results of modern isolation technique and

pharmacological screening procedure, new plant drugs usually find their way into modern

medicines. Now a days maximum world’s population depends on herbal medicines.

Medicinal plants often contain additional active principles other than the major active

principles and physiologically inert substances like cellulose and starch. Unlike the

chemical entities, which contains one active ingredient pulps a number of inert

substances, which makeup the dosage form (like tablet, capsules and syrups).

Indian system of medicines comprises of Ayurveda, Unani, Siddha, Homeopathy,

Naturopathy, and Yoga. Each of which uses the herbal constituents in some or the other

form, crude drug are not so effective because they have not been tested for efficacy

according to rigid pharmacological standards. As the constituents derived from the

medicinal plants proved to cure the human disorders they isolated and used for their

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pharmacological action1-3

. The constituents having particular therapeutic effect are

identified and isolated. Natural product research has lead to a new physiological and

pharmacological concept, particular when a new compound is found to have specific

biological effects.

The plant kingdom represents an enormous reservoir of pharmacologically valuable

molecules to be discovered. Among the estimated 350,000 plant species on the earth,

only a small percentage has been pharmacologically investigated and the fraction

submitted to biological or pharmacological screening is even smaller. Over the last

decade, we have witnessed a substantial acceleration of changes in the drug discovery

process as a whole, and these changes have necessarily had a substantial impact in the

area of natural products. Compounds of natural play a major role as ‘drugs’ and as ‘lead

structure’ for the development of synthetic molecules for the discovery and validation of

drug targets, herbal extracts and finished products or phytopharmaceuticals 4.

A WHO survey has reported that about 70-80% of world’s population rely chiefly on

traditional medicines, mainly on herbal resources, in their primary healthcare. Towards

the end of twentieth century herbal medicine became more main stream throughout the

world, partly as a result of the value of traditional medicine systems, particularly of Asian

origin. We have also seen an increase in the popularity and use of natural remedies in

developed countries, including herbs, herbal medicines, over-the-counter health food,

nutraceuticals, and herbal medicinal products. Overall, the world market for herbal

medicine and products is increasing rapidly, especially for Chinese, German, and Indian

herbal medicines.

Over the past decade there has been an explosion of interest in the antimicrobial,

particularly antibacterial and antifungal activity of natural products. This is driven by a

number of factors including increasing antibiotic resistance and fear of development of

even more infectious “superbugs”, the impact of infectious diseases on mortality and

morbidity, and increasing interest in “natural” therapies and a move to more self-care.

Traditional communities also wish to retain their ethanopharmacological heritage and

exploration of traditional treatments for a variety of diseases has the potential to empower

these communities and improve both their health and economy. This is particularly

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important in the developing nations where the use of conventional antibiotics may be

limited due to cost or other factors. In addition these communities often have a rich

tradition of use of herbal and other products for endemic infections; this serves as a

starting point for researchers interested in finding treatments for these diseases 5, 6

.

1.1Mouth ulcers:

Mouth ulcers are small, painful sores on the inside lining of the mouth. They

usually develop on the inside of the lips and cheeks and on the underneath and edge of the

tongue. Medicines from a pharmacist can reduce the pain and help mouth ulcers to heal.

Mouth ulcers include lesions, sores, laceration, abrasions, or any open break in the

mucosa of the mouth, lips or tongue. Mouth ulcers may also be called stomatitis and are a

symptom of a variety of mild to serious diseases, disorders and conditions. Mouth ulcers

can result from vitamin deficiencies, infection, inflammation, trauma, malignancy and

other diseases and abnormal processes. 7

Mouth ulcers can occur in any age group or population. Mouth ulcers can be the

result of a mild condition, such as a canker sore or excessive or overly aggressive tooth

brushing. Mouth ulcers can also be the result of a moderate condition, disorder or disease,

such as gingivitis or a cold sore. Mouth ulcers can also occur due to some diseases,

disorders and conditions that can be serious, even life-threatening. These include oral

cancer and leukoplakia.

1.1.1Causes: In many cases the underlying cause of mouth ulcers is not known, but they

may be associated with stress or tissue injury. Causes of mouth ulcers include:

Biting or chewing the inside of the cheek

Damage to the inside of the mouth from very hot food or drinks

Damage to the inside of the mouth from some foods (e.g., caffeine, tangy cheese,

chocolate, acidic, spicy or salty food)

Brushing the teeth and gums too hard

Some toothpastes and mouth rinses

Poorly fitting dentures, braces, rough dental fillings or sharp edges on teeth,

certain medicines, including herbal remedies.

Some medical conditions (e.g., HIV/AIDS, inflammatory bowel disease, coeliac

disease)

A dry mouth (which may be due to medicines or medical conditions)

Quitting smoking

Some nutritional deficiencies (e.g., low iron, folic acid, zinc, B-group vitamins)

Hormone changes (e.g., menstruation)

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Stress.

Depending on the cause, mouth ulcers can be short-term and disappear quickly,

such as when mouth ulcers occur due to ill-fitting dentures that are replaced by properly

fitting dentures. Mouth ulcers can also occur chronically or long-term, such as mouth

ulcers that happen with oral cancer or periodontal disease that is not treated.

Diagnosing mouth ulcers and their root cause begins with taking a thorough

personal and family medical history, including symptoms, and completing a physical

examination. This includes an oral examination and oral X-rays. A full dental

examination, performed by a dentist and/or periodontics (a specialist in periodontal

disease) may be recommended is the cause is believed to be due to periodontal disease.8

Diagnosing many common causes of mouth ulcers, such as oral thrush, cold sores

and canker sores, can often be made by the symptoms and the appearance of the mouth

ulcers. Making a diagnosis of mouth ulcers may also include performing a variety of tests

to help to diagnose potential underlying diseases, conditions or disorders, such as oral

cancer and leukoplakia. Tests can include biopsy of the mouth ulcers.

A diagnosis of mouth ulcers and their cause can easily be delayed or missed

because symptoms of mouth ulcers may be mild or intermittent and for other reasons.

Treatment of mouth ulcers varies based on the underlying cause. Some conditions can be

easily and successfully treated and cured, while others may require more intensive

treatment and may not have an optimal prognosis.

1.1.2Sign & Symptoms:

Some people feel a tingling or burning on the inside of the lips or cheeks, 1-2

days before an ulcer appears.

Mouth ulcers are:

Round or oval shaped, shallow sores, usually less than 1cm across

Yellow to grey-white in colour with a raised red rim; there may be redness

and swelling around them

Usually very painful.

Most mouth ulcers heal in 7-14 days without scarring. They are not contagious.

Blisters or sores on the lips and around the outside of the mouth are usually cold sores,

not mouth ulcers. A pharmacist or doctor can help you know the difference.

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1.1.3 Three main types of mouth ulcer:

1. Minor ulcer: This is the most common type of ulcer. It account for 80% of all mouth

ulcers. They are small (2-8mm in diameter) and normally heal naturally within 10-14

days. A minor ulcer will not cause any scarring.

2. Major ulcer: This type of ulcer is deeper and larger than a minor ulcer, and usually

has a raised or irregular border. A major ulcer is usually 1cm or more in diameter. This

type of ulcer will heal more slowly, over a period of several weeks, and can cause

scarring. Approximately 10% of mouth ulcers are major.

3. Herpetiform ulcers: These ulcers form as multiple, pinhead sized sores. These tiny

ulcers often fuse together to form larger, irregular shaped sores which are extremely

painful. Approximately 5-10% of mouth ulcers are herpetiform.

The mouth and tongue are compared to most other parts of the body - which explains the

amount of discomfort, caused by something so small .

Figure 1. Oral Mouth Ulcer

The current popular theory is that they are linked to the auto-immune system and an

allergic reaction, in that certain triggers (that may be different from one person to the

next) cause the mucosal lining (protective layer on the cheeks, gums, tongue, throat etc.)

to become compromised, such that it is attacked by one's own saliva, or unfriendly

bacteria within it.

Causes of mouth ulcer 9:

In many case cause of mouth ulcer is not known, but they may be associated with stress

or tissue injury. Most minor, single mouth ulcers are caused by damage to the mouth. For

example, you may accidentally bite the inside of your cheek while you are eating or burn

the inside of mouth with hot food, damage to your mouth can also occur if you use a

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toothbrush incorrectly, or from a sharp tooth, or filling..No specific single cause has yet

been isolated.

Table 1. Causes of Mouth Ulcer

SYSTEMIC CAUSES

Blood (haematological) disease

_ Anaemia

_ Leukaemias and

myelodysplastic

syndromes

_ Neutropenias

_ Hypereosinophilic syndrome

_ Hypoplasminogenaemia

SKIN DISEASE

_ Lichen planus

_ Pemphigus

_ Pemphigoid

_ Erythema multiforme

_ Dermatitis herpetiforme

_ Linear IgA disease

_ Epidermolysis bullosa

LOCAL CAUSES

_ Trauma

_ Burns

_ Necrotising sialometaplasia

INFECTIONS

_ Viral

_ HSV

_ VZV

_ EBV

_ CMV

_ HIV

_ Coxsackie viruses

_ ECHO viruses

_ Bacterial

_ Mycobacteria

_ Treponema pallidum

• Parasitic

_ Leishmania

VASCULITIDES

_ Lupus erythematosus

_ Behçet’s disease

_ Wegener’s granulomatosis

_ Sweet’s syndrome

_ Reiter’s syndrome

_ Periarteritis nodosa

APHTHAE and

APHTHOUS-LIKE

ULCERS

_ PFAPA (periodic fever,

aphthae, pharyngitis,

adenitis)

_ Other periodic syndromes

OTHER-

-Food :include chocolate,

coffee, peanuts, almonds,

strawberries, cheese,

tomatoes

- Family history

-vitamin B12 Iron deficiency

GASTROINTESTINAL

DISEASE

_ Coeliac disease

_ Crohn’s disease

_ Ulcerative colitis

MALIGNANT DISEASE

_ Oral carcinoma

_ Antral carcinoma

_ Lymphomas

_ Kaposi’s sarcoma

_ Salivary neoplasms

DRUGS

_ Cytotoxic agents

_ Alendronate

_ Nicorandil

_ Phenytoin

_ NSAIDs

_ Lamotrigine

The current popular theory is that they are linked to the auto-immune system and an

allergic reaction, in that certain triggers cause the mucosal lining (protective layer on the

cheeks, gums, tongue, throat etc.) to become compromised, such that it is attacked by

one's own saliva, or unfriendly bacteria within it.

Sign and Symptoms: Some people feel a tingling or burning on the inside of the lips or

cheeks, 1-2 days before an ulcer appears. A mouth ulcer will be round or oval in shape.

7

Figure 2. : Major Aphthous Ulceration, Soft Palate Complex

It is white, yellow, or grey in colour, and will be inflamed around the edge. Most mouth

ulcers will only last between 10-14 days, although in more severe cases, they may last for

several weeks.

Mouth ulcer is:

Round or oval shaped, shallow sores, usually less than 1 cm across.

Yellow to grey-white in colour with a raised red rim there may be redness and

swelling round.

Usually vary painful.

An inflammatory halo present highlights ulcer is red halo around the yellow or grey

ulcer.

1.1.4 Treatment:

Symptomatic treatment is the primary approach to dealing with oral ulcers. If their cause

is known, then treatment of that condition is also recommended. Adequate oral hygiene

may also help in relieving symptoms. Topical antihistamines, antacids, corticosteroids or

applications meant to soothe painful ulcers may be helpful, as may be oral analgesics

such as paracetamol or ibuprofen and local anesthetic lozenges, paints or mouth rinses

such as benzocaine and avoiding spicy or hot foods may reduce pain. Rinsing the mouth

out with brine (warm salted water) or rubbing garlic on the sore area may help.

Amino acids are the building blocks of life; L-Lysine has been highly beneficial in

many cases. Supplements containing range of fundamental amino acids are also now

readily available.

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Avoid (don’t over-use) mouthwashes and toothpaste with a powerful anti-microbial

action. Products containing hyaluronic acid actively assist in tissue regeneration and

can prevent ulceration caused by physical damage.

The treatment of patients with mouth ulcers depends on the aetiology: the fundamental

cause should, where possible, be corrected. The medical history should exclude relevant

systemic disorders (haematological, infections, gastrointestinal, or skin diseases) or

causal drug use 10

.

Medicines:

Treatment of mouth ulcers begins with prevention. This includes seeking regular

dental care (twice yearly) and maintaining good oral hygiene, such as brushing the teeth

at least twice a day and flossing once a day. Most mouth ulcers heal by themselves

without treatment, but medicines can reduce the discomfort and help them to heal faster.

There is a range of non-prescription products available for mouth ulcers. Treatment plans

for mouth ulcers are individualized based on the underlying cause, the presence of

coexisting diseases, the age and medical history of the patient

Pastes:

Form a protective, soothing layer over the mouth ulcer

Some pastes contain anti-inflammatory medicines to reduce pain and

swelling. These medicines may speed healing, especially if applied as

soon as the ulcer begins.

Mouthwashes and lozenges:

Some products contain an antiseptic to stop bacteria in the mouth from

infecting the ulcer

Some products contain a medicine to reduce pain and swelling

Are helpful for treating mouth ulcers that are in hard to reach places

Help keep the mouth clean if it is too painful to brush teeth properly.

Gels and paints:

Some products contain a medicine to reduce pain and swelling

Some products contain a local anaesthetic (e.g., lignocaine, benzocaine) to

numb the ulcer

Some products contain an antiseptic to stop bacteria from infecting the

ulcer.

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1.2 Bacteriology:

Bacterial processes leading to ulceration can be caused by Mycobacterium

tuberculosis (tuberculosis) and Treponema pallidum (syphilis). Opportunistic activity by

combinations of otherwise normal bacterial flora, such as aerobic streptococi, Neisseria,

Actinomyces, spirochetes, and Bacteroides species can prolong the ulcerative process

Coccidioides immitis (valley fever), Cryptococcus neoformans (cryptococcosis),

Blastomyces dermatitidis ("North American Blastomycosis") are some of the fungal

processes causing oral ulceration.

In the mouth there are many good and bad micro-organisms and bacteria, which

now have access to the wound surface and produce toxins which in turn promote further

cell death causing the ulcer to get larger. Also at this stage the bacteria lining the ulcer.

This situation now continues until the causative agent is gone, and the body’s immune

system comes up with the solution and the bad bacteria are quashed. How long this takes

depends on many factors. Staphylococcus, Pseudomonas, Bacillus, E.coli and Candida

species are an important component normal flora of the Oropharynx 11

.

1.2.1Bacteria:

1. Escherichia coli.

2. Pseudomonas aeruginosa.

3. Staphylococcus aureus.

4. Bacillus subtilis.

5. Fungi: Candida albicans.

i. Escherichia coli: E. coli is Gram-negative, facultative anaerobic and non-sporulating.

Cells are typically rod-shaped, and are about 2.0 micrometres (μm) long and 0.5 μm in

diameter, with a cell volume of 0.6 – 0.7 (μm) 3

. Optimal growth of E. coli occurs at 37°C

(98.6°F) but some laboratory strains can multiply at temperatures of up to 49°C (120.2°F)

.However, E. coli are extremely sensitive to such antibiotics as streptomycin or

gentamicin.

If E. coli bacteria escape the intestinal tract through a perforation (for example from an

ulcer, a ruptured appendix, or due to a surgical error) and enter the abdomen, they usually

cause peritonitis that can be fatal without prompt treatment. In the bowel, it adheres to the

mucus of the large intestine. Growth can be driven by aerobic or anaerobic respiration

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Escherichia coli encompass an enormous population of bacteria exhibit a very high

degree of both genetic & phenotypic diversity.

ii. Pseudomonas aeruginosa: It is a Gram-negative, aerobic, rod-shaped bacterium with

unipolar motility. An opportunistic human pathogen, P. aeruginosa is also an

opportunistic pathogen of plants. P. aeruginosa is the type species of the genus

Pseudomonas (Migula). P. aeruginosa secretes a variety of pigments, including

pyocyanin (blue-green), pyoverdine (yellow-green and fluorescent), and pyorubin (red-

brown) it classified as an aerobic organism, P. aeruginosa is considered by many as a

facultative anaerobe, as it is well adapted to proliferate in conditions of partial or total

oxygen depletion. Pseudomonas aeruginosa is a common bacterium that can cause

disease in animals, including humans. It is found in soil, water, skin flora, and most man-

made environments throughout the world. It thrives not only in normal atmospheres but

also in hypoxic atmospheres, and has, thus, colonized many natural and artificial

environments.

iii. Staphylococcus aureus: Staphylococci (staph) are Gram-positive spherical bacteria

that occur in microscopic clusters resembling grapes. Bacteriological culture of the nose

and skin of normal humans invariably yields staphylococci. S. aureus colonizes mainly

the nasal passages, but it may be found regularly in most other anatomical locales,

including the skin, oral cavity and gastrointestinal tract. S epidermidis is an inhabitant of

the skin. Staphylococci are perfectly spherical cells about 1 micrometer in diameter. The

enterotoxin produce by s. aureus is a heat-stable Protein which heating at 1000.

c for 30 -

70 minutes.

S. aureus infections may spread through contact with pus from an infected wound, skin-

to-skin contact with an infected person by producing hyaluronidase that destroys tissues,

and contact with objects such as towels, sheets, clothing, athletic equipment used by an

infected person. S. aureus can cause a range of illnesses from minor skin infections, such

as pimples, impetigo,boils cellulitis folliculitis.

iv. Bacillus subtilis: Bacillus subtilis cells are rod-shaped, Gram-positive bacteria that

are naturally found in soil and vegetation. Bacillus subtilis grow in the mesophilic

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temperature range. The optimal temperature is 25-35 degrees Celsius. Bacillus subtilis

bacteria have been considered strictly aerobic, meaning that they require oxygen to grow

and they cannot undergo fermentation

The most optimal activity occurs at a temperature of 37 degrees Celsius and a basic pH of

8. Bacillus subtilis bacteria use their flagella for swarming motility.

v. Candida albicans: Candida albicans is a diploid fungus (a form of yeast) and a causal

agent of opportunistic oral and genital infections in humans. Systemic fungal infections

(fungemias) have emerged as important causes of morbidity and mortality in immuno-

compromised patients (e.g., AIDS, cancer chemotherapy, organ or bone marrow

transplantation). C. albicans biofilms readily form on the surface of implantable medical

devices.

C. albicans is commensal and is among the gut flora, the many organisms that live in the

human mouth and gastrointestinal tract. Under normal circumstances, C. albicans lives in

80% of the human population with no harmful effects, although overgrowth results in

candidiasis. Candidiasis also may occur in the blood and in the genital tract.

1.3 Transmucosal drug delivery system:

Amongst the various routes of administration tried so far in the novel drug delivery

systems, localized drug delivery to tissues of the oral cavity has been investigated for the

treatment of periodontal disease, bacterial and fungal infection. Over the decades

mucoadhesion has become popular for its potential to optimize localized drug delivery,

by retaining a dosage form at the site of action (e.g. within the gastrointestinal tract) or

systemic delivery by retaining the formulation in intimate contact with the absorption site

(e.g. buccal cavity).

Well defined bioadhesion is the ability of a material (synthetic or biological) to adhere to

a biological tissue for an extended period of time. The biological surface can be epithelial

tissue or it can be the mucus coat on the surface of a tissue. If adhesion is to a mucous

coat, the phenomenon is referred to as a mucoadhesion. The use of mucoadhesive

polymers in buccal drug delivery has a greater application. Various mucoadhesive

devices, including tablets, films, patches, disks, strips, ointments and gels, have recently

been developed.

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However, buccal patch offer greater flexibility and comfort than the other devices. In

addition, a patch can circumvent the problem of the relatively short residence time of oral

gels on mucosa, since the gels are easily washed away by saliva. Buccal route of drug

delivery provides the direct access to the systemic circulation through the jugular vein

bypassing the first pass hepatic metabolism leading to high bioavailability. Other

advantages such as excellent accessibility, low enzymatic activity, suitability for drugs or

that mildly and reversibly damage or irritate the mucosa, painless administration, easy

withdrawal, facility to include permeation enhancer/ enzyme inhibitor or pH modifier in

the formulation, versatility in designing as multidirectional or unidirectional release

system for local or systemic action.12,13

1.3.1Buccal Drug Delivery: The buccal region of oral cavity is an attractive site for the

delivery of drugs owing to the ease of the administration. Buccal drug delivery involves

the administration of desired drug through the buccal mucosal membrane lining of the

oral cavity. This route is useful for mucosal (local effect) and transmucosal (systemic

effect) drug administration. In the first case, the aim is to achieve a site-specific release of

the drug on the mucosa, whereas the second case involves drug absorption through the

mucosal barrier to reach the systemic circulation. Based on current understanding of

biochemical and physiological aspects of absorption and metabolism of many

biotechnologically produced drugs, they cannot be delivered effectively through the

conventional oral route. Because after oral administration many drugs are subjected to

pre-systemic clearance extensive in liver, which often leads to a lack of significant

correlation between membrane permeability, absorption, and bioavailability. Direct

access to the systemic circulation through the external jugular vein by pass the drugs

from the hepatic first pass metabolism which may lead to higher bioavailability. 14, 15

1.3.2Advantages of Buccal Drug Delivery System: 16, 17, 18

Excellent accessibility

Presence of smooth muscle and relatively immobile mucosa, hence suitable for

administration of retentive dosage forms

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Direct access to the systemic circulation through the internal jugular vein bypasses

drugs from the hepatic first pass metabolism leading to high bioavailability

Low enzymatic activity

Suitability for drugs or excipients that mildly and reversibly damages or irritates the

mucosa

Painless administration

Facility to include permeation enhancer/enzyme inhibitor or pH modifier in the

formulation

Versatility in designing as multidirectional or unidirectional release systems for local

or systemic actions etc.

Oral mucosal drug delivery systems are easy and painless to administer and well

accepted by the patient.

Precise dosage form localization is possible and there is ability to terminate delivery

when required

Flexibility in physical state, shape, size and surface.

For patient suffering with nausea or vomiting or in the state of unconsciousness, with

an upper gastrointestinal tract disease or surgery which affects oral drug absorption, the

oral cavity a useful site for drug delivery for upper symptoms.

Maximized absorption rate due to intimate contact with the absorbing membrane and

decreased diffusion barriers.

Excellent route for the systemic delivery of drug with high first pass metabolism,

thereby offering a greater bioavailability.

A significant reduction in dose can be achieved, thereby reducing dose dependent

side effects.

Drugs which are unstable in the acidic environment of the stomach or are destroyed

by the enzymatic or alkaline environment of the intestines can be administered by this

route.

It offers a passive system for drug absorption and does not require any activation.

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It allows for the local modification of tissue permeability, inhibition of protease

activity or reduction in immunogenic response. Thus, selective use of therapeutic agents

like peptides, proteins and ionized species can be achieved.

The oral mucosa lacks prominent mucus secreting goblets cells and therefore there is

no problem of diffusion limited mucus buildup beneath the applied dosage form. The

presence of saliva ensures relatively large amount of water for drug dissolution unlike in

case of rectal and transdermal routes.

It satisfied several features of the controlled release system.

It can be made unidirectional to ensure only buccal absorption.

Bioadhesion prolongs the residence time at the site of drug absorption, and thus

improves bioavailability and dosing interval.

Rapid onset of action.

1.3.3 Limitation:

Drug administration via this route has certain limitations

Drugs which irritate the mucosa or have a bitter or unpleasant taste or an obnoxious

odour cannot be administered by this route.

Drugs which are unstable at buccal pH cannot be administered by this route.

Only those drugs which are absorbed by passive diffusion can be administered by

this route.

1.3.4 Types of dosage form for buccal delivery: In the past decades, to till now,

different drug delivery systems intended for buccal administration have been developed.

The most common buccal dosage forms are tablets and patches. Such type of form must

be of a small size and a suitable geometry so as to not interfere with physiological

function of the mouth, even after their hydration in the oral cavity. One of the

requirements is that they do not adhere too tightly because it is undesirable to exert too

much force to remove the formulation/ dosage form after use, otherwise the mucosa

could be injured. An alternative is the use of formulations that dissolve or disintegrate

completely during the application period. Moreover, in the case of Transmucosal

administration, Drug release should be unidirectional (towards the mucosa), and the

release into the saliva should be avoided.

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Matrix type: The buccal patch designed in a matrix configuration contains drug,

adhesive, and additives mixed together.

Reservoir types: The buccal patch designed in a reservoir system contains a cavity for

the drug and additives separate from the adhesive. An impermeable backing is applied to

control the direction of drug delivery; to reduce patch deformation and disintegration

while in the mouth; and to prevent drug loss. Additionally, the patch can be constructed

to undergo minimal degradation in the mouth, or can be designed to dissolve almost

immediately.

Patches: Patches are laminated and generally consist of an impermeable backing layer

and a drug-containing layer that has mucoadhesive properties and from which the drug is

released in a controlled manner. Moreover, buccal patches for systemic delivery of

tyrotropin-releasing hormone, octreotide, oxytocin, buserelin, calcitonin and

leuenkephalinhave been studied.

Novel drug delivery system: Novel drug delivery systems, such as lipophilic gel, buccal

spray and phospholipids vesicles have been recently proposed to deliver peptides via the

buccal route. A novel liquid aerosol formulation (Oralin, Generex Biotechnology) has

been already developed. This system allows precise insulin dose delivery via a metered

dose inhaler in the form of fine aerosolized droplets directed into the mouth. This oral

aerosol formulation is rapidly absorbed through the buccal mucosal epithelium, and it

provides the plasma insulin levels necessary to control postprandial glucose rise in

diabetic patients. This novel, pain-free, oral insulin formulation has a number of

advantages including rapid absorption, a simple (user-friendly) administration technique,

precise dosing control (comparable to injection within one unit) and bolus delivery of

drug. 19

1.4 Peptic Ulcer Introduction

Gastric hyperacidity and gastro duodenal ulcer is a very common global problem today. It

is now generally agreed that gastric lesions develop when the delicate balance between

some gastro- protective and aggressive factors are lost. Major aggressive factors are acid,

pepsin, Helicobacter pylori and bile salts. Defensive factors mainly involve mucus

16

bicarbonate secretion and prostaglandins. Hyper secretion of gastric acid is a pathological

condition, which occurs due to uncontrolled secretion of hydrochloric acid from the

parietal cells of the gastric mucosa through the proton pumping H+K+ATPase. Even the

normal rate of acid secretion may cause ulceration in the breached mucosa when some

gastroprotective factors are lost. The modern approach to control gastric ulceration is to

inhibit gastric acid secretion, to promote gastro protection, block apoptosis and stimulate

epithelial cell proliferation for effective healing. Most of the antisecretory drugs such as

proton pump inhibitors (omeprazole, lansoprazole, etc.) and histamine H2-receptor

blocker (ranitidine, famotidine, etc.) are extensively used to control increased acid

secretion and acid related disorders caused by stress, NSAID’s and H. pylori; but there

are reports of adverse effects and relapse in the long run. On the contrary most of the

herbal drugs reduces the offensive factors and are proved to be safe clinically effective,

having better patient tolerance, relatively less expensive and globally competitive 20-

21.Exogenous aggressive factors such as smoke, anti-inflammatory drugs, alcohol, stress,

fatty foods and Helicobacter pylori infections triggered tissue necrosis through mucosal

ischemia, free radical generation and cessation of nutrient delivery, hydrochloric acid

together with pepsin, pancreatic enzymes and bile decreased the defense mechanisms of

gastrointestinal mucosa such as the intercellular junctions, local blood flow,

mucus/bicarbonate secretion and cellular growth. 22

Pathophysiology of ulcer is due to an imbalance between aggressive factors (acid, pepsin,

h. pylori and non-steroidal anti-inflammatory agents) and local mucosal defensive factors

(mucus bicarbonate, blood flow and prostaglandins). Integrity of gastroduodenal mucosa

is maintained through a homeostatic balance between these aggressive and defensive

factors23

.

Infection of the stomach mucosa with helicobacter pylori – a Gram-negative

spiral-shaped bacterium – is now generally considered to be a major cause of gastro-

duodenal ulcer24

. Helicobacter pylori were the first isolated microaerophilic gram-

negative bacteria from the gastric mucosa of gastritis patients by Marshall and Warren in

1980s. It is a spiral-shaped, highly motile organism with a unipolar flagellum that harbors

within and beneath the mucous layer of the stomach and often found attached to gastric

mucosa. It is a worldwide common infection with prevalence rates in the general

17

population ranges from not only 30-40% in United States, 80-90% in South America and

70-90% in Africa but also in developing countries like India, China from the age of

teenagers 20% to 50-60% of elderly subjects. 25, 26

According to the statistics, it causes peptic ulcer disease approximately one in six

(17%) persons and each year 1% to 2% of these will experience a major or life

threatening complication, such as bleeding or gastric outlet obstruction27

. H pylori is such

a threat that the World Health Organization's (WHO) International Agency for Research

into Cancer (IARC) in 1994 has classified as a “Class-I-Carcinogen” 28

.

The series of steps or pathogenic mechanisms of H pylori in the stomach are29

Attachment - The H pylori bacteria enter into the stomach and attach themselves to the

lining of the stomach to establish an environment in which to grow.

Toxin production - H pylori produce poisonous substances to increase the secretion of

water and electrolytes in the stomach and cause cell death in the cells of the stomach

lining. This will help the bacteria take over the stomach environment and will lessen the

competition for required nutrients.

Cell invasion - The bacteria will enter into the stomach lining cells for protection and

then kill the cells they are in (their host cells) so that they can move on to invade more

stomach-lining cells. This process will continue, thus creating tissue damage. This tissue

damage will become the ulcer formation in the stomach.

Loss of microvilli/villi – The substances released into the host cell during the ‘Cell

Invasion’ step cause a change in the stomach-lining cells. This change results in fewer

calories getting absorbed by the stomach. The body will get fewer nutrients from the food

eaten at every meal.

A common causative factor for gastric ulceration is an invasion of Helicobacter pylori, a

micro-aerophilic, gram-negative, flagellated, spiral-shaped bacterium. Half of all gastric

ulcer cases are associated with infection by H. pylori. The bacterium's spiral shape and

high motility allow it to penetrate the deep portions of the mucus gel layer, restrict gastric

emptying and survive in the grooves between epithelial cells under the protective gastric

mucosal layer of the stomach. There, it causes local damage by inducing inflammatory

mediator influx. Prostaglandins are involved in promoting the defense mechanisms of the

stomach, and H. pylori may promote gastric mucosal prostaglandin secretion by up to

18

50% to maintain its preferred environmental conditions. Because prostaglandin levels in

the gastric mucosa are decreased in elderly patients, ageing is associated with a

diminished epithelial cell turnover rate and a reduced capacity to repair the gastric

mucosa. Advanced age is therefore a major risk factor for complicated peptic ulcer

disease. According to an estimate by the World Health Organization (WHO), half of the

world's population is infected with H. pylori, but the infection has no detectable

symptoms in most cases. However, over the past two decades, there has been a decrease

in reported H.pylori-related peptic ulcer disease. This decrease is due to early detection

using several sophisticated diagnostic tools and early treatment of the infection 30

.

1.4.1 Pathogenesis of Helicobacter pylori :

H. pylori colonization itself is not a disease, but an infection can lead to various clinical

disorders in the upper gastrointestinal tract. In most cases, H. pylori colonization induces

histological gastritis, but pronounced clinical signs seldom develop. It is estimated that H.

pylori-positive patients have a 10% to 20% lifetime risk of developing ulcer disease and a

1% to 2% risk of developing distal gastric cancer . This infection depends on different

factors that relate primarily to the pattern and severity of gastritis. H. pylori bacteria

mainly adhere to gastric epithelial cells and release cytotoxins causing duodenal ulcer.

Several infection-associated factors of H. pylori, such as urease, catalase, lipase, adhesion

molecules, cytotoxin-associated gene protein (CagA), a homologue of the Bordetella

pertussis toxin secretion protein (picB) and vacuolating cytotoxin (VacA), contribute to

gastric ulceration. 31

H. pylori produces a variety of enzymes and is characterized by high urease activity.

Urease breaks urea into bicarbonate and ammonia, which help to neutralize gastric

hydrochloric acid (HCl) and protect the bacterium in the acidic environment of the

stomach. Hydroxide ions generated by the equilibration of water and ammonia may

contribute to gastric mucosal epithelium damage. Conversely, H. pylori infection reduces

epithelial cell bicarbonate secretion, which leads to excessive diffusion of HCl into the

mucosa, causing damage of the gastro-duodenal lining and leading to ulcer formation. It

appears that H. pylori infection activates the vago-vagal reflexes (gut-brain axis) in the

gastroduodenal mucosa that damage the mucosal cells directly and enhance the secretion

of gastric HCl, which ultimately leads to ulcerogenesis 32, 33

. Two other types of enzymes

19

produced by H.pylori, proteases and phospholipases, also participate in the breakdown of

the glycoprotein lipid complex of the mucous gel layer; this can cause severe gastric

ulceration. In elderly persons, the integrity of the gastric mucosal surface becomes

impaired and progressively susceptible to damage by factors that can overwhelm the

protective barriers of the stomach.

Another class of proteins, termed heat shock proteins (HSPs),also plays a crucial role in

H.pylori-induced gastric ulceration.HSPs are a class of functionally-related proteins

whose expression is increased when exposed to elevated temperatures or other stress 34

.

H. pylori appears to bind gastric epithelial cells and mucin via HSP 60. Adaptive

immunity targeting HSP60 was found to be induced in H. pylori-infected patients.

A 62K urease-associated protein belonging to the HSP60 family of stress proteins

participates in extracellular assembly and/or protection of urease inactivation in the

hostile environment of the stomach35

. H. pylori infection activates both epithelial and

immunomodulatory cells, including monocytes and mononuclear phagocytes, which in

turn secrete a number of pro-inflammatory cytokines, including TNF-α, IL-1/, IL-6,

interferon (IFN)- and granulocyte-macrophage colony stimulating factor 36

. Activated

monocytes overexpress interleukin-2 receptors on their surfaces and produce superoxides

and other inflammatory factors that ultimately damage mucus epithelial cells 37

.

The H. pylori genome study is centered on attempts to understand pathogenesis.

Approximately 29% of the loci in the genome database are categorized as pathogenic. A

specific region of the bacterial genome encodes the virulence factor CagA. The cagA

gene codes for one of the major H. pylori virulence proteins. The bacterium physically

interacts with gastric epithelial cells and introduces CagA protein into the host cells.

Bacterial strains that possess the CagA gene are associated with an ability to cause ulcers

through inhibition of mucin synthesis 38

. This finding may suggest that cooperation

among different H. pylori proteins is necessary to induce cell-cycle alterations in infected

cells 39

. H.pylori induces mitogenic signals and proto-oncogene expression in gastric

epithelial cells.

1.4.2 Treatment of H. pylori

To date, the most effective therapies of H. pylori infection require a minimum of two

antibiotics in combination with a gastric acid inhibitor. Both Triple Therapy (levofloxacin

20

/ Clarithromycin + amoxicillin + proton pump inhibitor) and Bismuth Quadruple Therapy

(bismuth + tetracycline + metronidazole + proton pump inhibitor) are well known for H.

pylori eradication as well as for H.pylori-induced gastropathy prevention. Complete

eradication of H.pylori infection improves symptoms, including dyspepsia, gastritis and

peptic ulcers, and may prevent gastric cancer. However, these treatments may cause

nausea, drug resistance 40

, infection recurrence, stomach upset and diarrhea. Rising levels

of acquired antimicrobial resistance necessitate the search for an effective H. pylori

infection prevention strategy. Alternatively, there is a growing interest in and need to find

non-toxic, safe and inexpensive anti-ulcer formulations from medicinal plants.

1.4.3 H. pylori and natural medicines

Currently available treatments for peptic ulcers include antacids (systemic and

nonsystemic) and drugs which reduce acid secretion such as H2 anti-histaminics, proton

pump inhibitors, anticholinergics, prostaglandin analogues, ulcer protectives, ulcer

healing drugs and anti-H.pylori drugs . These drugs have decreased the morbidity rates,

but produce many adverse effects including relapse of the disease, and are often

expensive for the poor. In light of the above, it is pertinent to study natural products from

food/plants as potential anti-ulcer compounds. Due to less side effects compared to

synthetic drugs, currently 80 % of the world population depends on plant-derived

medicine for the first line of primary health care.41

For centuries, herbals have been used in traditional medicine to treat a wide range of

ailments, including gastrointestinal (GI) disorders, such as dyspepsia, gastritis and peptic

ulcer disease (PUD).Natural antioxidants are usually considered safe by most consumers,

and safety tests are not typically required by legislation because natural products are

generally recognized as safe (GRAS). The medicinal properties of folk plants are

attributed mainly to the presence of natural antioxidants (mainly polyphenols and

flavonoids).

Flavonoids and other polyphenols present in the plant materials are beneficial for human

health. Several mechanisms may account for their antioxidant activity. Flavonoids and

polyphenols are efficient in trapping superoxide anion (O2-), hydroxyl (OH·), peroxyl

(ROO·) and alcohoxyl (RO·) radicals, decreasing acid mucosal secretion, inhibiting the

production of pepsinogen, promoting gastric mucosa formation and decreasing

21

ulcerogenic lesions 42

. In addition, they have membrane stabilizing properties, inhibit

lipid peroxidation in different systems and affect some processes of intermediary

metabolism. Any clinical trial of a putative herbal drug should be accompanied by a

measurement of oxidative damage to show whether any benefit of that drug is correlated

with its antioxidant activity. Recent studies have suggested that H. pylori infection can be

suppressed through the use of medicinal plants.

1.5 In situ gel: Introduction

In situ gel forming systems have been widely investigated as vehicles for sustained drug

delivery. This interest has been sparked by the advantages shown by in situ forming

polymeric delivery systems such as ease of administration and reduced frequency of

administration, improved patient compliance and comfort. In situ gel formation occurs

due to one or combination of different stimuli like pH change, temperature modulation

and solvent exchange. So, In situ gelling system via different route such as oral, nasal,

ophthalmic etc can be formulated.

Various natural and synthetic polymers such as gellan gum, alginic acid, xyloglucan,

pectin, chitosan, poly (DL lactic acid), poly (DL-lactide-co-glycolide) and

polycaprolactone are used for formulation development of in situ forming drug delivery

systems. Gastro retentive in situ gelling system helps to increase bioavailability of drug

compared to conventional liquid dosage form. The gel formed from in situ gelling

system, being lighter than gastric fluids, floats over the stomach contents or adhere to

gastric mucosa due to presence of bioadhesive nature of polymer and produce gastric

retention of dosage form and increase gastric residence time resulting in prolonged drug

delivery in gastrointestinal tract. 43

In situ gel forming drug delivery systems are in principle capable of releasing drug

molecule in a sustained manner affording relatively constant plasma profiles. These

hydrogels are liquid at room temperature but undergo gelation when in contact with body

fluids or change in pH. These have a characteristic property of temperature dependent,

pH dependent and cation induced gelation. Compared to conventional controlled release

formulations, in situ forming drug delivery systems possess potential advantages like

simple manufacturing processes and ease of administration. 44, 45

22

Intimate contact of a delivery system at the absorbing site maximizes not only drug

absorption, but also influences the rate of drug absorption. These in situ gel preparations

can be easily formulated in bulk and these formulations give homogeneity of drug

distribution when compared to other conventional suspensions. These in situ gels also

have good mucoadhesion property, which helps in coating of the ulcer lining once the sol

comes in contact with the gastric pH. 46

1.5.1 Approaches of In situ drug delivery 47, 48

There are four broadly defined mechanisms used for triggering the in situ gel formation

of biomaterials:

Physiological stimuli (e.g., temperature and pH),

Physical changes in biomaterials (e.g., Diffusion of solvent and swelling),

Chemical reactions (e.g., enzymatic, ionic and photo-initiated polymerization).

A. In situ formation based on physical mechanism

Swelling and Diffusion

Swelling of polymer by absorption of water causes formation of gel certain biodegradable

lipid substance such as myverol 18-99 (glycerol mono-oleate) forms in situ gel under

such phenomenon. Solution of polymer such as N – methyl pyrrolidone (NMP) involves

diffusion of solvent from Polymer solution into surrounding tissue and results in

precipitation or solidification of polymer matrix.

B. In situ gelling based on chemical stimuli

Ionic crosslinking

Certain ion sensitive polysaccharides such as carrageenan, Gellan gum (Gelrite®), Pectin,

Sodium Alginate undergo phase transition in presence of various ions such as k+ , Ca+,

Mg+, Na+. For eg.alginic acid undergoes gelation in presence of divalent/polyvalent

cations e.g. Ca2+ due to the interaction with guluronic acid block in alginate chains.

Enzyamatic crosslinking

Certain natural enzymes which operate efficiently under physiologic conditions without

need for potentially harmful chemicals such as monomers and initiators provides a

convenient mechanism for controlling the rate of gel formation, which allows the

mixtures to be injected before gel formation insitu.

23

Photo-polymerisation

A solution of monomers such as acrylate or other polymerizable functional groups and

initiator such as 2,2 dimethoxy-2-phenyl acetophenone, camphorquinone and ethyl erosin

can be injected into a tissues site and the application of electromagnetic radiation used to

form gel designed readily to be degraded by chemical or enzymatic processes or can be

designed for long term persistence in vivo. Typically long wavelength ultraviolet and

visible wavelengths are used. A photopolymerizable, biodegradable hydrogels as a tissue

contacting material.

C. In situ gel formation based on physiological stimuli

Temperature dependant in situ gelling: These are liquid aqueous solutions before

administration, but gel at body temperature. These hydrogels are liquid at room

temperature (20ºC -25ºC) and undergo gelation when in contact with body fluids (35ºC -

37ºC), due to an increase in temperature This approach exploits temperature-induced

phase transition. Some polymers undergo abrupt changes in solubility in response to

increase in environmental temperature (lower critical solution temperature, LCST). At the

LCST, hydrogen bonding between the polymer and water becomes unfavorable,

compared to polymer–polymer and water–water interactions, and an abrupt transition

occurs as the solvated macromolecule quickly dehydrates and changes to a more

hydrophobic structure.

pH dependant gelling

Another formation of in situ gel is based on Change in pH. Certain polymers such as

PAA (Carbopol®, carbomer) or its derivatives, polyvinylacetal diethylaminoacetate

(AEA), Mixtures of poly (methacrylic acid) (PMA) and poly (ethylene glycol) (PEG)

shows change from sol to gel with change of pH. The polymers with a large number of

ionizable groups are known as polyelectrolytes. Swelling of hydrogel increases as the

external pH increases in the case of weakly acidic (anionic) groups, but decreases if

polymer contains weakly basic (cationic) groups.

1.5.2 Advantages of floating drug delivery system 49, 50

The gastroretentive systems are advantageous for drugs absorbed through the

stomach, e.g. ferrous salts,antacids

24

Acidic substances like aspirin cause irritation on the stomach wall when come in

contact with it. Hence, HBS formulation may be useful for the administration of

aspirin and other similar drugs.

Administration of prolongs release floating dosage forms, tablet or capsules, will

result in dissolution of the drug in the gastric fluid. They dissolve in the gastric

fluid would be available for absorption in the small intestine after emptying of the

stomach contents. It is therefore expected that a drug will be fully absorbed from

floating dosage forms if it remains in the solution form even at the alkaline pH of

the intestine.

The gastro retentive systems are advantageous for drugs meant for local action in

the stomach. e.g. antacids.

When there is a vigorous intestinal movement and a short transit time as might

occur in certain type of diarrhea, poor absorption is expected. Under such

circumstances it may be advantageous to keep the drug in floating condition in

stomach to get a relatively better response.

FDDS improves patient compliance by decreasing dosing frequency.

Bioavailability enhances despite first pass effect because fluctuations in plasma

drug concentration are avoided; a desirable plasma drug concentration is

maintained by continuous drug release.

Better therapeutic effect of short half-life drugs can be achieved.

Gastric retention time is increased because of buoyancy.

Enhanced absorption of drugs which solubilize only in stomach

Superior to single unit floating dosage forms as such microspheres releases drug

uniformly and there is no risk of dose dumping.

Avoidance of gastric irritation, because of sustained release effect, floatability and

uniform release of drug through multi particulate system.

1.5.3 Advantages of floating in situ gel 51

In situ gel forms a low density viscous layer on the gastric contents and hence

provides more effective surface area than a tablet. This leads to more drug release

and improve the bioavailability.

Floating obtained is faster than the tablets.

25

CHAPTER 2: LITERATURE REVIEW

2.1 A. K. Nadkarni (1976) Symplocos racemosa (Lodhra) Family Styraceae. The bark is

considered cooling & mild astringent. Useful in diarrhea .Native Indian Preparations-a

decoction of wood as gargle for giving firmness to spongy & bleeding gums

(shushruta).In bleeding gums a paste composed of lodhra bark , rasot ,& tubers of

Cyprus rotundus & honey is applied to gums.(chakradatta). 52

2.2 Rustomjee Naserwanjee Khory (1999) reported that the bark contains tannins

27.4%, resin and Calcium oxalate crystals. The bark of white guava is astringent, and the

decoction is used along with other astringents for chronic diarrhea of children. It is also

uesd as wash in Prolapsus. The leaves are astringent and stomachic and are used to arrest

vomiting in diarrhea.53

2.3 Viqar Uddin Ahmad et al. (2003) reported one new phenolic glycoside named

benzoylsalireposide along with one known phenolic glycoside named salireposide have

been isolated from Symplocos racemosa. Four other known compounds i.e. b-amyrin,

oleonolic acid, β-sitosterol and b-sitosterol glycoside were also isolated from this plant.

The structure elucidation of the isolated compounds was based primarily on 1D- and 2D-

NMR analysis, including COSY, HMQC, and HMBC correlations. The compound 1 and

2 showed inhibitory activity against snake venom phosphodiesterase I.54

2.4 Lyudmila Boyanova et al. (2003) reported evaluation of the inhibitory effect of

Bulgarian propolis on Helicobacter pylori growth in vitro. Activity of 30% ethanolic

extract of propolis (EEP) against 38 clinical isolates of H. pylori was evaluated by using

the agar-well diffusion method. Ethanol was used as a control. In addition, the effect of

propolis on the growth of 26 H. pylori and 18 campylobacter strains was tested by the

disc diffusion method. Mean diameters of H. pylori growth inhibition by the agar-well

diffusion method, using 30, 60 or 90 µl EEPor 30µl ethanol per well, were 17·8, 21·2,

28·2 and 8·5 mm, respectively. EEP was significantly more active than ethanol against H.

pylori (P≤ 0·001).

26

The results obtained by the disc diffusion method were similar. The use of moist propolis

discs resulted in mean diameters of growth inhibition of 21·4 mm for H. pylori and 13·6

mm for Campylobacter spp. Dried propolis discs exhibited antibacterial effect against

73·1%of H. pylori isolates, with a considerable zone of growth inhibition (> 15 mm) in

36·4%of isolates. Using dried propolis discs resulted in mean diameters of growth

inhibition of 12·4 mm for H. pylori and 11·6 mm for Campylobacter spp. In conclusion,

Bulgarian propolis possesses considerable antibacterial activity against H. pylori, and can

also inhibit the growth of Campylobacter jejuni and Campylobacter coli. The potential of

propolis in the prevention or treatment of H. pylori infection is worth further extensive

evaluation. 55

2.5 S. Kambhoja et al. (2004) reported phytochemical analysis, Thin layer

chromatography and antiinflammatory activity. The bark was collected and dried for four

days under sun and powdered. It was extracted with different solvents ethanol, methanol

and ethyl acetate by soxhlet hot extraction process. Preliminary phytochemical analysis

was carried out for different extracts. It was found that saponin glycosides and

carbohydrate were present in the extracts. Thin layer chromatography studies were

carried out using Methanol: Chloroform and Methanol: Ethyl acetate. Spraying reagents

a) 5% Alcoholic sulphuric acid, b) 1% Vanillin in alcohol. Rf values were calculated for

the different spots. Extracts were screened for antiinflammatory activity by carageenan

induced rat paw oedema method by using ibuprofen as standard drug. The methonolic

and ethyl acetate extract has shown significant anti-inflammatory activity when compared

to that of control.56

2.6 In the present study Bhutani KK et al. (2004) reported in vivo effect of aqueous

extracts of Symplocos racemosa Roxb. (Fam. Symplocaceae) on serum FSH and LH

levels in immature female Sprague-Dawley rats under basal conditions. Aqueous extract

on oral administration significantly stimulated serum FSH level along with the rise in

serum LH level. Moreover, histopathological studies revealed enhanced folliculogenesis.

These results are in concordance with the traditional use of Symplocos racemosa for

female disorders.57

27

2.7 QIAN He et al. (2004) reported that the leaves of P. guajava Linn contain an

essential oil rich in cineol, tannins and triterpenes. In addition, three flavonoids

(quercetin, avicularin, and guaijaverin) have been isolated from the leaves. The

antioxidant activity of phenolic compounds is determined by their molecular structure

and, more specifically, by the position and degree of hydroxylation of the ring structure.

Phenolic compounds are typical active oxygen scavengers in foods and have evaluated by

several methods.58

2.8 Peroioli L. et al. (2004) reported development & evaluation of mucoadhesive buccal

patch. New formulation for topical administration of drugs in the oral cavity has been

developed using several film-forming and mucoadhesive polymers. The films have been

evaluated in terms of swelling, mucoadhesion and organoleptic characteristics. The best

film, containing polyvinylpyrrolidone (PVP) as film-forming polymer and

carboxymethylcellulose sodium salt (NaCMC) as mucoadhesive polymer, was loaded

with ibuprofen as a model compound and in vitro and in vivo release studies were

performed. In vivo studies showed the presence of ibuprofen in saliva (range 70-210

microg/ml) for 5 h and no irritation was observed. These mucoadhesive formulations

offer many advantages in comparison to traditional treatments and can be proposed as a

new therapeutic tool against dental and buccal diseases and disturbs.59

2.9 Bardonnet P.L.et al. (2006) reported the real difficulty of increasing the gastric

residence time of a dosage form,Bardonnet PL et al. have first summarized the important

physiologic parameters, which act upon the gastric residence time. Afterwards, they have

reviewed the different drug delivery systems designed until now, i.e. high-density,

intragastric floating, expandable, superporous hydrogel, mucoadhesive and magnetic

systems. Finally, focused on gastroretentive dosage forms especially designed against H.

pylori, including specific targeting systems against this bacterium.60

2.10 P.S. Rajinikanth et al.(2007) reported development of gellen based new intra-

gastric floating in situ gelling system for controlled delivery of amoxicillin for the

treatment of peptic ulcer disease caused by Helicobacter pylori (H. pylori). Gellan based

28

amoxicillin floating in situ gelling systems (AFIG) were prepared by dissolving varying

concentrations of gellan gum in deionized water containing sodium citrate, to which

varying concentrations of drug and calcium carbonate, as gas-forming agent, was added

and dissolved by stirring. The formulation variables like concentration of gellan gum and

calcium carbonate significantly affected the in vitro drug release from the prepared

AFIG. The in vivo H. pylori clearance efficacy of prepared AFIG in reference to

amoxicillin suspension following repeated oral administration to H. pylori infected

Mongolian gerbils was examined by polymerase chain reaction (PCR) technique and by a

microbial culture method. AFIG showed a significant anti-H. pylori effect in the in vivo

gerbil model. It was noted that the required amount of amoxicillin for eradication of H.

pylori was 10 times less in AFIG than from the corresponding amoxicillin suspension.

The results further substantiated that the prepared AFIG has feasibility of forming rigid

gels in the gastric environment and eradicated H. pylori from the gastrointestinal tract

more effectively than amoxicillin suspension because of the prolonged gastrointestinal

residence time of the formulation.61

2.11 Moin k.et al.(2007) reported in design and evaluation in situ gelling system for oral

sustained release drug delivery of Famotidine, which was selected as a model drug due to

its short biological half-life (2-3 hrs) and as an H2 receptor antagonist to be released in

stomach. On the basis of the preliminary trials, a 32 full factorial design was employed to

study the effect of independent variables, concentration of pectin (X1) and concentration

of CaCl2 (X2) on dependent variables like viscosity, drug content, Q50, Q80 and similarity

factor. Main effects and interaction terms of the formulation variables could be evaluate

quantitatively by a mathematical model. It was found that both the pectin and

concentration of CaCl2 had significant impact on viscosity, drug content, Q50, Q80 and

similarity factor (f2) of the system. In-vitro release study revealed that drug released from

the insitu gel followed non-fickian diffusion. In vivo study for the selected batch of

sodium alginate was carried out by pylorus legation method in rats, which showed gel

formation in gastric juice and reduction in ulcer index. Stability study was also carried

out for three months, which showed no major changes from their initial state.62

29

2.12Ahmad et al. (2007) reported that phytochemical investigation of the n-butanol

soluble fraction of the bark of stem of Symplocos racemosa Roxb yielded two new

phenolic glycosides of salirepin series, symplocuronic acid and sympocemoside, while

salirepin was isolated for the first time from this plant. The bark of stem of Symplocos

racemosa Roxb yielded has phenolic glycosides of salirepin series, symplocuronic acid,

sympocemoside, 1-ethyl brachiose-3'-acetate along with ketochaulmoogric acid,

nonaeicosanol, triacontyl palmitate, methyl triacontanoate, symplocomoside,

symponoside, symplososide, symploveroside. Benzoyl salireposide and salireposide. An

investigation of the kinetic and anti-angiogenic properties of plant glycoside inhibitors of

thymidine phosphorylase.63

2.13 Thimmasetty J. et al. (2008) reported buccal absorption studies of a carvedilol

solution in human volunteers showed 32.86% drug absorption. FTIR and UV

spectroscopic methods revealed that there was no interaction between carvedilol and

polymers. Carvedilol patches were prepared using HPMC, carbopol 934, eudragit RS

100, and ethylcellulose. The patches were evaluated for their thickness uniformity,

folding endurance, weight uniformity, content uniformity, swelling behaviour, tensile

strength, and surface pH. In vitro release studies were conducted for carvedilol-loaded

patches in phosphate buffer (pH, 6.6) solution. Patches exhibited drug release in the range

of 86.26 to 98.32% in 90 min. Data of in vitro release from patches were fit to different

equations and kinetic models to explain release profiles. Kinetic models used were zero

and first-order equations, Hixon-Crowell, Higuchi, and Korsmeyer-Peppas models. In

vivo drug release studies in rabbits showed 90.85% of drug release from HPMC-carbopol

patch while it was 74.63 to 88.02% within 90 min in human volunteers. Good correlation

among in vitro release and in vivo release of carvedilol was observed.64

2.14 Hussain S et al. (2009) reported the potential of symplocomoside (1) and

symponoside (2), glycosides isolated from the bark of to inhibit thymidine

phosphorylase (TP) activity and associated angiogenesis. Compound 1 was a reversible,

noncompetitive inhibitor of deoxythymidine binding to TP and 2 was a reversible,

uncompetitive inhibitor. Both compounds were active in in vitro angiogenic assays

inhibiting endothelial cell migration and invasion in Matrigel, but did not inhibit growth

30

factor-induced proliferation and were not cytotoxic. Compound 1 may have potential as

an anti-angiogenic and anti-tumor agent.65

2.15 Elekwa et al. (2009) reported phytochemical screening ,Thin layer chromatography

and antibacterial activity of extracts (ethanol, methanol and aqueous) of the leaves and

stem barks of Psidum guajava L. Results of the phytochemical screening revealed the

presence of alkaloids (all the extracts), saponins (ethanol and methanol), cardenolides

with steroided rings, and cardenolides with deoxy sugar (all the extracts). Thin layer

chromatographic separation of ethanol and methanol extracts gave three spots each with

Rf values ranging from 0.60 – 0.70. Only the aqueous extract inhibited Bacillus subtilis

and Fusarium spp. The presence of these constituents tends to support the uses of this

plant medicinally.66

2.16 Ahmed Abd El-Meguid Mostafa et al. (2009) reported the topical application of

quercetin. Quercetin is a useful therapeutic agent for the treatment of colitis and gastric

ulcer. The objective of this study was to determine the effect of topical application of

quercetin in the treatment of the most prevalent form of aphthous ulcers. Topical

application 3 times daily of Quercetin cream to minor mouth ulcers relieved pain and

produced complete healing in the majority of patients (35%) between 2 to 4 days and in

90% between 4 to 7 days, compared with no ulcer healing within 7 days in control

group.67

2.17 Subhash V. Deshmane et al. (2009) reported characterization of effect of chitosan

with PVK. Objective of present work was to characterize the effect of chitosan with PVP-

K on water soluble drug by preparing mucoadhesive patch. Each formulated batch was

subjected to various evaluation parameters. The swelling % was found to be function of

solubility of drug & PVK-30. The mucoadhesive strength, vapour transmission & in vitro

release of water soluble drug through water insoluble chitosan base matrix were found

satisfactory. The physical appearance of buccal patch was examined by scanning electron

microscopy. The released kinetics model best to fit for optimized batch was Hixoon

31

crowell, indicating that drug release from systems in which there is change in surface

area & diameter of particles in dosage form. 68

2.18 P. Thirunavukkarasu et al. (2009) reported determination of the gastro protective

effect of E. agallocha in a model of NSAID induced ulcer rat. The lyophilized extract

was given by oral gavages (125 and 62.5mg/kg) three times at 12 h intervals before

administering dicolofenac 100mg/kg. Pretreatment with the extract resulted in a

significant decreased of the ulcerated area. The volume and acidity of the gastric juice

decreased in the pretreated rats. The plant extract was elevated in the gastric juice of

untreated rats, showed hear normal levels in the pretreated rats.The E. agallocha was able

to decrease the acidity and increase the mucosal defense in the gastric areas, there by

justifying its use as an antiulcerogenic agent.69

2.19 M. Vijaya et al. (2010) reported anti-pyretic activity of ethanol extract of Symplocos

racemosa (EESR) bark in experimental models. Subcutaneous injection of 20% aqueous

suspension of Brewer’s yeast in wistar rats leads to pyrexia. Intraperitoneal

administration of EESR at the dose of 100 and 200 mg/kg were shown dose dependent

decrease in body temperature in brewer’s yeast induced hyperthermia in rats. EESR

significantly decrease in body temperature (p<0.05) at 200mg/kg when compared to

control.These findings suggest that the ethanol extract of Symplocos racemosa possessed

good antipyretic activity. Preliminary phytochemical screening of the extract showed the

presence of carbohydrates, triterpenoids, tannins, flavonoids, anthocyanins, steroids and

glycosides which may be responsible for antipyretic activity.70

2.20 Devmurai V.P. (2010) reported in Preliminary phytochemical screening of

petroleum ether and alcohol extract of Symplocos Racemosa Roxb that alcoholic extract

contains carbohydrate, glycoside, saponin and terpenoid & alkaloid. Ether extract

indicated presence of glycoside, phytosterol and steroid. An antibacterial evaluation of

the petroleum ether and ethanolic extract was carried out and it is found that ethanolic

extract possess a good antibacterial activity.71

32

2.21 M. Vijayabaskaran et al. (2010) reported antitumor and antioxidant status of

ethanol extract (100 and 200mg/kg) of Symplocos racemosa (EESR).Extracts were

evaluated against Ehrlich ascites carcinoma (EAC) bearing swiss albino mice. Acute and

short term toxicity studies were performed initially in order to ascertain the safety of

EESR. After 24 h of tumor inoculation, the extract was administered daily for 14 days

intraperitoneally. After administration of last dose followed by 18 h fasting, the mice

were sacrificed for observation of antitumor activity. The effect of EESR on the growth

of transplantable murine tumor, life span of EAC bearing mice, hematological profile and

liver biochemical parameters (lipid peroxidation, antioxidant enzymes) were estimated.

Treatment with EESR decreased the tumor volume and viable cell count thereby

increasing the lifespan of EAC bearing mice and brought back the hematological

parameter more or less normal level. The effect of EESR also decreases the level of lipid

peroxidation and increased the levels of catalase (CAT). The present work indicates that

the ethanol extract of Symplocos racemosa exhibited antitumor effect by modulating lipid

peroxidation and augmenting anti-oxidant defense system in EAC bearing mice.72

2.22 Pongsak Rattanachaikunsopon et al. (Jan. 2010) reported isolation of four

flavonoids from Psidium guavaja leaves.Four antibacterial flavonoids (morin-3-O-

lyxoside, morin-3-O-arabinoside, quercetin, and quercetin-3-Oarabinoside) were isolated

from fresh and dried Psidium guajava leaves, and their concentrations were determined.

Among them, quercetin and morin-3-O-arabinoside were the most and the least abundant,

respectively. 73

2.23 A. M. Metwally Sohafy et al. (2010) reported the antimicrobial testing of guava

glycosides.Quercetin is the main flavonoidal nucleus of guava glycosides. Meanwhile,

the antimicrobial testing showed that the extracts and the isolated compounds possess

antibacterial and antifungal activities. These findings explain the folkloric use of the

extracts as bactericide, in cough, diarrhea, gargles to relieve oral ulcers and inflamed

gums wound.74

33

2.24 Metwally A.M. et al. (2010) reported Phytochemical investigation and

antimicrobial activity of Psidium guajava L. leaves. Psidium guajava L. leaves were

subjected to extraction, fractionation and isolation of the flavonoidal compounds. Five

flavonoidal compounds were isolated which are quercetin, quercetin-3-O-α-L-

arabinofuranoside, quercetin-3-O-β-D-arabinopyranoside, quercetin-3-O-β-D-glucoside

and quercetin-3-O-β-D-galactoside. Quercetin-3-O-b-D-arabinopyranoside was isolated

for the first time from the leaves. Fractions together with the isolates were tested for their

antimicrobial activity. The antimicrobial studies showed good activities for the extracts

and the isolated compounds.75

2.25 C.H.Durry (2010) reported that white guava bark, especially of the root is much

valued as an astringent. Dr.Wwitz employed it with success in chronic diarrhea in

children. He administered it in the form of decoction, in doses of one or more

teaspoonful, three or four times daily. The root and young leaves are astringent and are

esteemed useful in strengthening the stomach. During the cholera epidemic at Mauritius,

decoction of the leaves, according to M. Bouton ,was frequently used for arresting the

vomiting and diarrhea.76

2.26 T.M. Kalyankar et al. (2010) reported review of Bioadhesive Drug Delivery

System. The term ‘bioadhesive’ describes materials that bind to biological substrates,

such as mucosal membranes and in bioadhesive drug delivery systems. The bioadhesive

drug delivery formulation highlights the fact that readily accessible sites are utilised, with

the eye, oral cavity and being targeted. The GI tract and the nasal cavity have also been

extensively examined as a site for bioadhesive drug delivery. The present review explains

the success achieved with the bioadhesive formulationas adhesion of bioadhesive drug

delivery devices to mucosal membranes leads to an increased drug concentration gradient

at the absorption site and therefore improved bioavailability of systemically delivered

drugs and also used to target local disorders at side-effects that may be caused by

systemic administration of drugs.77

34

2.27 Harshad G. Parmar et al. (2010) reported review article on Buccal patch. Rapid

development in the field of molecular biology & gene technology resulted in generation

of many macromolecular drugs peptides, proteins, polysaccharides in great number

possessing superior pharmacological efficacy with desire specificity & devoid of

untoward & toxic effects. The need for research into drug delivery systems extends

beyond ways to administer new pharmaceutical therapies.Buccal adhhsive system offer

innumerable advantages in terms of accessibility, administration & withdrawl, retentivity,

low enzymatic activity, economy & high patient compliance.78

2.28 Amit Khairnar et al. (2010) reported preparation of Mucoadhesive buccal patch of

Aceclofenac using polymer like Gelatin, Poly Sodium CMC and Poly Vinyl Alcohol.

Eight formulations were prepared with varying the concentration of Poly Sodium CMC

and evaluated for various parameters like weight variation, patch thickness, volume

entrapment efficiency %, and measurement of % elongation at break, folding endurance,

in vitro mucoadhesive time, invitro release and stability study.The formulations showed a

sustained release. The F5 formulation containing Aceclofenac 6%, Gelatin 4.5%, Poly

Sodium CMC 5.5%, Propyleneglycol 5%, Poly vinyl Alcohol 2.5% and Distilled Water

up to 100%, showed a release of 88.4% after 8 hours. The Aceclofenac stability studies

were performed at 40 ± 20 C / 75 ± 5% RH. Among the eight formulations, F5

formulation showed maximum stability.79

2.29 Patel R.P.et al. (2010) reported preparation, optimization & evaluation of in situ

gelling system of Renitidine. HCL based on ranitidine that retains in the stomach by

adherence to gastric wall providing increased gastric residence time resulting in

prolonged drug delivery in gastrointestinal tract. Sodium alginate was used as polymer

CaCo3 as cross linking agent. The insitu formulation exhibited the expectations, viscosity,

drug content & sustained drug release. The study reports that oral admission of oral

solutions containing sodium alginate results in the formulation of in situ gel and such

formulations are homogeneous liquids when administered orally and become gel at

contact site. The results of 32 factorial designs reveals that concentrations of sodium

35

alginate & CaCo3 significantly affected the dependent variables of viscosity, Q50 & Q80.

These in situ gel, are, thus suitable for sustained release of ranitidine HCL.80

2.30 Ramachandran S, et al. (2010) reported development of floating drug delivery

system for improving the drug bioavailability by prolongation of gastric residence time of

famotidine in stomach. The floating micro balloons were prepared using polymer

Eudragit L-100 by solvent evaporation and diffusion technique. The prepared famotidine

loaded microspheres were characterized for drug loading, entrapment, encapsulation

efficiency, particle size distribution, surface morphology, differential scanning

calorimetric, test for buoyancy, in-vitro release and in-vivo antiulcer studies.

The results showed an increased drug loading, encapsulation and entrapment efficiency.

The thermo gram of the DSC showed that the drug was encapsulated in amorphous form

and SEM studies revealed the discrete, spherical shaped spheres with rough surface and

presence of holes on floating microspheres due high entrapment of PEG which are

responsible for drug release and floating ability. The sizes of spheres were found between

20-120 micron which exhibited prolonged release (In-vitro > 8 h) and remained buoyant

for > 10 h. The mean particle size increased and the drug release rate decreased at higher

Eudragit L-100 polymer concentration. The in-vivo results showed significant antiulcer

property of famotidine loaded microspheres when compared to control and standard

group of rats by using pyloric ligation method. The mean volume of gastric secretion,

mean pH and mean total acid for formulation treated group was calculated as 3.45+/-0.88

ml, 5.65+/-0.74, and 114.15+/-1.80 mEq/L respectively.81

2.31 Dr. Soumendra Darbar et al. (2010) reported evaluation of the gastro protective

effect of Livina, a polyherbal formulation on ethanol (50%) induced gastric ulcers in

mice. Forty young white male Swiss albino mice were divided to five groups

(8mice/group). Three case groups received Livina (50, 100, 200 mg/kg) and control

negative and positive groups received distilled water and ranitidine respectively. Animals

were killed and their stomachs were removed and macroscopic and microscopic ulcer

index were determined. Data were subjected to one-way ANOVA followed by Dennett’s

36

t-test. The results indicated that polyherbal formulation, Livina (50,100,150 mg/kg)

significantly decreased the ulcer index (p<0.05) and these doses of formulation exerted

macroscopic curative ratios of 67.63%, 75.11% and 81.09% respectively. However,

Livina at doses of 100 and 200 mg/kg significantly (P< 0.05) showed an antiulcer effect

characterized by reduction of acid volume (AV), free acidity (FA), total acidity (TA), and

increasing rate of pH, when compared to the control group. The present findings

demonstrate that, Livina has gastro protective effect on ethanol induced gastric ulcer in

mice model.82

2.32 Ch. Santhosh Kumari et al. (2010) reported antimicrobial and antiulcer activities

of Aloe vera plant extract were evaluated against H. pylori strains. According to several

studies, oral consumption of Aloe Vera works effectively to soothe conditions like

heartburn, arthritis and rheumatism pain and asthma. Therefore the current study is aimed

to evaluate the anti-H. pylori and antiulcer properties of Aloe vera.

The antimicrobial activity was detected by using disc diffusion method. In vivo activities

were also studied in albino rats by ethanol induced ulcers and the treatment regimens.

The results showed that Aloe vera exhibited strong antimicrobial activity against H.

pylori at two different concentrations of 250, 500mg/mL in comparison with standard

Clarithromycin. In vivo studies showed a very good response in ulcer healing properties.

Study found that use of Aloe vera may act as complementary and alternative medicine for

gastrointestinal diseases. 83

2.33 Anju Dhiman et al. (2011) reported Antibacterial activity of metahnolic extract P.

guajava. The methanolic extract exhibited antibacterial activity against E. coli with

minimum inhibitory concentration, 0.78 μg/ml, minimum bactericidal concentration of 50

μg/ml, and appreciable antifungal activity with minimum inhibitory concentration of 12.5

μg/ml. Preliminary phytochemical analysis of methanolic extract revealed the presence of

antimicrobial compounds such as flavonoids, steroids, and tannins, which may contribute

for the antimicrobial action of P. guajava.84

37

2.34 Narasimha Rao R et al. (2011) reported in the present work collection of plant

material, extraction of the crude drug, Successive solvent extraction, phytochemical tests

of plant extract, Thin layer chromatography, HPTLC and In vitro anthelminthic activity.85

2.35 Hetangi Rathod et al. (2011) reported efficacy of In situ gelling system. Among

oral dosage form, liquid dosage forms are more prone to low bioavailability because of

their quick transit from the gastrointestinal tract. Sustained release liquid formulation

with efficacy can be produced using approach of In situ gel. The purpose of the present

work was to develop oral in situ gelling system using Galan gum for in situ gelation of

ambroxol-HCl. The formulation variables like concentration of polymer and calcium

chloride will be optimized using factorial design. Optimized formulations were prepared

having desirability and evaluated for various parameter.86

2.36 Dasharath M. Patel et al. (2011) reported in an attempt to develop a new floating

in situ gelling system of amoxicillin with increased residence time using sodium alginate

as gelling polymer to eradicate H. pylori. Floating in situ gelling formulations were

prepared using sodium alginate, calcium chloride, sodium citrate, hydroxy propyl methyl

cellulose K100, and sodium bicarbonate. The prepared formulations were evaluated for

solution viscosity, floating lag time, total floating time, and in vitro drug release. The

formulation was optimized using a 32 full factorial design. Dissolution data were fitted to

various models to ascertain kinetic of drug release. Regression analysis and analysis of

variance were performed for dependent variables. All formulations (F1–F9) showed

floating within 30 s and had total floating time of more than 24 h. All the formulations

showed good pourability. It was observed that concentration of sodium alginate and

HPMC K100 had significant influence on floating lag time, cumulative percentage drug

release in 6 h and 10 h. The batch F8 was considered optimum since it showed more

similarity in drug release (2=74.38 ) to the theoretical release profile. Floating in situ

gelling system of amoxicillin can be formulated using sodium alginate as a gelling

polymer to sustain the drug release for 10 to 12 h with zero-order release kinetics.87

38

2.37 Biplab Adhikary et al. (2011) reported the healing activities of black tea (BT) and

the theaflavins (TF) against the indomethacin-induced stomach ulceration were studied in

a mouse model. Indomethacin (18mg/kg, p.o.) administration induced maximum

ulceration in the glandular portion of the gastric mucosa on the 3rd day, accompanied by

increased lipid peroxidation and protein oxidation, depletion of thiol-defense and mucin,

as well as reduced expressions of cyclooxygenases (COX) and prostaglandin (PG) E

synthesis in the gastric tissues, and plasma total antioxidant status of mice. Treatment

with BT (40mg/kg), TF (1 mg/kg), and omeprazole (3 mg/kg) produced similar (74%–

76%) ulcer healing, as revealed from the histopathological studies. Treatment with all the

above samples reversed the adverse oxidative effects of indomethacin significantly. BT

and TF also enhanced the PGE synthesis by augmenting the expressions of COX 1 and 2,

but did not modulate acid secretion.88

2.38 Shanthi A. et al. (2011) reported the anti‐ulcer activity of the polyherbal

formulation. It was investigated by ethanol induced gastric ulcer model in wistar rats. In

this evaluation the ulcer index was measured using histopathological sections. The

formulation with 500mg/kg per oral produced significant inhibition of the gastric lesions

in ethanol induced ulcer model with respect to standard 20mg/kg of Omeprazole (P.O)

administration. And the dose fixation was made with the help of acute toxicity studies

with varying doses in wistar rats. And the result shows that the formulation might be

useful in severe gastric ulcer, antiulcerogenic and as well as ulcer healing properties,

which might be due to its antisecretory activity. The formulation is non‐toxic even at

relatively high concentration.89

2.39 CH. Prasanthi et al. (2011) reported review of herbal formulations used in

treatment of H. Pylori infection. This review focuses the issues related to the diagnosis

and treatment of H pylori infection including herbal formulations. One of the most

vulnerable gastro intestinal tract infections affecting the human population worldwide are

H pylori infections. It causes complicated gastric problems such as gastritis, gastro

duodenal ulcers, gastric cancer and primary B-cell gastric lymphoma. The current dosage

regimens proposed by the international guidelines which are in practice to abate this

39

chronic destructive bacterial infection (combination of two antibiotics (clarithromycin

plus amoxicillin or metronidazole) with a PPI for at least 7 days for the eradication of H

pylori) were still found to be unsatisfactory. So there is a need of hour to design and

develop alternative drug delivery systems viz. gastro retentive delivery systems, site

specific delivery systems and probiotics. 90

2.40 Jayvadan K. Patel et al. (2012) reported development of Alginate based floating In

situ gelling system of famotidine. It was prepared by dissolving varying concentration of

alginate in deionized water containing sodium citrate, to which varying concentration of

drug & Calcium chloride was added & dissolved by stirring. Results of preliminary trials

indicate that concentration of sodium alginate, calcium chloride & sodium citrate affected

the characterization of In situ gel. A 32 full factorial design was developed to study the

effect of independent variables, concentration of sodium alginate (X1) & concentration of

calcium chloride (X2) as dependent variable ; i.e. viscosity, drug content, drug release at 4

hrs. (Q50)& drug release at 8 hrs (Q80). A subsequent drug release was obtained for more

than 8 hrs. In vivo testing of FIGF to albino Wistar rats demonstrated significant antiulcer

effect of famotidine.91

2.41Ehsan Mirkamandar et al. (2012) reported evaluation of the invitro antimicrobial

activity of a methanolic extract of Salvadora persica solution on Helicobacter pylori

isolated from duodenal ulcer. Minimum inhibitory concentration (MIC) and minimal

bactericidal concentration (MBC) of the extract were determined by the agar dilution

method. At concentrations of 10, 100, 200, 500 μg/mL,no zone of inhibition around the

ditches was observed while a clear zone of inhibition (12 mm) was detected at 1000

μg/mL concentration for all the isolates. The best antimicrobial activity was observed at

MIC 1000 μg/mL (P≤0.05). The MBC results showed that at a concentration of 1000

μg/mL all cells were dead while at a concentration of 750 μg/mL of S. persica a few

H.pylori cells were still able to form colonies on Brucella agar supplemented with sheep

red blood cells and antibiotics.

40

From the above results it can be concluded that high concentration of S.persica could

inhibit the growth of H. pylori and MIC and MBC were similar at that concentration.

92

2.42 Ananya Chatterjee et al. (2012) presented a Review of the pathophysiology of

H.pylori infection and its potential herbal remedy. Natural medicines and plant products,

such as tea, resveratrol, curcumin, garlic, cinnamon, etc. can heal H. pylori induced

gastric ulcers by scavenging the reactive oxygen and nitrogen species, boosting the host

immune system, modulating host-pathogen heat shock proteins interactions. They are

nontoxic in nature and hence can be used safely. Therefore, it is concluded that inclusion

of natural antioxidants in the normal, daily diet may be the best remedial measure for

continued protection from H. pylori infection.93

41

CHAPTER 3: AIM AND OBJECTIVES

Traditional herbal medicine and their preparations have been widely used for the

thousands of years in developing and developed countries owing to its natural origin and

lesser side effects or dissatisfaction with the results of synthetic drugs.

Mouth contains a wide variety of micro-organism. Actinomyces, Neisseria and

Candida species are the important components of flora of the oropharynx. The micro-

organisms frequently identified in root caries are Lactobacillus acidophilus, Actinomyces

viscosus, Nocardia spp., Neisseria spp., streptococcus mutans, Staphylococcus aureus

and fungi like Candida albicans and Aspergillus spp.

Staphylococcus aureus, Pseudomonas aureginosa and Candida albicans are

frequently encountered in high proportions in smooth tooth surfaces and gingiva, in

moderate proportions in pits and fissure of teeth, periodontal ligament, saliva, cheek and

tongue. In the oral cavity they have been commonly implicated as a cause of

periodontitis. So the herbal approach is needed to face the problem.

Recent interest has been expressed in the delivery of drug to or via mucosal

membrane by the use of adhesive materials. Several mucoadhesive formulations are

available or in developmental stages. Drug delivery through the buccal mucosa offers a

novel route of drug administration.

Various synthetic polymers are under investigation as carrier for buccal drug delivery. In

the present study, polymers such as, chitosan, hydroxypolymethyl cellulose (HPMC)

hydroxypropyl cellulose, hydroxyethyl cellulose (HEC), polyvinyl alcohol (PVA) and

polyvinyl pyrollidone (PVP) were employed. These polymers seen to have potential and

are comparatively economical.

Recently there has been a rapid progress in the understanding of peptic ulcer most of the

studies focus on newer and better drug therapy. This has been the rationale for the

development of new antiulcer drugs which include the herbal drugs. Herbal prescriptions

and natural remedies are commonly employed in developing countries for the treatment

of various diseases, this practice being an alternative in orthodox pharmacotherapy.

Natural products of plant origin are still a major part of global traditional medicine

especially in gastrointestinal disorders. Some studies have shown that drugs commonly

42

used for peptic ulcers such as H2-blockers (ranitidine, famotidine etc.), M1-blockers

(pirenzepine, telenzepine etc), proton pump inhibitors (omeprazole, pantoprazole etc.)

have danger of drug interaction, adverse effect and increased incidence of relapses during

ulcer therapy. The herbal medicines derived from various plant extracts are being

increasingly utilized to treat a wide variety of clinical diseases.

Literature review indicated that antiulcer activity of these plants (Psidium guajava &

Symplocos racemosa) have not been evaluated so far. In view of this, the present study

was aimed to formulate & evaluating the antibacterial & antiulcer activity of extracts of

Psidium guajava & Symplocos racemosa.

3.1 The aim of this research project is to develop & characterize herbal based

1. Mucoadhesive buccal patch for mouth ulcer

2. In-situ Gel for peptic ulcer.

• To Formulate & evaluate mucoadhesive lipid based herbal buccal patch for

mouth ulcer as novel device to decrease sign & symptoms associated with dry

mouth that can be effectively used in mouth ulcers.

• To formulate & evaluate herbal In situ gel as a novel formulation for peptic ulcer

caused by H.pylori & other factor.

• 2 plant species selected for the development of buccal patch & Insitu gel

1. Psidium guajava linn

2. Symplocos racemosa Roxb

3.2 Objectives

• Collection, identification and authentication of plant material.

• Pharmacognostic and phytochemical evaluation of plant material.

• Evaluation of extracts for Total Phenolic content & total flavonoid content.

• Evaluation for antimicrobial activity against pathogenic organism.

• To design and evaluate polymeric system as patches for transmucosal drug

delivery.

• To design and evaluate In-situ gel as floating drug delivery for treatment of peptic

ulcer .

43

• To study in vitro release kinetics from selected polymeric patches & Insitu gel.

• Evaluation of antiulcer activity on animal (In-vivo).

44

CHAPTER 4: PLAN OF WORK

PHASE I: Literature survey Ist year (0 – 3 months):-

1. Exhaustive & updated review of literature for selected plants.

2. Literature review on plants used for ulcer, methods of screening & mode of

action.

3. Literature review on Transmucosal Drug Delivery system & In situ Drug Delivery

system.

PHASE II: Collection of plant material & extraction: Ist year (4-8 months):-

1. Collection & authentication of plant material.

2. Preliminary Phytochemical screening, Standardization of plant material as per

WHO guidelines.

3. Preparation of extracts with polar & non polar solvents viz. pet.ether, chloroform,

methanol, alcohol, water.

4. Qualitative phytoprofiles of extracts by chemical tests & TLC.

PHASE III (A): Preliminary pharmacological studies (9-12months):-

1. Evaluation of antimicrobial activity of plant extracts by determination of MIC

2. Evaluation of antimicrobial activity of plant extracts by zone of inhibition

PHASE III (B): II year (1-6 months):-

1. Quantitative determination of bio active fraction & HPTLC finger prints.

2. Determination of Antibacterial activity of extracts against H. Pylori

PHASE IV: Formulation development of extracts: II year (6-12 months):-

1. Development of Bioadhesive buccal patches

2. Antimicrobial evaluation of prepared formulations

45

3. Physical Evaluation

4. In-vitro & In-vivo characterization of Bio adhesive patches.

• PHASE V: (A)Formulation & characterization of In-situ gel: III year (0 – 6

months )

1. Development of In situ gel using extracts.

2. Characterization & optimization of In situ gel.

3. Determination of antiulcer activity of optimized batch

• PHASE V: (B) Stability studies as per ICH guidelines (6-12 months)

• PHASE VI : Documentation of results ( 6-12 months )

1. Systematic documentation

2. Evaluation of statistical significance of results by using computer aided software.

46

CHAPTER 5: MATERIALS & METHODS

5.1 Plant Profile:

5.1.1.Psidium guajava : The apple guava or common guava (Psidium guajava; known

as goiaba in Portuguese and guayaba in Spanish) is an evergreen shrub or

small tree native to Mexico, the Caribbean, and Central and South America.94

It is

easily pollinated by insects; in culture, mainly by the common honey bee, Apis mellifera.

Kingdom: Plantae

Order: Myrtales

Family: Myrtaceae

Subfamily: Myrtoideae

Tribe: Myrteae

Genus: Psidium

Species: P. guajava

Common names: Guava, goiaba, guayaba, djamboe, djambu, goavier, gouyave, goyave,

goyavier, perala, bayawas, dipajaya jambu, petokal, tokal, guave, guavenbaum, guayave,

banjiro, goiabeiro, guayabo, guyaba, goeajaaba, guave, goejaba, kuawa, abas, jambu batu,

bayabas, pichi, posh, enandi

Part Used: Fruit, leaf, bark

Called guayaba in Spanish-speaking countries and goiaba in Brazil, guava is a common

shade tree or shrub in door-yard gardens in the tropics. It provides shade while the guava

fruits are eaten fresh and made into drinks, ice cream, and preserves. In the richness of

the Amazon, guava fruits often grow well beyond the size of tennis balls on well-

branched trees or shrubs reaching up to 20 m high. Cultivated varieties average about 10

meters in height and produce lemon-sized fruits. The tree is easily identified by its

distinctive thin, smooth, copper-colored bark that flakes off, showing a greenish layer

beneath 95

.

47

Guava fruit today is considered minor in terms of commercial world trade but is widely

grown in the tropics, enriching the diet of hundreds of millions of people in the tropics of

the world. Guava has spread widely throughout the tropics because it thrives in a variety

of soils, propagates easily, and bears fruit relatively quickly. The fruits contain numerous

seeds that can produce a mature fruit-bearing plant within four years. In the Amazon

rainforest guava fruits are much enjoyed by birds and monkeys, which disperse guava

seeds in their droppings and cause spontaneous clumps of guava trees to grow throughout

the rainforest.

Tribal and herbal medicine uses

Guava may have been domesticated in Peru several thousand years ago; Peruvian

archaeological sites have revealed guava seeds found stored with beans, corn, squash, and

other cultivated plants. Guava fruit is still enjoyed as a sweet treat by indigenous peoples

throughout the rainforest, and the leaves and bark of the guava tree have a long history of

medicinal uses that are still employed today.

The Tikuna Indians decoct the leaves or bark of guava as a cure for diarrhea. In fact, an

infusion or decoction made from the leaves and/or bark has been used by many tribes for

diarrhea and dysentery throughout the Amazon, and Indians also employ it for sore

throats, vomiting, stomach upsets, for vertigo, and to regulate menstrual periods 96

.

Tender leaves are chewed for bleeding gums and bad breath, and it is said to prevent

hangovers (if chewed before drinking). Indians throughout the Amazon gargle a leaf

decoction for mouth sores, bleeding gums, or use it as a douche for vaginal discharge and

to tighten and tone vaginal walls after childbirth. A decoction of the bark and/or leaves or

a flower infusion is used topically for wounds, ulcers and skin sores. Flowers are also

mashed and applied to painful eye conditions such as sun strain, conjunctivitis or eye

injuries.

Centuries ago, European adventurers, traders, and missionaries in the Amazon Basin took

the much enjoyed and tasty fruits to Africa, Asia, India, and the Pacific tropical regions,

so that it is now cultivated throughout the tropical regions of the world. Commercially the

48

fruit is consumed fresh or used in the making of jams, jellies, paste or hardened jam, and

juice. Guava leaves are in the Dutch Pharmacopoeia for the treatment of diarrhea, and

the leaves are still used for diarrhea in Latin America, Central and West Africa, and

Southeast Asia. In Peruvian herbal medicine systems today the plant is employed for

diarrhea, gastroenteritis, intestinal worms, gastric disorders, vomiting, coughs, vaginal

discharges, menstrual pain and hemorrhages, and edema. In Brazil guava is considered an

astringent drying agent and diuretic and is used for the same conditions as in Peru. A

decoction is also recommended as a gargle for sore throats, laryngitis and swelling of the

mouth, and used externally for skin ulcers, and vaginal irritation and discharges.

Plant chemicals

Guava is rich in tannins, phenols, triterpenes, flavonoids, essential oils, saponins,

carotenoids, lectins, vitamins, fiber and fatty acids. Guava fruit is higher in vitamin C

than citrus (80 mg of vitamin C in 100 g of fruit) and contains appreciable amounts of

vitamin A as well. Guava fruits are also a good source of pectin - a dietary fiber. The

leaves of guava are rich in flavonoids, in particular, quercetin. Much of guava's

therapeutic activity is attributed to these flavonoids. The flavonoids have demonstrated

antibacterial activity. Quercetin is thought to contribute to the anti-diarrhea effect of

guava; it is able to relax intestinal smooth muscle and inhibit bowel contractions. In

addition, other flavonoids and triterpenes in guava leaves show antispasmodic activity.

Guava also has antioxidant properties which is attributed to the polyphenols found in the

leaves97

.

Guava's main plant chemicals include: alanine, alpha-humulene, alpha-hydroxyursolic

acid, alpha-linolenic acid, alpha-selinene, amritoside, araban, arabinose,

arabopyranosides, arjunolic acid, aromadendrene, ascorbic acid, ascorbigen, asiatic acid,

aspartic acid, avicularin, benzaldehyde, butanal, carotenoids, caryophyllene, catechol-

tannins, crataegolic acid, D-galactose, D-galacturonic acid, ellagic acid, ethyl octanoate,

essential oils, flavonoids, gallic acid, glutamic acid, goreishic acid, guafine,

guavacoumaric acid, guaijavarin, guajiverine, guajivolic acid, guajavolide, guavenoic

acid, guajavanoic acid, histidine, hyperin, ilelatifol D, isoneriucoumaric acid,

49

isoquercetin, jacoumaric acid, lectins, leucocyanidins, limonene, linoleic acid, linolenic

acid, lysine, mecocyanin, myricetin, myristic acid, nerolidiol, obtusinin, octanol,

oleanolic acid, oleic acid, oxalic acid, palmitic acid, palmitoleic acid, pectin, polyphenols,

psidiolic acid, quercetin, quercitrin, serine, sesquiguavene, tannins, terpenes, and ursolic

acid 98,99

.

Biological activities and clinical research

The long history of guava's use has led modern-day researchers to study guava extracts.

Its traditional use for diarrhea, gastroenteritis and other digestive complaints has been

validated in numerous clinical studies. A plant drug has even been developed from guava

leaves (standardized to its quercetin content) for the treatment of acute diarrhea. Human

clinical trials with the drug indicate its effectiveness in treating diarrhea in adults. Guava

leaf extracts and fruit juice has also been clinically studied for infantile diarrhea. In a

clinical study with 62 infants with infantile rotaviral enteritis, the recovery rate was 3

days (87.1%) in those treated with guava, and diarrhea ceased in a shorter time period

than controls. It was concluded in the study that guava has "good curative effect on

infantile rotaviral enteritis."

Guava has many different properties that contribute to its antidiarrheal effect: it has been

documented with pronounced antibacterial, antiamebic and antispasmodic activity. It has

also shown to have a tranquilizing effect on intestinal smooth muscle, inhibit chemical

processes found in diarrhea and aid in the re-absorption of water in the intestines. In other

research, an alcoholic leaf extract was reported to have a morphine-like effect, by

inhibiting the gastrointestinal release of chemicals in acute diarrheal disease. This

morphine-like effect was thought to be related to the chemical quercetin. In addition,

lectin chemicals in guava were shown to bind to E-coli (a common diarrhea-causing

organism), preventing its adhesion to the intestinal wall and thus preventing infection

(and resulting diarrhea).

The effective use of guava in diarrhea, dysentery and gastroenteritis can also be related to

guava's documented antibacterial properties. Bark and leaf extracts have shown to have in

50

vitro toxic action against numerous bacteria. In several studies guava showed significant

antibacterial activity against such common diarrhea-causing bacteria as Staphylococcus,

Shigella, Salmonella, Bacillus, E. coli, Clostridium, and Pseudomonas. It has also

demonstrated antifungal, anti-yeast (candida), anti-amebic, and antimalarial actions 100

.

In a recent study with guinea pigs (in 2003) Brazilian researchers reported that guava leaf

extracts have numerous effects on the cardiovascular system which might be beneficial in

treating irregular heat beat (arrhythmia). Previous research indicated guava leaf provided

antioxidant effects beneficial to the heart, heart protective properties, and improved

myocardial function. In two randomized human studies, the consumption of guava fruit

for 12 weeks was shown to reduce blood pressure by an average 8 points, decrease total

cholesterol levels by 9%, decrease triglycerides by almost 8%, and increase "good" HDL

cholesterol by 8%. The effects were attributed to the high potassium and soluble fiber

content of the fruit (however 1-2 pounds of fruit was consumed daily by the study

subjects to obtain these results!) 101

. In other animal studies guava leaf extracts have

evidenced analgesic, sedative, and central nervous system (CNS) depressant activity, as

well as a cough suppressant actions 102

. The fruit or fruit juice has been documented to

lower blood sugar levels in normal and diabetic animals and humans. Most of these

studies confirm the plant's many uses in tropical herbal medicine systems 103

.

Current practical uses

Guava, known as the poor man's apple of the tropics, has a long history of traditional use,

much of which is being validated by scientific research. It is a wonderful natural remedy

for diarrhea - safe enough even for young children. For infants and children under the age

of 2, just a cup daily of guava fruit juice is helpful for diarrhea. For older children and

adults, a cup once or twice daily of a leaf decoction is the tropical herbal medicine

standard. Though not widely available in the U.S. market, tea-cut and powdered leaves

can be obtained from larger health food stores or suppliers of bulk botanicals. Newer in

the market are guava leaf extracts that are used in various herbal formulas for a myriad of

purposes; from herbal antibiotic and diarrhea formulas to bowel health and weight loss

51

formulas. Toxicity studies with rats and mice, as well as controlled human studies show

both the leaf and fruit to be safe and without side effects.

5.1.2. Symplocos racemosa :

Latin Name: Symplocos racemosa Roxb. (Symplocaceae)

Sanskrit /Indian Name: Lodhra, Tilva, Shavara, Lodh

General information:

Lodh Tree bark has been traditionally used as a uterine tonic. Additionally, the tree and

its formulations have been effective in controlling bleeding and digestive disorders.

Kingdom: Plantae

Order: Ericales

Family: Symplocaceae

Genus: Symplocos

Distribution- found in north- east India, Assam and Pegu regions of India.

Lodhra is botanically named as Symplocos racemosa it belongs to the genus Symplocos

and family Symplocaceae. Symplocos is a genus of flowering plants in the order Ericales,

containing about 250 species native to Asia, Australia and the Americas. Symplocos

racemosa is an evergreen tree or shrub. Leaves are dark green above, orbicular, elliptic

oblong, and glabrous above. Flowers are white, turning yellow, fragarant, simple or

compound racemes, the droops are purplish black, subcylindric, smooth and 1-3 seeded.

Chemical Constituents

Symplocos racemosa is a medicinal plant. Its bark is used to treat various ailments. The

chemical constituents of the Symplocos racemosa bark led to the isolation of two new

phenolic glycosides, Symconoside A and Symconoside B 104

. The important chemical

constituents of Symplocos racemosa are flavonoids, tannins, loturine, loturidine, and

colloturine. Symplocos racemosa is proven to contain the dithiadiazetidin namely

symploate, as well as linoleic acid, oleic acid, salireposide, symplocososide, betasito-

52

glycoside, symponoside, symplososide, symploveroside, benzoylsalireposide and

salireposide 105

. The scientific literature data also proves the presence of phenolic

glycosides, salireposide and benzoyl salireposide, which are found to be present in ethyl

acetate extract.

Pharmacology

Symplocos racemosa (Fam. symplocaceae) is a widely used ayurvedic remedy for various

ailments. It is also known as lodhra and is used as a single drug or in multicomponent

preparations. It possesses cardiotonic, antipyretic, antihelmintic and laxative properties. It

is beneficial in billow fever, urinary discharge, blood troubles, burning sensations,

leucoderma, and jaundice. In Indian traditional medicine the bark is also useful in bowel

complaints such as diarrhoea, dysentery, liver complaints, fever, ulcer etc 106

. The bark of

this plant also possesses anticancer activity.

A study has been also carried out to evaluate the antitumor activity of the ethanol extract

of Symplocos racemosa against Ehrlich’s Ascites Carcinoma (EAC) in mice. Treatment

with ethanolic extract decreased the tumor volume and viable cell count thereby

increasing the lifespan of EAC bearing mice. The study indicates that the ethanol extract

of Symplocos racemosa exhibited antitumor effect by modulating lipid peroxidation and

augmenting anti-oxidant defense system in EAC bearing mice 107

.

In addition, researchers have evaluated the antibacterial effect of S. racemosa extracts

against acne inducing bacteria. Symplocos racemosa is used in Indian System of

Medicine for various female disorders. In vivo effect of aqueous extracts of Symplocos

racemosa on serum FSH and LH levels in immature female Sprague–Dawley rats under

basal conditions has been observed. There are also lots of scientific literature data

proving the different pharmacological activity of Symplocos racemosa extract, e.g.

gonadotropin releasing, antioxidant, antiarthritic and antibacterial.

Health Benefits

Symplocos racemosa is a widely used as ayurvedic remedy for various ailments. Lodhra

bark is acrid, digestible, and astringent to bowels. It is useful in treatment of fever, eye

diseases, for spongy gums and bleeding. It cures diseases of the blood, leprosy, dropsy

and liver complaints. It is also useful in abortions and miscarriages and for ulcers of

vagina. Traditionally bark is given in menorrhagia and other uterine disorders 108

. Unani

53

medicine uses it as emmenogogue, aphrodisiac. It is also a potent remedy for

inflammation and cleaning uterus. It contains salireposide and benzoyl salireposide which

are inhibitors of phosphodiesterase I and have showed its depressant action on blood

pressure and instestinal movements 109

. Symplocos racemosa also works as a natural

antipyretic agent with reduced or no toxicity.

5.2 EXCIPIENT PROFILES:

5.2.1: Hydroxy propyl methylcellulose110, 111

:

Nonproprietary Names: BP: Hypromellose, USP: Hypromellose.

Synonyms: Benecel MHPC; E464; hydroxypropyl methylcellulose; HPMC; Methocel;

methylcellulose propylene glycol ether; methyl hydroxypropylcellulose; Metolose;

Tylopur.

Chemical Name and CAS Registry Number: Cellulose hydroxypropyl methyl ether

[9004-65]

Structural Formula:

Functional Category:

Coating agent; film-former; rate-controlling polymer for sustained release; stabilizing

agent; suspending agent; tablet binder; viscosity-increasing agent.

Applications in Pharmaceutical Formulation or Technology:

Hypromellose is widely used in oral, ophthalmic and topical pharmaceutical

formulations. In oral products, hypromellose is primarily used as a tablet binder, in

film-coating, and as a matrix for use in extended-release tablet formulations. Depending

upon the viscosity grade, concentrations of 2.20% w/w are used for film forming

solutions to film-coat tablets. Lower-viscosity grades are used in aqueous film coating

solutions, while higher-viscosity grades are used with organic solvents.

Description: Hypromellose is an odorless and tasteless, white or creamy-white fibrous

or granular powder.

54

Physical Properties:

Acidity/alkalinity : pH = 5.5.8.0 for a 1% w/w aqueous solution.

Ash : 1.5.3.0%, depending upon the grade and viscosity.

Auto ignition temperature : 360°C

Density (bulk) : 0.341 g/cm3

Density (tapped) : 0.557 g/cm3

Density (true) : 1.326 g/cm3

Melting point : Browns at 190.200°C; chars at 225.230°C. Glass

transition temperature is 170.180°C.

Solubility: Soluble in cold water, forming a viscous colloidal solution, practically

insoluble in chloroform, ethanol (95%), and ether, but soluble in mixtures of

ethanol / methanol and dichloromethane, and mixtures of water and alcohol.

Specific gravity: 1.26

Moisture content: Hypromellose absorbs moisture from the atmosphere; the

amount of water absorbed depends upon the initial moisture content and the

temperature and relative humidity of the surrounding air.

Viscosity (dynamic): Typical viscosity values for 2% (w/v) aqueous solutions of

Methocel .nominal viscosity (mPa s), methocel K4M Premium 4000. Typical

viscosity values for 2% (w/v) aqueous solutions of Methoce.

Methocel product USP 28 designation Nominal viscosity (mPa s)

Methocel K4M Premium 2208 4000

Methocel K15M Premium 2208 15000

Methocel E4M Premium 2910 4000

Stability and Storage Conditions:

Hypromellose powder is a stable material, although it is hygroscopic after drying.

Solutions are stable at pH 3.11. Hypromellose undergoes a reversible sol.gel

transformation upon heating and cooling, respectively. The gel point is 50.90°C,

depending upon the grade and concentration of material. Aqueous solutions are

comparatively enzyme-resistant, providing good viscosity stability during long-term

storage. However, aqueous solutions are liable to microbial spoilage and should be

preserved with an antimicrobial preservative aqueous solutions may also be sterilized

55

by autoclaving; the coagulated polymer must be redispersed on cooling by shaking.

Hypromellose powder should be stored in a well-closed container, in a cool, dry place.

Incompatibilities:

Hypromellose is incompatible with some oxidizing agents. Since it is nonionic,

hypromellose will not complex with metallic salts or ionic organics to form insoluble

precipitates.

Method of Manufacture:

A purified form of cellulose, obtained from cotton linters or wood pulp, is reacted

with sodium hydroxide solution to produce swollen alkali cellulose that is chemically

more reactive than untreated cellulose. The alkali cellulose is then treated with chloro

methane and propylene oxide to produce methyl hydroxypropyl ethers of cellulose.

The fibrous reaction product is then purified and ground to a fine, uniform powder or

granules.

Safety:

Hypromellose is widely used as an excipient in oral and topical pharmaceutical

formulations. It is also used extensively in cosmetics and food products. Hypromellose

is generally regarded as a nontoxic and nonirritant material, although excessive oral

consumption have a laxative effect.

Handling Precautions;

Hypromellose dust may be irritant to the eyes and eye protection is recommended.

Excessive dust generation should be avoided to minimize the risks of explosion.

Hypromellose is combustible.

Related Substances:

Hydroxyethyl cellulose; hydroxyethylmethyl cellulose; hydroxypropyl cellulose;

hypromellose phthalate; methyl cellulose.

5.2.2: Carbopol 940:110,111,112

Nonproprietary Names: BP: Carbomers, PhEur: Carbomer, USPNF: Carbomer

Synonyms: Acritamer; acrylic acid polymer; Carbopol; carboxy polymethylene,

polyacrylic acid;carboxyvinyl polymer; Pemulen; Ultrez

56

Chemical Name and CAS Registry Number: Carbomer [9003-01-4]

Empirical Formula and Molecular Weight: Carbomers are synthetic high-molecular-

weight polymers of acrylic acid that are crosslinked with either allyl sucrose or allyl

ethers of pentaerythritol. They contain between 56% and 68% of carboxylic acid

(COOH) groups calculated on the dry basis. The BP 2004 and PhEur 2005 have a

single monograph describing carbomer; the USPNF 23 contains several monographs

describing individual carbomer grades that vary in aqueous viscosity and in labeling

for oral or non-oral use.

Structural Formula:

Carbomer polymers

Carbomer polymers are formed from repeating units of acrylic acid. The monomer unit is

shown above. The polymer chains are crosslinked with allyl sucrose or allyl pent

aerythritol.

Functional Category:

Bioadhesive; emulsifying agent; release-modifying agent; suspending agent; tablet

binder; viscosity- increasing agent.

Applications in Pharmaceutical Formulation or Technology:

Carbomers are mainly used in liquid or semisolid pharmaceutical formulations as

suspending or viscosity-increasing agents. Formulations include creams, gels, and

ointments for use in ophthalmic, rectal, and topical preparations. Carbomer resins have

also been investigated in the preparation of sustained-release matrix beads, as enzyme

inhibitors of intestinal proteases in peptide-containing dosage forms, as a bioadhesive for

cervical patch and for intranasally administered microspheres, in magnetic granules for

57

site-specific drug delivery to the esophagusand in oral mucoadhesive controlled drug

delivery systems.

Carbomers are also used in cosmetics. Therapeutically, carbomer gel formulations have

proved efficacious in improving symptoms of moderate-to-severe dry eye syndrome.

Emulsifying agent 0.1.0.5, gelling agent 0.5.2.0, suspending agent 0.5.1.0, and tablet

binder 5.0.10.0.

Description: Carbomers are white-colored, .fluffy. Acidic, hygroscopic powders

with a slight characteristic odor.

Physical Properties:

Acidity/alkalinity : pH = 2.7.3.5 for a 0.5% w/v aqueous dispersion; pH =

2.5.3.0 for a 1% w/v aqueous Dispersion.

Density (bulk) : 1.76.2.08 g/cm3

Density (tapped) : 1.4 g/cm3

De Glass transition temperature: 100.105°C

Melting point : Decomposition occurs within 30 minutes at 260°C.

Specific gravity : 1.41

Moisture content:

Normal water content is up to 2% w/w. However, carbomers are hygroscopic and

typical equilibrium moisture content at 25°C and 50% relative humidity is 8.10%

w/w.

Particle size distribution:

Primary particles average about 0.2 µ #m in diameter. The flocculated powder

particles average 2.7 µ Lm in diameter and cannot be broken down into the primary

particles.

Solubility:

Soluble in water and, after neutralization, in ethanol (95%) and glycerin. Although

they are described as .soluble.Carbomers do not dissolve but merely swell to a

remarkable extent, since they are three-dimensionally crosslinked microgels.

58

Viscosity (dynamic):

Carbomers disperse in water to form acidic colloidal dispersions of low viscosity that,

when neutralized produce highly viscous gels. Carbomer powders should first be

dispersed into vigorously stirred water, taking care to avoid the formation of

indispersible lumps, then neutralized by the addition of a base agents that may be used

to neutralize carbomer polymers include amino acids, borax, potassium hydroxide,

sodium bicarbonate, sodium hydroxide, and polar organic amines such as

triethanolamine.

Stability and Storage Conditions:

Carbomers are stable, hygroscopic materials that may be heated at temperatures below

104°C for up to 2 hours without affecting their thickening efficiency. However,

exposure to excessive temperatures can result in discoloration and reduced stability.

Complete decomposition occurs with heating for 30 minutes at 260°C. At room

temperature, carbomer dispersions maintain their viscosity during storage for

prolonged periods. Exposure to light causes oxidation that is reflected in a decrease in

dispersion Carbomer powder should be stored in an airtight, corrosionresistant

container in a cool, dry place. Packaging in aluminum tubes usually requires the

formulation to have a pH less than 6.5, and packaging in other metallic tubes or

containers necessitates a pH greater than 7.7 to prolong carbomer stability.

Incompatibilities:

Carbomers are discolored by resorcinol and are incompatible with phenol, cationic

polymers, strong acids, and high levels of electrolytes. Trace levels of iron and other

transition metals can catalytically degrade carbomer dispersions. Certain amino

functional actives form water-insoluble complexes with carbomer; often adjusting the

solubility parameter of the fluid phase using appropriate alcohols and polyols can

prevent this. Carbomers also form pH-dependent complexes with certain polymeric

excipients.

Safety:

Carbomers are used extensively in nonparenteral products, particularly topical liquid

and semisolid preparations. They may also be used in oral formulations. Carbomers

59

are generally regarded as essentially nontoxic and nonirritant materials; there is no

evidence in humans of hypersensitivity reactions to carbomers used topically. In

humans oral doses of 1.3 g of

Carbomer have been used as a bulk laxative.

Handling Precautions: Observe normal precautions appropriate to the circumstances

and quantity of material handled. Carbomer dust is irritating to the eyes, mucous

membranes, and respiratory tract.

Related Substances: Polycarbophil.

5.2.3 : Sodium alginate 113

Physical and Chemical Properties:

Colour : White or white yellow

Nature : Hygroscopic

Odour : Odourless

Taste : Tasteless

Chemical structure:

Functional Category

Stabilizing agent; suspending agent; tablet and capsule disintegrate; tablet binder;

viscosity increasing agent

Solubility:

Practically insoluble in ethanol (95%), ether, chloroform, and ethanol/water

mixtures in which the ethanol content is greater than 30%. Also, practically insoluble in

60

other organic solvents and aqueous acidic solutions in which the pH is less than 3. Slowly

soluble in water, forming a viscous colloidal solution.

Viscosity (dynamic):

Various grades of sodium alginate are commercially available that yield

aqueous solutions of varying viscosity. Typically, a 1% w/v aqueous solution, at 20°C,

will have a viscosity of 20–400 mPa s (20–400 cP). Viscosity may vary depending

upon concentration, pH, temperature, or the presence of metal ions.26–28.Above pH

10, viscosity decreases.

Incompatibilities

Sodium alginate is incompatible with acridine derivatives, crystal violet,

phenyl mercuric acetate and nitrate, calcium salts, heavy metals, and ethanol in

concentrations greater than 5%. Low concentrations of electrolytes cause an increase in

viscosity but high electrolyte concentrations cause salting- out of sodium alginate;

salting-out occurs if more than 4% of sodium chloride is present.

Stability and Storage Condition

Sodium alginate is a hygroscopic material, although it is stable if stored

at low relative humidity and a cool temperature. Aqueous solutions of sodium alginate

are most stable at pH4–10. Below pH 3, alginic acid is precipitated. A 1% w/v aqueous

solution of sodium alginate exposed to differing temperatures had a

viscosity60–80% of its original value after storage for 2 years. Solutions should not be

stored in metal containers.

Solutions are ideally sterilized using ethylene oxide, although filtration using a 0.45 mm

filter also has only a slight adverse effect on solution viscosity.Heating sodium alginate

solutions to temperatures above 708 C causes depolymerization with a subsequent loss

of viscosity. Autoclaving of solutions can cause a decrease in viscosity, which may vary

depending upon the nature of any other substances present. Gamma irradiation should not

be used to sterilize sodium alginate solutions since this process severely reduces solution

viscosity. Preparations for external use may be preserved by the addition of 0.1%

chlorocresol, 0.1% chloroxylenol, or parabens. If the medium is acidic, benzoic acid may

also be used. The bulk material should be stored in an airtight container in a cool, dry

place.

61

Safety

Sodium alginate is widely used in cosmetics, food products, and

pharmaceutical formulations, such as tablets and topical products, including wound

dressings. It is generally regarded as a nontoxic and nonirritant material, although

excessive oral consumption maybe harmful. A study in five healthy male volunteers fed a

daily intake of 175 mg/kg body-weight of sodium alginate for 7 days, followed by a daily

intake of 200 mg/kg body-weight of sodium alginate for a further 16 days, showed no

significant adverse effects. The WHO has not specified an acceptable daily intake for

alginic acid and alginate salts as the levels used in food do not represent a hazard to

health. Inhalation of alginate dust may be irritant and has been associated with industrial-

related asthma in workers involved in alginate production.

Handling Precautions

Observe normal precautions appropriate to the circumstances and quantity of

material handled. Sodium alginate may be irritant to the eyes or respiratory systemif

inhaled as dust. Eye protection, gloves, and a dust respirator are recommended. Sodium

alginate should be handled in a well- ventilated environment.

Regulatory Status

GRAS listed. Accepted in Europe for use as a food additive. Included in the

FDA Inactive Ingredients Database (oral suspensions and tablets).Included as an

excipient in non-parenteral medicines (oral capsules, modified release tablets, enteric-

coated tablets and lozenges) licensed in the UK.

5.2.4:CalciumChloride: CaCl2,is a salt of calcium and chlorine. It behaves as a

typical ionic halide, and is solid at room temperature. Common applications

include brine for refrigeration plants, ice and dust control on roads, and desiccation.

Because of its hygroscopic nature, anhydrous calcium chloride must be kept in tightly

sealed, air-tight containers. Calcium chloride can serve as a source of calcium ions in an

aqueous solution, as calcium chloride is soluble in water. This property can be useful for

displacing ions from solution. For example, phosphate is displaced from solution by

calcium:

62

3 CaCl2 (aq) + 2 K3PO4 (aq) → Ca3 (PO4)2 (s) + 6 KCl (aq)

Molten calcium chloride can be electrolysed to give calcium metal and chlorine gas:

CaCl2 (l) → Ca (s) + Cl2 (g)

Calcium chloride has a very high enthalpy change of solution. A considerable

temperature rise accompanies its dissolution in water. The anhydrous salt is deliquescent;

it can accumulate enough water in its crystal lattice to form a solution.

CAS number: 10043-52-4

5.2.5: Glycerin : (C3H8O3), also known as glycerol and glycerine, is an odorless,

colorless, oily, viscous liquid that has a sweet taste. Glycerol (or glycerine, glycerin) is a

simple polyol (sugar alcohol) compound. It is a colorless, odorless, viscous liquid that is

widely used in pharmaceutical formulations. Glycerol has three hydroxyl groups that are

responsible for its solubility in water and its hygroscopic nature. The glycerol backbone

is central to all lipids known as triglycerides. Glycerol is sweet-tasting and generally

considered non-toxic.

Glycerol is completely soluble in water and alcohol. It is slightly soluble in ether, ethyl

acetate, and dioxane and insoluble in hydrocarbons. Glycerol has useful solvent

properties similar to those of water and simple aliphatic alcohols because of its three-

hydroxyl groups. Glycerol is a useful solvent for many solids, both organic and inorganic

which is particularly important for the preparation of pharmaceuticals. The solubility of

gases in glycerol, like other liquids is temperature and pressure dependent.

Synthetic glycerin is used in food products, nutritional supplements, pharmaceutical

products, personal-care products, and oral-care products. In the pharmaceutical industry,

glycerin is used as a sweetener in syrups, lozenges, and as an excipient in eyewash

solutions. It may also be found in eardrop products, jellies and creams for topical use, in

expectorants for congestion, suppositories, and gel capsules.

As an individual prescription product, glycerin has uses as a hyperosmotic, osmotic

diuretic, and ophthalmic agent. It may be used as eye drop in the treatment of glaucoma

to reduce intraocular pressure, as a solution or suppository for short-term treatment of

constipation, to evacuate the bowel prior to colonoscopy, and in some ocular surgeries. It

may be given intravenously to reduce pressure inside the brain, and used externally on

63

the skin as a moisturizer. Glycerin has many other uses in the agricultural, food and

pharmaceutical industry.

The U.S. Food and Drug Administration (FDA) classify glycerin as “generally

recognized as safe” (GRAS). The overall risk of toxicity from glycerin found in

pharmaceutical products is low. If one were to come into contact with large, bulk

quantities of glycerin, eye irritation may occur. Skin irritation is unlikely unless the skin

is damaged where contact occurs. Inhalational toxicity is low due to low volatility, but

prolonged, excessive ingestion can cause elevated blood sugar or fat levels in the blood.

114

64

5.3METHODS:

5.3.1 List of equipments & software used:

Equipments:

Brookfield Viscometer. Model No. CAP-2000, Brookfield Engineering Lab.

Middleboro, MA-02346, USA.

Double Beam UV Spectrophotometer. Model No. V-530, Jasco Corporation Tokyo,

Japan.

DSC – Shimadzu DSC60

Electronic weighing balance - Model No. AB54-S, Mettler Toledo, Switzerland.

Fourier Transform Infrared Spectrophotometer Model No. FT/IR-4100, Jasco

Corporation Tokyo, Japan.

Franz type diffusion cell - Fabricated by Sai Enterprises, Pune

Homogenizer – IKA T25, ULTRA- TURRAX.

HPTLC- CAMAG Linomet 5

Incubator

Magnetic stirrer - Whirlmatic Mega, 9 stations, Spectralab, India.

Microscope (Binocular compound):Micron Optic Model kg5

Microscope (Binocular Motic):B3 Professional series

Muffle furnace

Precision balance - Model No. CB-330, Contech.

Precision melting point apparatus - Model No. VMP-I, Veego.

Thermocycler( Eppendorf, USA)

Ultrasonicator - Entertech

Softwares:

PCP Disso

Design Expert Software

65

5.3.2 Collection and authentification of plants:

Bark of Symplocos racemosa was purchased from Astha Herbal, Pune and

authenticated from Agharkar Research Institute, Pune. Barks were coarsely

pulverized separately after sufficient shade drying. Materials were passed through

120 meshes to remove fine powders and coarse powder was used for extraction.

Leaves of Psidium guajava were collected from Garden of Allana College of

Pharmacy and authenticated from Botanical Survey of India, Pune , Maharashtra.

Leaves were dried in shade and pulverized. Materials were passed through 120

meshes to remove fine powders and coarse powder was used for extraction.

5.3.3. Pharmacognostic evaluation:

I) Macroscopic and Microscopic evaluation:

The macroscopic and microscopic features were studied according to the standard

methods. For macroscopy, the colour, odour, taste, size, shape was studied.

Microscopical studies were done by preparing a thin section of the specimen. The

sections were cleared, stained with Phloroglucinol and Hydrochloric acid and mounted in

glycerine and observed under microscope using 10X lense. For powder characteristics,

the same staining reagent was used.

II) Determination of foreign organic matter: 115

Procedure:

5 gm of air dried coarsely powdered drug was spreaded in a thin layer. The

sample was inspected with the unaided eye. The foreign organic matter was separated

manually as completely as possible. Sample was weighed and percentage of foreign

organic matter was determined from the weight of the drug taken (Indian Pharmacopoeia,

2007).

III) Determination of Loss on Drying:

Procedure: Accurately weighed glass stoppard, shallow weighing bottle was dried and

2g of sample was transferred to the bottle and covered, the weight was taken and sample

66

was distributed evenly and poured to a depth not exceeding 10 mm. Then loaded bottle

was kept in oven and stopper was removed. The sample was dried to constant weight.

After drying it was collected to room temperature in a desiccator. Weighed and calculated

loss on drying in terms of percent w/w (Indian Pharmacopoeia, 2007).

5.3.4 Determination of ash values:

Procedure: Ash value is used to determine quality and purity of crude drug. Ash value

contains inorganic radicals like phosphates carbonates and silicates of sodium, potassium,

magnesium, calcium etc. Sometimes inorganic variables like calcium oxalate, silica,

carbonate content of the crude drug affects ‘total ash value’. Such variables are then

removed by treating with acid and then acid insoluble ash value is determined.

I) Total Ash:

Procedure: Accurately weighed 2g of the air-dried crude drug was taken in a tared silica

dish and incinerated at a temperature not exceeding 4500C until free from carbon, cooled

in a desiccator and weight was taken. The process was repeated till constant weight was

obtained. The percentage of ash was calculated with reference to air-dried drug (Indian

Pharmacopoeia, 2007).

II) Water-Soluble Ash:

Procedure: The ash, obtained as per the method described above boiled for 5 minutes

with 25 ml of water, filtered, and collected the insoluble matter in a Gooch crucible,

washed with hot water and ignited for 15 minutes at a temperature not exceeding 4500C

and weight was taken. Subtracted the weight of the insoluble matter from the weight of

the ash; the difference in weight represents the water-soluble ash. The percentage of

water-soluble ash was calculated with reference to air-dried drug (Indian Pharmacopoeia,

2007).

III) Acid-Insoluble Ash:

Procedure: The ash obtained as per method described above and boiled with 25 ml of 2

M hydrochloric acid for 5 minutes, filtered, and collected the insoluble matter in a Gooch

67

crucible or on an ash less filter paper, washed with hot water, ignited, and cooled in a

desiccator and weighed. The percentage of acid-insoluble ash was calculated with

reference to the air-dried drug (Indian Pharmacopoeia, 2007).

5.3.5 Determination of extractive values:

Different extractive values like alcohol soluble extractive, water soluble extractive

values were performed by standard method (Indian Pharmacopoeia, 2007).

I) Determination of water-soluble extractive value:

Procedure:

5 gm of air dried coarsely powdered drug was macerated with 100 ml of

chloroform water in a closed flask for 24 hours, and it was shaken frequently during first

6 hours and allowed to stand for 18 hours. Then it was filtered, 25 ml of the filtrate was

evaporated in a flat shallow dish, and dried at 1050C and weighed. Percentage of water-

soluble extractive value was calculated with reference to air-dried drugs (Indian

Pharmacopoeia, 2007).

II) Determination of alcohol-soluble extractive value:

Procedure: 5 gm of air-dried coarsely powdered drug was macerated with 100 ml of

ethanol of specified strength in a closed flask for 24 hours, and it was shaken frequently

during first 6 hours and allows standing for 18 hours. Then it was filtered, during

filtration precaution was taken against loss of ethanol, 25 ml of the filtrate was

evaporated in a flat shallow dish, and dried at 1050C and weighed. Percentage of ethanol

soluble extractive value was calculated with reference to air-dried drugs (Indian

Pharmacopoeia, 2007).

III) Determination of chloroform soluble extractive value:

Procedure: 5 grams of air-dried drug was macerated with 100 ml of chloroform in a

closed flask, shaking frequently during the first 6 hours and allowed to stand for 18 hours

separately. Thereafter, it was filtered rapidly taking precaution against loss of

Chloroform. Evaporated 25ml of filtrate to dryness in a tarred flat bottom shallow dish

68

dried at 1050C and weighed. Percentage chloroform soluble extractive was calculated

with reference to the air-dried drug (Indian Pharmacopoeia, 2007).

IV) Determination of Methanol soluble extractive value: 5 grams of air-dried drug

was macerated with 100 ml of methanol in a closed flask, shaking frequently during the

first 6 hours and allowed to stand for 18 hours separately. Thereafter, it was filtered

rapidly taking precaution against loss of methanol. Evaporated 25ml of filtrate to dryness

in a tarred flat bottom shallow dish dried at 1050C and weighed. Percentage chloroform

soluble extractive was calculated with reference to the air-dried drug (Indian

Pharmacopoeia, 2007).

5.3.6. Extraction of plant constituents:

I) Technique: Soxhlet extraction method (Contineous hot Extraction)

Solvents: Solvent or extraction agents used in the preparation of phytopharmaceuticals

must be suitable for dissolving the important therapeutic drug constituents and thus for

separating them from the substance containing the drug which are to be extracted. The

powder of bark and leaves defatted with petroleum ether (60-80 c).The extraction was

carried out by using water & methanol as a solvent and employing soxhlet extraction

method.

5.3.7. Preliminary phytochemical screening:

The extracts were concentrated and subjected to phytochemical screening using st

andard procedures. The compounds were analysed for alkaloids, glycosides, flavonoids,

tannins, proteins, carbohydtares, amino acids and steroids 116,117

I) Test for Carbohydrates:

a) Molisch test: Two ml of extract solution was treated with few drops of 15 percent

ethanolic α- napthol solution in a test tube and 2 ml of concentrated Sulphuric acid was

added carefully along the side of tubes. The formation of reddish violet ring at the

junction of two layers indicates the presence of carbohydrates.

69

b) Test for reducing sugars:

i) Benedict’s test: To 2 ml of Benedict’s reagent, 1 ml of extract solution was added,

warmed, and allowed to stand. Formation of red precipitate indicates presence of sugars.

ii) Fehling’s test: 5 ml of extract solution was mixed with 5 ml Fehling’s solution (equal

mixture of Fehling’s solution A and B) and boiled. Development of brick red precipitate

indicates the presence of reducing sugars.

c) Test for monosaccharides:

Barfoed’s test: Test solution was treated with equal volume of Barfoed’s reagent. Heated

for 1–2 min. in boiling water bath and cooled. Red precipitate indicates presence of

monosaccharides.

II) Test for Proteins:

a) Biuret test: The extract was treated with 1 ml of 10 percent sodium hydroxide

solution and heated. A drop of 0.7 percent copper sulphate solution was added to the

above mixture. The formation of purple violet colour indicates the presence of proteins.

b) Million’s test: The extract was treated with 2 ml of Million’s reagent. Formation of

white precipitate indicates the presence of proteins and amino acids.

III) Test for amino acids:

Ninhydrin test: The extract was treated with Ninhydrin reagent at pH range of 4-8 and

boiled. Formation of purple colour indicates the presence of amino acids.

IV) Test for Steroids:

a) Salkowski test: One ml of concentrated Sulphuric acid was added to 10 mg of extract

dissolved in 1 ml of chloroform. A reddish brown colour exhibited by chloroform layer

and green fluorescence by the acid layer suggests the presence of steroids.

b) Liebermann – Burchard reaction: 2 ml of extracts was treated with chloroform.1 – 2

ml acetic anhydride and two drops of conc.H2SO4 was added from the side of the test

70

tube. First red, then blue and finally green colour appeared indicates the presence of

steroids.

V) Test for Cardiac Glycosides:

a) Test for deoxysugars (Keller-Killiani test): To 2 ml of extract, glacial acetic acid,

one drop 5 % Ferric chloride and conc. Sulphuric acid was added. Presence of cardiac

glycosides is indicated by formation of reddish brown colour at junction of the two liquid

layers and upper layer appeared bluish green.

b) Legal’s test (Test for cardenoloids): To the extract 1 ml pyridine and 1 ml sodium

nitroprusside was added .Pink to red colour appears.

VI)Test for Anthraquinone Glycosides:

a) Borntrager’s test: To 3 ml extract, dil. H2SO4.was added boiled and filtered. To cold

filtrate, equal volume benzene or chloroform was added. Organic layer was

separated.Strong ammonia solution was added..Ammonical layer turns pink or red.

b) Modified Borntrager’s test: To 5 ml extract, 5 ml 5 % FeCl3 and 5 ml dil. HCL was

added. Heated for 5min in boiling water bath. Cooled and benzene was added. Organic

layer was separated, Equal volume dilute ammonia was added. Ammonal layer shows

pinkish red colour.

VII) Test for Alkaloids: To the extract, add dilute HCL. Shake well and filter. With

filtrate perform following tests.

a) Dragendorff’s test: To 2-3 ml filtrate, add few drops Dragendorff’s reagent. Orange

brown ppt is formed.

b) Mayer’s test: 2-3 ml.filtrate with few drops Mayer’s reagent gives ppt.

c) Hager’s test: 2-3 ml. filtrates with few drops Hager’s reagent gives yellow ppt.

d) Wagner’s test: 2-3 ml. filtrates with few drops Wagner’s reagent gives reddish brown

ppt.

71

VIII) Test for Tannins and Phenolic compounds:

a) Ferric Chloride test: 5 ml of extract solution was allowed to react with 1 ml of 5 %

ferric chloride solution. Greenish black colouration indicates the presence of tannins.

b) Lead acetate test: 5 ml of extract solution was allowed to react with 1 ml of 10

percent aqueous lead acetate solution. Development of yellow coloured precipitate

indicates the presence of tannins.

IX) Test for Flavonoids :

117

a) Lead acetate test: Few drops of 10 percent lead acetate are added to the extract.

Development of yellow coloured precipitate confirms the presence of flavonoids.

b) Sodium Hydroxide test: To the extract increasing amount of Sodium Hydroxide was

added gives yellow colour, which disappeared after addition of acid.

c) Shinoda test (Magnesium Hydrochloride reduction test): To the test solution few

fragments of Magnesium turning and cone. Hydrochloric acid was drop wise.Pink scarlet,

crimson red or occasionally green to blue colour appears after few minutes.

5.3.8. Thin layer chromatography of plant extracts: 118,119

Steps involved in performing TLC of extracts:

Precoated TLC plate: (silica gel- G60F254)

Activation of TLC plate:

Heating in oven for 30 min. at 105OC activated TLC plate.

Sample application: Dipping the capillary into the solution to be examined and

applied the sample by capillary touched to the thin layer plate at a point about 2

cm from the bottom. Air-dried the spot.

Chamber saturation: The glass chamber for TLC should be saturated with

mobile phase. Mobile phase was poured into the chamber and capped with lid.

Allowed saturating about 30 min.

72

Chromatogram development: After the saturation of chamber and spotting of

samples on plate, it was kept in chamber. The solvent level in the bottom of the

chamber must not be above the spot that was applied to the plate, as the spotted

material will dissolve in the pool of solvent instead of undergoing

chromatography. Allowed the solvent to run around 10-15 cm on the silica plate.

Visualization:

Plates were removed and were examined visually, under UV and suitable

visualizing agent (Vanillin-sulphuric acid, Methanolic ferric chloride solution)

after that Rf was calculated by following formula.

Rf =

Distance traveled by solute from origin line

Distance traveled by solvent from origin line

5.3.9 HPTLC of plant extrats:

From the pharmacopoeial perspective, a better quality control of raw material can be

achieved by specifying quantitative test procedure for the determination of the range or a

minimum content of the active ingredient or marker substances. A chromatographic

finger profile represents qualitative/ quantitative determination of various components

present in a complex plant extract, irrespective whether or not their exact identity is

known. For quantitative analysis of active ingredients or marker substances with

simultaneous separation and detection High Pressure liquid chromatography is the best

technique 120, 121

.

Stationary phase: Silica gel aluminum plate

Mobile phase: CHCL3: Methanol: Water (65:35:10), n-hexane-ethyl acetate, n-

butanol-water.

Sample applicator: CAMAG Linomet 5

Saturation time: 15 min

Length of chromatogram run: 8cm

Development time: approximately 15 min

73

Detection: at 200nm, 220nm, 270nm, 290nm.

Scanning speed: 20mm/sec

Scanner: CAMAG thin layer chromatography scanner

Software: WINCATS software version 1.4.2

5.3.10 Characterization of plant extracts:

PGME of leaves of plant Psidium Guajava l. & SRME of Bark powder of Symplocos

racemosa):

Organoleptic properties: The plant extracts were analyzed for color and odour

& consistency.

Physical Characterization: Markers of both plant drugs were analyzed for

colour, odour, taste, melting point & solubility.

UV Spectroscopy: The UV Spectrum provides a useful means of detecting

conjugated unsaturated chromophores within a molucle. UV absorption is

characteristically broad.The UV spectrum obtained shows absorption bands which

gives valuable information regarding the nature of compound.

1. Ultra violet spectroscopy of Metahnolic extracts: Ultra violet spectroscopy of

Metahnolic extracts of Psidium guajava & Symplocos racemosa were performed & ƛmax

was determined.

2. Ultra violet spectroscopy of Marker compound of Psidium guajava (Quercetin):

0.1g of Quercetin is dissolved in the 100ml Phosphate buffer (1000µg/ml).0.1ml of that

solution is removed and diluted 100ml (10µg/ml).And then max was determined. 122

3. Ultra violet spectroscopy of Marker compound of Sympolcos racemosa(Gallic

acid): 0.1g of gallic acid is dissolved in the 100ml Phosphate buffer (1000µg/ml).0.1ml

of that solution is removed and diluted 100ml (10µg/ml).And then max was

determined. 123

4. Fourier transform infrared spectroscopy (FTIR): IR Spectrum is highly

characterstic to establish the identification of compounds.FTIR spectroscopy was

conducted using Jasko FT-IR 4100 Spectrophotometer and the spectrum was recorded in

the wavelength region of 4000-400 cm-1.FTIR spectrum of Plant extracts & their marker

compounds were determined using kbr dispersion method.

74

5.3.11 Determination of total phenolic content: 124,125

By Folin-ciocalteau calorimetric reaction:

Principle:

The Folin–Ciocalteu reagent (FCR)or Folin's phenol reagent or Folin–Denis reagent, is a

mixture of phosphomolybdate and phosphotungstate used for the colorimetric assay of

phenolic and polyphenolic antioxidants It works by measuring the amount of the

substance being tested needed to inhibit the oxidation of the reagent.However, this

reagent does not only measure total phenols and will react with any reducing substance.

The reagent therefore measures the total reducing capacity of a sample, not just the level

of phenolic compounds. Folin&Ciocalteu’s phenol reagent does not contain phenol.

Rather, the reagent will react with phenols and nonphenolicreducing substances to form

chromogens/phenolates that can be detected spectrophotometrically. The color

development is due to the transfer of electrons at basic pH to reduce the

phosphomolybdic/phosphotungstic acid complexes to form chromogens in which the

metals have lower valency. Addition of Folin & Ciocalteu’s phenol reagent generates

chromogens that give increasing absorbance between 550 nm and 750 nm the phenolates

are only present in alkaline solution but the reagent and products are alkali unstable.

Hence a moderate alkalinity and a high reagent concentration are used in the procedure

below.

Chemicals:

Folin-ciocalteau- [reagent: distill water] in(1:10)proportion.

Na-carbonate 7.5% in H2O (stable for week)

Distilled water

Standard:

Gallic acid-Prepare stock solution of 100ppm (stable for few days at 40c)

Make further dilutions of 10, 20, 40, 50, 60, 80ppm of Gallic acid.

Extract sample preparation:

0.01 gm diluted to 10ml with distil water.

Take 2ml and dilute to 25ml with distil water.

75

Equipment:

Spectrophotometer - at 740 nm.

Normal laboratory glassware – including volumetric flasks and test tubes.

Dispensing pipettes – to deliver 1, 4 and 5 ml volumes.

Procedure:

1 ml. test solution (extract / Gallic acid)

5 ml. folin-ciocalteall reagent.

Mix well & wait for 3-8 min.

Add 4 ml 7.5 % Na – carbonate.

Cover tube for 2 hour at room temperature.

Absorbance at 740 nm against reagent blank.

Calibration curve of Gallic acid absorbance.

Extrapolate test reading on graph.(gives Gallic acid equivalent).

76

5.3.12: Determination of total flavonoid content: 124,125

By Aluminium Chloride colorimetric assay

Principle:

The basic principle of Aluminium chloride colorimetric method is that Aluminium

chloride forms acid stable complexes with the C-4 keto group and either the C-3 or C-5

hydroxyl group of flavones and flavonols. In addition it also forms acid labile complexes

with the ortho-dihydroxyl groups in the A- or B-ring of flavonoids, in presence of

alkaline medium(NaOH,NaNO2) to give color intensity as per concentration of flavonoid

present in that, which can be detected spectrophotometricaly.

Chemicals:

AlCl3 10%

NaNO2 5%

NaOH 1M

Distilled water

Standard: Catechin

Prepare stock solution of 100 ppm.

Make further dilutions of 10, 20, 40, 50, 60, 80 ppm of catechin.

Extract sample preparation:

0.01gm dilutes to 10ml with distil water.

Take 2ml and dilute to 25ml with distilled water.

Equipment:

Spectrophotometer - at 510 nm.

Normal laboratory glassware – including volumetric flasks and test tubes.

Dispensing pipettes – to deliver 0.3ml, 1.0ml and 2.0 ml volumes.

77

1 ml. test solation (extract /catechin)

4 ml. D. H2O

0.3 ml. 5% NaNO2

keep it for 5 min

add 0 .3 ml 10 % AlCl3

2 ml 1 M NaOH

make up volume upto 10 ml with distill H2O

absorbance at 510 nm

Calibration curve of catechin absorbance

Extrapolate test reading on graph. (gives catechin

equivalent)

78

5.3.13 Atimicrobial assay: 126,127

By Cup plate diffusion method

Requirements:

Nutrient broth

Cultured microorganism (Pseudomonas auerogenosa, Staphylococcus aureus,

E.coli, Candida albicans)

Distilled water

Extract solutions

Apparatus:

Petri plates

Pipette

Conical flask

Spreader

Cork borer

Cotton plug

Procedure:

1. Preparation of nutrient agar & sabarode broth

2. Sterilization.

3. Preparation of extract solution.

4. Process

5. Incubation of Petri plates at 37oc for 24 hr.

6. Measurements of ZOI by zone reader.

I) Preparation of nutrient & sabarode broth broth:

Beef extract: 5gm

Pepton: 5gm

NaCl: 2.5gm

Agar powder: 12.5gm

Distill water: Up to 500ml

79

Procedure:

Beef extract, peptone and NaCl was dissolved into D.W.in 500ml conical flask.

It was gently heated form homogeneous solution.

Agar powder was added at the end.

Conical flask was covered with cotton plug.

1. Sterilization:

Nutrient agar was sterilized by autoclave at 15lb pressure, 121oc, for 30 min.

Petri plates, pipette was wrapped in to paper and sterilized in to hot air oven at

1600c for 30 min.

Spreader and cork borer was sterilized by ethanol.

2. Preparation of Extract solution:

0.5%, 1.0%, 1.5%, 2.0%, 2.5% and 3.0% extract solutions were made in D.W.

Further dilutions were made from 50-500µg/ml of extract.

3. Process:

Nutrient agar was aseptically poured into Petri plates.

2-3 drops of standard culture were added on nutrient agar.

It was spreads on nutrient agar in Petri plate with spreader.

Wells were made in Petri plate with cork borer (7mm).

Extract solution was added into well.

4. Incubation:

Petri plates are labelled and incubated for 24hr.at 370c in incubator.

ZOI was measured by zone reader.

II) Collection and preservation of culture

Candida albicans(MTCC227) freeze dried culture wad obtained from IMITECH

Microbial type Culture Collection & Gene Bank, Chandigarh), Whereas ,Staphylococcus

aureus (MTCC 737), Pseudomonas aeruginosa (MTCC 2642), Escherichia coli (MTCC

1687) were obtained from Abeda Inamdar Senior College , Pune. Nutrient agar (pH 7.2-

7.4) was used for routine susceptibility testing of nonfastidious bacteria.0.5-3% solutions

of all extracts were made in Distilled Water & zone of inhibition were measured. All

extracts showed good antibacterial activity. Further dilutions were made from 50 µg-500

µg/ml. Ampicillin (10µg/disc) was used as a standard. 20 % v/v WFI in DMSO was used

80

as a control. Antibacterial assay was carried out by agar well diffusion method. After 16

to 18 hours of incubation, each plate is examined.

5.3.14 Determination of Antibacterial activity against H.Pylori (in-vitro) 92,128

Evaluation of the inhibitory effect of Psidium guajava & Symplocos racemosa extracts

was determined on Helicobacter pylori growth in vitro. Activity of Methanolic &

aqueous extract of P. guajava against clinical isolate of H. pylori was evaluated by using

the agar-well diffusion method14

. Amoxycillin and clarithomycin was used as a control.

Mean diameters of H. pylori growth inhibition was determined.

5.3.15 Acute oral toxicity test:

The acute oral toxicity study for SRME & PGME was carried out according to OECD

guidelines 423. Swiss albino mice were fasted overnight, water also being withheld. The

SRME & PGME was administered at a dose of 2000 mg/kg. Animals were observed

individually during the first 30 minutes and periodically during 24 hours, with special

attention given during the first 4 hours and daily thereafter, for a total 14 days.(Animal

Ethical Committee No:Ref/ACP/IACE/11-12/12-05)

5.3.16 Determination of Antibacterial activity against H. Pylori (in-vivo) 27, 29

Animal groups (n=12): The treatment regimen as follows:

Group 1: Vehicle control (VC): Ulcerated non infected Vehicle treated.

Group 2: Standard drug treated group (CAO): (clarithromycin 25 mg/kg + amoxicillin

50 mg/kg + omeprazole 20 mg/kg) p.o.

Group 3: SRME treated group: (SRME 50 mg/kg/day) p.o.

Group 4: SRME treated group: (SRME 100 mg/kg/day) p.o.

Group 5: SRME treated group: (SRME 200 mg/kg/day) p.o.

Group 6: SRME treated group: (SRME 200 mg/kg/day) + CAO (clarithromycin 25

mg/kg + amoxicillin 50 mg/kg + omeprazole 20 mg/kg) p.o.

Group 7: PGME treated group: (PGME 100 mg/kg/day) p.o.

Group 8: PGME treated group: (PGME 200 mg/kg/day) p.o.

Group 9: PGME treated group: (PGME 400 mg/kg/day) p.o.

Group 10: PGME treated group: (PGME 400mg/kg/day) + CAO (clarithromycin 25

mg/kg + amoxicillin 50 mg/kg + omeprazole 20 mg/kg) p.o.

Group 11: Healthy Control (HC): Non ulcerated non infected Vehicle treated.

81

I) Induction of unhealed ulcers:

Chronic ulcers were induced in the rats. The rats were fasted for 24 hours before the

induction of ulcers. The rats were anesthetized with ketamine (60 mg/kg i.p). An

epigastric incision was made through midline and stomach was exposed. 0.3 ml of a 20%

solution of acetic acid was injected into the sub serosal layer of the glandular portion of

the stomach with the aid of a tuberculine syringe. Subsequently stomach was re-

internalized; the abdomen was closed and sutured. The animals were maintained in

individual cages with meshed bottom to prevent coprophagy. The size of the mesh (4 x 4

mm) allowed feces to fall to the floor of the cage below the mesh. After the induction of

ulcers, five days were required for the ulcers to develop fully. The fifth day after ulcer

induction was considered day 0. These ulcerated animals were administered

indomethacin1mg/kg/day p.o. for 4 weeks to produce unhealed ulcers. High mortality of

rat after subcutaneous indomethacin necessiated change of route from subcutaneous to

per oral. Thus a modification was made in the original protocol. Unhealed ulcers were

produced after oral administration of indomethacin. To infect the pyloric antrum tissue of

the animals with H. pylori, a broth of H. pylori (1 ml p.o.) was administered three times a

week for four weeks. During this period indomethacin administration was uninterrupted.

H. pylori was resurrected from the cryopreserved stage onto brucella blood agar culture

plates using fresh sheep blood and blood agar media. H.pylori was grown in

microaerophillic conditions in a desiccator which was maintained at 370C in an incubator.

Thereafter, the bacterial colonies were scraped from the culture plates and transferred

aseptically into H. pylori broth consisting of brucella broth and fetal calf serum in

laminar air flow. The bacterial broth administered to rats was adjusted to Mc-Farland

turbidity standard 1 using brucella broth and bacterial colonies in order to ascertain the

bacterial load in the broth. Broth was prepared each week and during these four weeks,

H. pylori inoculum (9x108

cfu/ml) with bacteria in the mid log phase were thrice a week

to infect the pyloric antrum mucosa by oral gavage. In the normal group of animals, 1 mL

of sterile brucella broth and 1mL of 1% tween 80 solution was administered 3 times.

After four weeks of administration of indomethacin and H. pylori, infection status was

determined by randomly sacrificing two animals from each group and determining their

ulcer area and presence of H. pylori in the pyloric antrum tissue by polymerase chain

reaction.

82

The treatment of the standard drug regimen (CAO) and test drug (piperine) was initiated

and continued for 4 weeks. CAO regimen consisted of clarithromycin 25 mg/kg +

amoxicillin 50 mg/kg + omeprazole 20 mg/kg in 1% tween 80. Piperine was administered

at 10, 20 and 40 mg/kg in 1% tween 80. Piperine and CAO were administered by per oral

route every day for four weeks. The vehicle treated animals were treated with 1ml of 1%

solution of tween 80. The dose of piperine (10, 20 and 40 mg/kg p.o) was selected on the

basis of the pilot study performed on the unhealed ulcer induced rats.

At the end of the treatment regimen of four weeks, after 24 hour fasting, the rats were

euthanized under deep ether anesthesia. A midline incision was performed. The stomachs

were rapidly removed and opened along the greater curvature. It was washed with normal

saline and each stomach was photographed using a crystal clear display (CCD) scanner at

a magnification of 2400 dots per inch (DPI). The lesion was localized and measured

along the external (comprising the regenerative tissue) and internal (only exposed sub

mucosa) borders for area determination. The fundic portion of each stomach was excised.

The contents were washed off and fundic area was excised off. The pyloric antrum area

of stomach was selected and used for all investigations as it has been previously

investigated that H. pylori is favorably harbored in this region. The image of each excised

stomach was captured at a magnification of 2400 D.P.I and then it was processed for

nucleic acid extraction, RUT, biochemical studies and western blot to determine the H.

pylori infection status, histopathological and gene expression studies.

The pyloric antrum region of each stomach was divided into three parts in each group of

animals. Out of the twelve animals in each group, the excised stomachs of six animals

were utilized for mitochondrial assays, RUT (Rapid Urease Test) and histopathological

studies. The rest of the six animals were used for nucleic acid extraction to be further

used for infection status determination, reverse transcriptase PCR and western blot

analysis.

II) Preparation of genomic DNA for PCR

DNA isolation from pyloric antrum tissue was performed according to phenol chloroform

C-TAB method.Briefly, 200 mg of the pyloric antrum tissue sample was suspended in

250 μL of digestion buffer II {0.1M NaCl, 0.01M Tris-HCl (pH 8.0), 0.25M EDTA (pH

8.0), 1% SDS} containing 100μg/ml of proteinase k. To this, 250 μl of digestion buffer I

83

{0.1M NaCl, 0.01M Tris-HCl (pH 8.0), 0.25M EDTA (pH 8.0)} was added and

incubated at 56oC overnight. DNA was extracted with an equal volume of phenol

chloroform and precipitated with 0.6 volume iso propanol. The DNA pellets were washed

thrice with 80%, 75% and 70% ethanol, respectively, and finally re suspended in 100μl of

sterile water for injection. All the steps were performed in aseptic conditions to minimize

contamination. The DNA was extracted and preserved at -20oC until amplification was

performed.

III) Preparation of genomic DNA for polymerase chain reaction (PCR)

DNA isolation from gastric autopsy samples was performed according to phenol

chloroform C-TAB method. Briefly, the salivary samples were suspended in 250 μL of

digestion buffer II. {0.1M NaCl, 0.01M Tris-HCl (pH 8.0), 0.25M Ethylene diamine tetra

acetic acid (EDTA, pH 8.0), 1% sodium dodecyl sulphate (SDS)} containing 100 μg/ml

of proteinase k .To this, 250 μl of digestion buffer I {0.1M NaCl, 0.01M Tris-HCl (pH

8.0), 0.25M EDTA (pH 8.0)} was added and incubated at 56°C overnight. DNA was

extracted with an equal volume of phenol chloroform and precipitated with 0.6 volume

iso propanol. The DNA pellets were washed thrice with 80%, 75% and 70% ethanol,

respectively, and finally re suspended in 50μl-100μl of sterile water for injection. All the

steps were performed in aseptic conditions to minimize contamination. DNA was

extracted and preserved at -20oC until amplification was performed.

IV) PCR Sensitivity assay

The detection limits of the PCR assay was determined by preparation of 10-fold serial

dilution, from 50 nanogram to 1 femtogram of the isolated genomic DNA from H. pylori

strain ATCC 26695 in sterile water for injection. An aliquot of each dilution was

amplified by PCR, and the amplicons visualized on 1.5% agarose gel stained with

ethidium bromide. Sensitivity of this PCR assay was ascertained based on the maximum

dilution of genomic DNA in which the primers were able to amplify their specific gene

sequences.

V) PCR Specificity assay

DNA isolated from an entirely sequenced H. pylori reference strain DNA (ATCC 26695)

was used as a positive control. The specificities of the PCR method was evaluated for

84

three different bacteria (Staphylococcus aureus) strains were obtained from NCIM

(National Centre for Industrial Microbes) Pune, Staphylococcus aureus NCIM 2079,

Escherichia coli NCIM 2345, Bacillus subtilis NCIM 2063.

VI) Amplification of virulent and non-virulent genes of H. pylori

H. pylori specific genes were amplified in a programmable thermal cycler. The template

DNA (1 µL) was added to 19 µL of the reaction mixture containing PCR buffer (50 µmol

KCl, 10 µmol Tris-HCl (pH 8.3), 1.5% [v/v] Triton X-100), 1.5 µmol MgCl2, 200 µmol

concentrations of each dNTP, 10 pmol of each primer (forward and reverse), and 1 U of

Taq polymerase (Takara, USA).

PCR amplification was carried out, which included initial denaturation at 950C for 5

minute, 40 cycles with 1 cycle consisting of 30 seconds at 940C, 30 seconds at 52

0C, 1

minute at 720C. The final cycle included a 10 min extension step to ensure full extension

of the PCR products. Amplification was carried out in a thermocycler.The PCR products

were analyzed on a 1.5% agarose gel stained with ethidium bromide for visualization.

The DNA of H. pylori (type ATCC 26695) served as a positive control. Water instead of

DNA template was used as a negative control. To avoid false positive results because of

contamination, extreme care, such as using fresh disposable devices, preparing template

DNA and pre- and post-PCR materials in separate places, changing gloves frequently and

other measures outlined by Kwok and Higushi, 1989 was taken. At each amplification, H.

pylori DNA obtained from a fully sequenced standard strain (ATCC 26695) was used as

a positive control, while sterile water for injection instead of DNA served as a negative

control. The products were analyzed by agarose gel electrophoresis and the image of the

gel was captured using gel documentation. The DNA isolated from all the samples were

amplified to get a particular base pair fragment corresponding to the specific H. pylori

gene. The annealing temperature was optimized according to melting point (Tm) of a

particular primer pair and was unique for each gene.

VII) Rapid Urease test

Weighed quantity of the excised pyloric antrum tissue (50 mg) was immersed in 5 mL

RUT solution. The colour change was recorded. The colour changed from yellow to pink

in one hour if H. pylori was present in the pyloric antrum tissue.

85

VIII) Calculation of ulcer area

The images were processed using Image J and Adobe photoshop softwares to determine

the ulcer area of the stomach. The software was calibrated at 95 pixels = 1 mm

IX) Culture broth techniques

H. pylori and E. coli culture media and culture were prepared in millipore water.

X) DNA extraction

Pyloric antrum tissue 200 mg was homogenized in 200ml of digestion buffer II. Then the

H. pylori DNA was extracted from the homogenate according to phenol chloroform

CTAB method .

XI) Biochemical estimations 34, 38

A) Determination of Protein

Protein concentration was estimated using BSA (bovine serum albumin) as a standard.

Lowry C reagent was prepared by following method. (a) Copper sulphate in 1% sodium

potassium tartarate (1 % w/v): 0.5 g of copper sulphate was dissolved in 1% sodium

potassium tartarate (prepared by dissolving 1gm of sodium potassium tartarate in 100ml

of distilled water). (b) Sodium carbonate in 0.1M sodium hydroxide (2% w/v): 2 g of

sodium carbonate was dissolved in 100ml of 0.1M sodium hydroxide. 2ml of solution (a)

was mixed with 100ml of solution (b) just before use. Sodium hydroxide (0.1 M): 4 g of

sodium hydroxide was dissolved in 400 ml distilled water and the final volume was made

up to 1000 ml with distilled water. Standard protein (bovine serum albumin): 20 mg of

bovine serum albumin was dissolved in 80ml of distilled water and few drops of sodium

hydroxide were added to aid complete dissolution of bovine serum albumin and to avoid

frothing. Final volume was made to 100ml with distilled water and stored overnight in a

refrigerator. Folin’s phenol reagent: Folin’s phenol reagent was diluted with distilled

water in the ratio of 1:2. (i.e. 1ml of Folin’s phenol reagent was mixed with 2ml of

distilled water). Briefly, dilute tissue fraction aliquots (0.1 ml) were taken in test tube. To

this, 0.8 ml of 0.1 M sodium hydroxide and 5.0 ml Lowry C reagent was added and the

86

solution was allowed to stand for 15 min. Then 0.5 ml of Folin’s phenol reagent was

added and the contents were mixed by vortex mixer. Colour developed was measure at

660 nm against reagent blank containing distilled water instead of sample. Different

concentrations (40-200 µg) of (bovine serum albumin) BSA were taken and process as

above for standard graph. The values were expressed as mg of protein/ gm of wet tissue

(mg/gm).

B) Mitochondrial Estimations

Mitochondrial isolation

Mitochondria from excised stomach tissue were isolated from male by the method

of Rosenthal et al. 1987. This method uses 0.02% digitonin to free mitochondria from the

synaptosomal fraction. The stomach was rapidly removed, washed with normal saline,

finely minced, and 250 mg of stomach tissue was homogenized in a pre sterelized plastic

homogenizer with a plastic mortar and pestle at 4°C in 10 ml of isolation medium (225

mM mannitol, 75 mM sucrose, 5 mM HEPES, 1 mM EGTA, 1 mg/ml bovine serum

albumin, pH 7.4) containing 5 mg of the bacterial protease nagarse. Stomach

homogenates were brought to 30 ml, using isolation medium and divided equally into

three tubes, each consisting of 10 mL and then centrifuged at 2,000 g for 3 min at 40C.

Pellets were collected and the supernatant discarded. Pellets were resuspended to 10 ml

and recentrifuged as above, and the supernatants were pooled and centrifuged in four

tubes at 12,000 g for 8 min. The pellets, including the fluffy synaptosomal layer, were

resuspended in two tubes to 10 ml each in isolation medium containing 0.02% digitonin

and centrifuged at 12,000 g for 10 min. The brown mitochondrial pellets without the

synaptosomal layer were then resuspended again in 10 ml of medium and recentrifuged at

12,000 g for 10 min. The mitochondrial pellets were resuspended in 50 ml of isolation

medium into an eppendorf tube and used for further evaluation.

Complex-I (NADH dehydrogenase activity)

Complex-I was measured spectrophotometrically by the method of King and

Howard (1967). The method involves catalytic oxidation of NADH to NAD+ with

87

subsequent reduction of cytochrome C. 0.2 M glycyl glycine was prepared by dissolving

335 mg in 10 mL of phosphate buffer saline having the pH equal to 8.5.6 mM of NADH

was prepared by dissolving 42.6 mg in 10 mL of glycyl glycine buffer. 1.05 mM

cytochrome C was prepared by dissolving 13.65 mg in 1mL double distilled water.

0.02M NaHCO3 was prepared by dissolving 16.8 mg in 10 mL of double distilled water.

The reaction mixture contained 350 µL of 0.2 M glycyl glycine buffer (pH 8.5), 100 µL 6

mM NADH in 2 mM glycyl glycine buffer, 100 µL of 10.5 mM cytochrome C and

2.4mL of double distilled water and 20 µL of NAHCO3. The reaction was initiated by

addition of 10µL of solubilized mitochondrial sample and followed absorbance change at

550 nm for 180 seconds.

Calculation:

Mitochondrial Complex I (n mole of NADH oxidized /min/mg

Protein = Change in optical density per minute x 0.262 x 3 x 103

Amount of protein (mg) in 10µL

Complex-II (succinate dehydrogenase (SDH) activity)

SDH was measured spectrophotometrically according to King (1967). The

method involves oxidation of succinate by an artificial electron acceptor, potassium

ferricyanide. 3.12 gram of NaH2PO4 was weighed and dissolved in 100 mL double

distilled water. 2.83 gram of Na2HPO4 was weighed and dissolved in 100 mL double

distilled water. They both were mixed and the pH was adjusted to 7.8. Succinic acid

(0.2M) was prepared by dissolving 700 mg in 10 mL double distilled water. Potassium

ferricyanide (0.03M) was prepared by dissolving 19.6 mL in 2mL of double distilled

water. Bovine serum albumin (1%) was prepared by 100mg /100 mL. The reaction

mixture contained1.5 mL of 0.2 M phosphate buffer (pH 7.8), 300 µL of 1% BSA, 200

µL of 0.6 M succinic acid, and 25 µL 0.03 M potassium ferricyanide and 1.75 mL of

double distilled water. The reaction was initiated by the addition of mitochondrial

sample and absorbance change was followed at 420 nm for 180 seconds.

88

Calculation

Mitochondrial Complex II (milli mole of succinate dehydrogenase /mg protein) = Change

in optical density per minute x 3.8 x 0.435 x 106

Amount of protein (mg) in 25µL x 1000

Complex-III (MTT ability)

This is indirect method to measure the activity of the complex-III. The principle

of MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide], a pale yellow

substrate produces a purple product when incubated with living cells and the number of

viable cells/well is directly proportional to product, which follows by solubilization with

DMSO, can be measured (Mosmann,1983).

The MTT assay was based on the reduction of (3- (4, 5- dimethylthiazol-2-yl)-

2,5-diphenyl-H-tetrazolium bromide (MTT) by hydrogenase activity in functionally

intact mitochondria. The MTT reduction rate was used to assess the activity of the

mitochondrial respiratory chain in isolated mitochondria by the method of Liu et al.

(1997). Solution of MTT was prepared by dissolving 10mg in 10 mL of phosphate buffer

saline. Briefly, 100 µL mitochondrial samples were incubated with 10 µl MTT for 3

hours at 370C. The incubation was carried out in ELISA plate in a humidified atmosphere

of 5% CO2+95% air at 37°C for 3 h. The blue formazan crystals were solubilized with

200 µL dimethylsulfoxide and measured by an ELISA reader at 580 nm filter.

Calculation = The value of the mitochondrial complex III (No. of viable cells /well) was

calculated for each sample using the standard curve by extrapolation of the optical

density values.

Complex IV (cytochrome oxidase assay): Cytochrome oxidase activity was

assayed in stomach mitochondria according to the method of Sottocasa et al.

(1967). Sodium phosphate buffer (0.05M) was prepared by dissolving 1.17 gram

NaH2PO4 in 100 mL of double distilled water. Thereafter, 1.06 gram of Na2HPO4

was dissolved in 100 mL of double distilled water. Then both the solutions were

89

mixed and the pH adjusted to 7.4.Cytochrome C(0.03mM) was prepared by

dissolving 39 mg in 10 mL phosphate buffer. 100mM HCl was prepared by

dissolving 0.43mL concentrated HCl in 50 mL double distilled water. 100 mL

Cytochrome C was reduced by addition of 10 mg of sodium borate crystals and

neutralized with 100mM HCl till the pH was 7. Then, 100 µL reduced

Cytochrome C was added to 700 µL phosphate buffer. To this solution 10 µL of

sample was added and change in optical density was measured at 550 nanometer

for 180 seconds.

Mitochondrial Complex IV (mMole of cytochrome-C oxidized /min/mg

Protein) = Change in optical density per minute x 3 x 108

60 x 29.5 x Amount of protein (mg) in 10µL

Determination of DNA content

The method described by Schneider, 1957 was used to determine the levels of

DNA in the stomach tissue. 100mg of pyloric antrum tissues was homogenized in 5.0 ml

of ice-cold distilled water using Polter–Elvehjem homogenizer with a Teflon pestle. Then

5.0 ml of 5% TCA was added to the homogenate and kept in ice for 30 min to allow

precipitation of protein and nucleic acids. The contents were centrifuged and precipitate

obtained was washed thrice with 1.0 ml of ice-cold 10% TCA. Then, it was treated with

3.0 ml of absolute ethanol to remove lipids. The supernatant was decanted and then

subjected to centrifugation. The final precipitate dissolved in 5.0 ml of 5% TCA was kept

in water bath maintained at 90o

C for15minute with occasional shaking, which facilitated

the quantitative separation of nucleic acid and 7mm proteins. The supernatant after

centrifugation was used for the estimation of nucleic acids and the results were expressed

in mg/g tissue. The convention that 1 O.D. at 260 equals 40 μg /ml DNA was applied and

quantity of DNA was determined.

90

Determination of Myeloperoxidase

The myeloperoxidase assay was performed according to Krawisz et al., (1984).

Briefly the mucosa was scrapped to remove the mucus layer using a glass slide and the

mucosal scrapings were homogenized in a solution containing 0.5% hexadecyl trimethyl

ammonium bromide dissolved in 50 mM potassium phosphate buffer (pH 6), before

sonication in an ice bath for 10 seconds. The homogenates were freeze-thawed three

times, repeating the sonication after which they were centrifuged for 15 minutes at

20,000 × g. The level of MPO activity was measured using spectrophotometer (Jasco

Japan). 0.1 ml of the supernatant was mixed with 2.9 ml of 50 mM phosphate buffer, pH

6.0, containing 0.167 mg/ml O-dianisidine dihydrochloride and 0.0005% hydrogen

peroxide.

Calculation:

MPO activity was measured using the following formula

Myeloperoxidase activity is expressed in mU/mg = 1000 × X/weight of tissue (mg) X =

10×change in absorbance per minute/volume of supernatant

Statistical analysis:One way ANOVA followed by Tukey's multiple range test.

5.3.17 :Development of mucoadhesive buccal patch:

A) Preformulation studies

1) Polymer characterization:

Name of Polymers: HPMC K15, Carbapol

Both polymers were characterized for description, acidity, ash value, density, melting

point, solubility & specific gravity.

2) IR Spectrum of Polymers: FTIR spectroscopy was conducted using Jasko FT-IR

4100 Spectrophotometer and the spectrum was recorded in the wavelength region

of 4000-400 m-1.

FTIR spectrum of polymers were taken using kbr dispersion

method.

91

3) Drug excipient studies: Drug-excipient interaction studies 129,130

:

The DSC analysis of extract, HPMC and Carbapol were carried using a Shimadzu DSC

60 to evaluate any possible drug–polymer interaction. Accurately weighed 1mg samples

were hermetically sealed in aluminium crucible & heated at constant rate at 10 C /min.

over a temperature range of 40-300 C. Inert atmosphere was maintained by nitrogen gas

at a flow rate of 50ml/min.

4) Caliberation curves of extracts:

Preparation of Phosphate buffer 6.8: Monobasic 0.2M phosphate buffer was

prepared. 50 ml was placed in volumetric flask & 22.4ml sodium hydroxide

(0.2M) was added to it. Water was added to make 200 ml.

Caliberation curve: Extract was weighed & a solution of 100 mg/l was poured in

phosphate buffer pH 6.8. Spectrawas run on UV Spectrophotometer. Various

concentrations made to obtain in range of 10-100µg/ml & caliberation curve was

plotted.

B) Formulation tables for buccal patches of SRME & PGME 131

Composition of different buccal mucoadhesive patch 132, 133

:

Table 2. A) : Composition of buccal patch(Preliminary trial batch)

Patch Code

Sr.no. Ingredient B1 B2 B3 B4 B5 B6

1. HPMC(50cps) 250 150 200 150 200 200

2. Eudragit RL-100 -- 100 50 -- -- --

3. Carbapol-934 -- -- -- 100 50 --

4. Ethylcellulose -- -- -- -- 50 50

5. Glycerine 0.0588 0.0588 0.0588 0.0588 0.0588 0.0588

6. Ethanol 10ml 8ml 8ml 7ml 7ml 10ml

7. Acetone -- 2ml 2ml -- -- --

8. Tween 80 0.1 0.1 0.1 0.1 0.1 0.1

9. Water -- -- -- 3ml 3ml --

10. Extracts 100mg 100mg 100mg 100mg 100mg 100mg

92

The buccal mucoadhesive patches of extract B1 were prepared by solvent casting method

using film forming polymers for the patches mentioned in table 3. HPMC polymer (250

mg) was weighed accurately and dissolved in 2 ml of ethanol. The beaker-containing

polymer was kept aside for 5 minutes for swelling of polymer.100mg of extract was

weighed and dissolved in 2 ml of ethanol. Further 6 ml of ethanol was added to the above

polymer solution and stirred the dispersion. Then one drop of (0.0294 g) glycerin was

added to the polymer solution. The drug solution was added to the polymer solution. The

whole solution was mixed thoroughly with the help of a magnetic stirrer. The glass

mould of size 5* 3 cm2

was placed over a flat surface. The whole solution was poured

into the glass mould. An inverted funnel was placed over the mould to avoid sudden

evaporation. Similarly patch B2, B3, B4, B5&B6 were prepared. For preparing patch B2

and B3, Eudragit was dissolved in 2 ml acetone and HPMC was dissolved in 6 ml ethanol

and kept for drying 24 hours. The two polymeric solutions were mixed. For preparing

patch B4 and B5, Carbopol 934 was placed in 3 ml of water, and stirred for 60 min.

HPMC was dissolved in 5 ml of ethanol. The two polymeric solutions were mixed. For

preparing patch B6 both Ethyl cellulose and HPMC were dissolved in ethanol. The

moulds were kept 24 hours for drying of patch for formulations, B1 B2, B3, and B6.

Whereas for formulations B4 and B5 moulds were kept aside for 72 hours.

C) Optimization of buccal mucoadhesive patch 133

:

This formulation was further optimized by varying HPMC K15 and Carbopol and other

variables and 9 new formulations F1 to F9 were prepared.In this study three factor

namely, amount of polymer (HPMC and carbopol), amount of Tween 80 and amount of

glycerin were selected as independent variables while thickness, weight uniformity,

surface pH, mucoadhesive strength, permeation studies and in vitro drug release were the

dependent variable used for optimization process.

93

Optimization of Batch containing Extracts:

Table 2.B): Composition of Ingredients in buccal Patch

Patch

code

Amount of

Drug (mg)

(SRME &

PGME)

Total

amount

of po-

lymer

(mg)

Amount of

HPMC

Amount of

carbopol

Amou

nt of

Tween

80(ml)

Amoun

t of gly-

cerin(m

l)

Solvents

% Mg % mg Wat

er

Alco

hol

Acet

one

F1 100mg 150 100% 150mg 0% 0mg 0.1 0.1 1 10 0

F2 100mg 150 90% 135mg 10% 15mg 0.2 0.3 1.5 15 0

F3 100mg 150 80% 120mg 20% 30mg 0.3 0.5 2 20 0

F4 100mg 150 70% 105mg 30% 45mg 0.1 0.1 1 8 2

F5 100mg 150 60% 90mg 40% 60mg 0.2 0.3 1.5 11 4

F6 100mg 150 50% 75mg 50% 75mg 0.3 0.5 2 14 6

F7 100mg 150 40% 60mg 60% 90mg 0.1 0.1 1 7 3

F8 100mg 150 30% 45mg 70% 105mg 0.2 0.3 1.5 9 6

F9 100mg 150 20% 30mg 80% 120mg 0.3 0.5 2 11 9

5.3.18 Evaluation of mucoadhesive buccal patch 134

The buccal patches were evaluated for the following properties:

I) Physical Properties:

Physical appearance and surface texture.

Thickness uniformity and Diameter

Swelling Index

Surface pH

Moisture uptake

Folding endurance

Viscosity

Uniformity weight of patch

II) Mechanical Properties:

In-vitro Bioadhesion Studies

94

III) Other properties:

Drug Content Uniformity:

In vitro release studies.-The USP rotating paddle method.

Ex vivo buccal permeation study- Franz diffusion cell.

Residence time.

Drug- excipient interaction studies.

Short-term stability studies

I) Physical Properties:

1. Physical appearance and surface texture:

All the buccal patches were visually inspected for colour, clarity, flexibility and surface

texture.

2. Thickness uniformity and Diameter:

The Three patches of each formulation were taken and thickness of each patch was

measured using digital micrometer screw gauge with a least count of 0.01 mm at different

spots of the patches at three different positions of the patch and the average value was

calculated. (vernier caliper) .

3. Swelling Index:

After determination of the original patch weight and diameter, the samples were allowed

to swell on the surface of agar plate kept in an incubator maintained at 37±0.2 o. Increase

in the weight of the patch (n=3) was determined at preset time intervals (1-3h). The

percent swelling of the patches was calculated using the formula

Sd (%) = [(Dt –D0)/D0] x100

Where Dt is the weight of swollen patch after time t, D0 is the initial patch weight at zero

time.

4. Surface pH:

Buccal patches were left to swell for 2 h on the surface of an agar plate, prepared by

dissolving 2% (m/V) agar in warmed isotonic phosphate buffer of pH 6.75 under stirring

and then pouring the solution into a Petri dish till gelling at room temperature. The

surface pH was measured by means of a pH paper placed on the surface of the swollen

patch. The mean of three readings was recorded.

95

5. Moisture uptake:

The polymer used for the formulation of mucoadhesive patches is hydrophilic polymer.

The moisture absorption studies give an indication about the relative moisture absorption

capacities of polymers and an idea whether the formulation maintains its integrity after

absorption of moisture. 5% w/v agar in distilled water, in hot condition, was transferred

into Petri plates and it was allowed to solidify. Six drug free patches of each formulation

were selected and weighed.

They were placed in desiccator overnight prior to the study to remove moisture if any and

laminated on one side with water impermeable backing membrane. They were placed on

the surface of the agar and incubated at 370C for one hour in incubator. The patches were

removed and weighed again.

6. Folding endurance:

Three patches of each formulation of size (2x2 cm) were cut by using sharp blade.

Folding endurance was determined by repeatedly folding a small strip of patch at the

same place up to maximum 300 times or till it broke. The number of times, the patch

could be folded at the same

place without breaking gave the value of folding endurance. The mean value was

calculated.

7. Viscosity: Aqueous solutions containing both polymer and plasticizer were prepared in

the same concentration as that of the patches. A model LVDV-II Brookfield viscometer

attached to a helipath spindle number 4 or 18 was used. The viscosity was measured at 20

rpm at room

II) Mechanical Properties 135

:

In-vitro Bioadhesion Studies:

Mucoadhesive strength of the buccal films was measured on the modified physical

balance. The test assembly was fabricated as shown in schematic presentation in Fig no

11. This method involves the use of porcine membrane as the model mucosal

membrane. The fresh porcine membrane was purchased from slaughter house and used

within 2hrs then it was washed in isotonic phosphate buffer (6.8). The two sides of the

balance were balanced with a 5gm weight on the right hand side. A piece of fresh

membrane was glued to a support (glass block) with cyanoacrylate adhesive. The block

96

was then lowered into the glass container, which was then filled with isotonic phosphate

buffer (pH 6.8) kept at 370C, such that the buffer just reaches the surface of mucosal

membrane, and keeps it moist. This was then kept below the left hand setup of the

balance. The test film was glued with the same adhesive to a rubber block hanging on

the left hand side and the balance beam raised with the 5gm weight on the right pan was

removed off the weight. This lowered the rubber block along with the film over the

mucosa with a weight of 5gms.The balance was kept in this position for 3 minutes and

then slowly water was added to the plastic container in the right pan by pipette. The

detachment of two surfaces was obtained. Weight of water was measured. Then the

Bioadhesive strength of the film was calculated. Three films were tested on each porcine

membrane. After each measurement, the tissues were gently and thoroughly washed

with phosphate buffer (pH 6.8) and left for 5 minutes before the next experiment. Fresh

membrane was used for each batch of films.

III) Other properties:

1. Drug Content 136

: The 1 cm² area of the medicated patch was allowed to dissolve in

100 ml IPB, pH 6.8. The amount of extract in the solution was measured

spectrophotometrically at max of 236 nm & 260nm for PGME & SRME respectively.

From the absorbance and the dilution factor, the drug content in the film was calculated.

2. In vitro release study

136, 137:

The USP rotating paddle method (Modified USP Apparatus):

The United States Pharmacopeia (USP) XXIII rotating paddle method used to study the

drug release from the buccal patch. The dissolution medium consisted of phosphate

buffer pH 6.8. The release was performed at 37-C ± 0.5-C, with a rotation speed of 50

rpm. The backing layer of buccal patch attached to glass disk with instant adhesive

(cyanoacrylate adhesive). The disk was allocated to the bottom of the dissolution vessel.

Samples (5mL) were withdrawn at predetermined time intervals and replaced with fresh

medium. The samples filtered through Whatman filter paper and analyzed after

appropriate dilution by UV spectrophotometry at suitable nm.

97

The USP rotating paddle method (Modified USP Apparatus):

1. Mucoadhesive buccal patch U.S.P Dissolution study:% Release (Average) with

model fitting

Name of the Drug : PGME

Loading Dose in mg :0.1g

Dissolution Medium :P.B pH6.8

RPM :50

Volume of Dissolution Medium (ml) :100

Volume of Sample removed (ml) :1ml

Dilution Factor :10

Slope of Calibration curve :0.9869

Constant of Calibration curve :1.00

2. Mucoadhesive buccal patch U.S.P Dissolution study: % Release (Average) with

model fitting

Name of the Drug : SRME

Loading Dose in mg :0.1g

Dissolution Medium :P.B pH6.8

RPM :50

Volume of Dissolution Medium (ml) :100

Volume of Sample removed (ml) :1ml

Dilution Factor :10

Slope of Calibration curve :0.9945

Constant of Calibration curve :1.00

3. In vitro buccal permeation study- (Franz diffusion cell)

138, 139,140 :

The in vitro buccal drug permeation study of Drugs through the buccal mucosa (sheep or

rabbit) performed using Keshary-Chien/Franz type glass diffusion cell at 37°C ± 0.2°C.

Fresh buccal mucosa mounted between the donor and receptor compartments. The buccal

Patch was placed with the core facing the mucosa and the compartments clamped

together. The donor compartment filled with 1 mL of phosphate buffer pH 6.8. The

receptor compartment was filled with phosphate buffer pH 7.4, and the hydrodynamics in

the receptor compartment maintained by stirring with magnetic bead at 50 rpm. A 1-mL

98

sample can be withdrawn at predetermined time intervals and analyzed for drug content

at suitable nm using a UV-spectrophotometer.

4. Residence time 140

:

The in-vitro residence time was determined employing a modified USP disintegration

procedure. The disintegration medium was composed of 800 ml isotonic phosphate buffer

of pH 7.4 (IPB) maintained at 370C. A piece of porcine buccal tissue was used for this

study. The tissue was attached to a rectangular glass piece using cynoacrylate adhesive

from nonmucosal surface. The patch was tuck to the mucosal surface by applying small

pressure. The glass piece with tissue and patch placed in the basket of disintegration

apparatus and set in motion. The time necessary for complete erosion or detachment of

the patch from the mucosal surface was observed and recorded (mean of three

determinations).

5. Short-term stability studies 140

:

This involves placing the formulation in accelerated conditions of temperature and

humidity in presence of air and determining the drug content at suitable intervals of time.

By the data so obtained two conclusions can be drawn. Firstly, the shelf-life of

formulation can be established, secondly any incompatibility within formulation, if

present can be detected changes in the appearance, residence time, release behavior and

drug content of the stored bioadhesive patches were investigated after 1, 2, 3, 4, 5, and 6

months. The data presented were the mean of three determinations. Fresh and aged

medicated patches, after 6 months storage, were investigated.

5.3.19 Formulation & Evaluation of in-situ gel:

Polymer Characterization: Sodium alginate was characterized for following properties.

1) Swelling Index: 141

Swelling index was calculated by introducing the specified quantity concerned,

previously reduced to the required fineness and accurately weighed, into a 25-ml glass-

stoppered measuring cylinder. 25 ml of water was added and the mixture was thoroughly

shaked for every 10 minutes for 1 hour. Then the mixture was allowed to stand for 3

99

hours at room temperature, or as specified. The volume in ml occupied by the polymer

was measured, including any sticky mucilage. The mean value of the individual was

calculated, related to 1 gm material.

2) Determination of viscosity: 142

Polymer solutions of 1% w/v were prepared in water. The viscosities of prepared

polymeric solution were determined using Brookfield viscometer CAP 2000.

Spindle used: Flat type spindle

Method:

The spindle was attached to the spindle coupling nut. The spindle was immersed

in the test material and while immersing it was tilted to avoid the air trapping. Parameters

were selected and the viscosity was recorded. The spindle was cleaned before the samples

were changed. The motor was turned off and the spindle was removed and cleaned.

3) Ash value: 143

2 g powdered was weighed and taken into porcelain dish. The dish was supported

on a pipe-clay triangle placed on a ring of retort stand. It was heated with a burner, using

a flame about 2 cm. high and supported the dish about 7 cm. above the flame, it was

heated till vapors almost cease to be evolved, then lower the dish and heat more strongly

until all the carbon was burnt off. It was cooled in desiccators. Obtained ash was weighed

and calculates the percentage of total ash with reference to the air dried sample of the

powder.

4) DSC Studies: The DSC analysis of extracts, Sodium alginate, sodium citrate &

calcium chloride were carried using a Shimadzu DSC 60 to evaluate any possible drug –

polymer interaction. Accurately weighed 1mg samples were hermetically sealed in

aluminium crucible & heated at constant rate at 10 C /min. over a temperature range of

40-300 C. Inert atmosphere was maintained by nitrogen gas at a flow rate of

50ml/min.144

100

5.3.20 Formulation of in-situ gel of SRME & PGME: 145,

1. Preparation insitu gelling system of SRME & PGME:

The different concentration of sodium alginate solutions was prepared in ultra pure water

containing sodium citrate at 60oC. Calcium chloride was added to the solution after

cooling at below 40oC with stirring. Extracts were dissolved separately in 0.1N HCL

solution and then added slowly to the above sodium alginate solution while stirring on a

magnetic stirrer to get the homogeneous dispersion of the drug. 0.1 N NaOH was added

to the above solution to neutralize the hydrochloric acid while stirring.The above

formulations were sonicated in a bath sonicator for 15 minutes & then checked the

viscosity of the solutions and then add the prepared solutions in pH 1.2 buffer, to see the

gel formation and checked its physical appearance and took the dissolutions of the

prepared gels.

2. Preliminary batches:

Table 3. A): Composition of Preliminary trail batches

Batch

No. Conc. of

Sodium

alginate

pH Characteristic of In

situ Gel

J1 0.25 7.4 Gel is not formed

properly J2 0.25 7.4

J3 0.25 7.3

J4 0.5 7.1 Gel formation

J5 0.5 7.1

J6 0.5 7.2

J7 1.0 7.0 Gel formation

J8 1.0 6.0

J9 1.0 7.0

J10 1.5 6.8 Gel formation

J11 1.5 6.7

J12 1.5 6.8

All batches prepared using 0.075% (w/v) Calcium Chloride & 0. 25%(w/v) Sodium

citrate.* Mean± S.D.(n=3)

3. Experimental design:

A factorial design experiment was conducted to study the effect two factors. The

levels of the two factors were selected on the basis of the preliminary studies carried out

before implementing the experimental design. All other formulation and processing

101

variable were kept constant throughout the study of 32 factorial experimental design

layout.

4. 32Full factorial design layout:

147

Independent variable: X1= Sodium alginate,

X2= Calcium Chloride

Variable levels: Low (-1), Medium (0), High (+1)

Dependent variable: Y1= Floating time

Y2= %Drug release

Table 3 B): Composition of Factorial design batches

Sr.no Formulation

code

Actual Units

X1 X2 X1(gm)

w/v X2 (gm)

w/v

1 F1 -1 -1 0.5 0.05

2 F2 -1 0 0.5 0.075

3 F3 -1 +1 0.5 0.1

4 F4 0 -1 1 0.05

5 F5 0 0 1 0.075

6 F6 0 +1 1 0.1

7 F7 +1 -1 1.5 0.05

8 F8 +1 0 1.5 0.075

9 F9 +1 +1 1.5 0.1

In situ oral gel formed by Floating system with sodium alginate.

Table 3C): Ingredients of Formulation of floating System

Ingredient (w/v) F1 F2 F3 F4 F5 F6 F7 F8 F9

SRME& PGME 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

Sodium alginate 0.5 0.5 0.5 1 1 1 1.5 1.5 1.5

Calcium

chloride

0.05 0.075 0.1 0.05 0.075 0.1 0.05 0.075 0.1

Sodium citrate 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25

Propyl paraben 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40

Methyl paraben 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16

Sweetner 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2

Water q.s. q.s. q.s. q.s. q.s. q.s. q.s. q.s. q.s.

102

5. Optimized method for Sodium Alginate in situ oral gel preparation: (Floating

system):

Sodium alginate solutions of different concentration were prepared by adding the alginate

to ultrapure water containing sodium citrate and different concentration of calcium

chloride and heating to 600C while stirring on a magnetic stirrer. SRME, PGME and

preservatives was then dissolved in the resulting solution after cooling to below 40°C.

Prepared sols finally stored in amber color bottles until further use.

5.3.21 Evaluation of formulations:

The evaluation parameter of the floating system was evaluated by following way

differently to compare all the characters.

1. Physical appearance 147

:

Clarity is one of the most important characteristic features of In-Situ Oral

Gel preparations. All developed formulations were evaluated for clarity by visual

observation against a black and white background.

2. pH of formulation148

: All the prepared sodium alginate based in situ solutions of

SRME & PGME were checked for the pH of the solutions at 25oC. pH is the essential

parameter that determines the sol-gel transition.

3. Determination of Rheological properties:

The flow characteristics were determined by using rheometer at 25oc and

pressure is maintained at 2.4.The flow characteristic measured by the selection of

different parameters. The flow of gel is measured after the formation of gel 0.1N HCL

and for sol in its original form.

Viscosity of the samples was determined using a Brookfield digital

viscometer (Model no CAP-2000) with spindle number 1 at a controlled temperature

25±1°C. The viscosity of the solutions prepared in water was determined at ambient

condition. Increasing the concentration of a dissolved or dispersed substance generally

gives rise to increasing viscosity (i.e. thickening), and also as molecular weight of a

solute increases viscosity increases.

4. In-vitro floating ability 147,148

:

The in-vitro floating study was carried out using 900 ml of 0.1N HCL, (pH

1.2) .The medium temperature was kept at 37oC. 10ml formulation was introduced into

103

the dissolution vessel containing medium without much disturbance. The time the

formulation took to emerge on the medium surface (floating lag time) and the time the

formulation constantly floated on surface of the dissolution medium (duration of floating)

were noted.

5. In-vitro gelling Studies 147

:

To evaluate the formulations for their in-vitro gelling capacity by visual

method, coloured solutions of in situ gel forming drug delivery system were prepared.

The in-vitro gelling capacity of prepared formulations was measured by placing five ml

of the gelation solution (0.1N HCL, pH 1.2) in a 15 ml borosilicate glass test tube and

maintained at 37±1ºC temperature. One ml of coloured formulation solution was added

with the help of pipette.

The formulation was transferred in such a way that places the pipette at

surface of fluid in test tube and formulation was slowly released from the pipette. As the

solution comes in contact with gelation solution, it was immediately converted into stiff

gel like structure. The gelling capacity of solution was evaluated on the basis of stiffness

of formed gel and time period for which they formed gel remains as such. Color was

added to give visualized appearance to formed gel. The in-vitro gelling capacity was

graded in three categories on the basis of gelation time and time period for which the

formed gel remains.

1] (+) Gels after few minutes dispersed rapidly

2] (++) Gelation immediate remains for 8 hours

3] (+++) Gelation immediate remains for more than 8 hours

6. Drug content: 147

Accurately, 5ml of in-situ gel from different batches (equivalent to 25 mg of extract)

were measured and transferred to 100 ml of volumetric flask. To this 50-70 ml of 0.1 N

HCl was added and sonicated for 30 min. Volume was adjusted to 100 ml. Complete

dispersion of contents were ensured, visually and filtered using Whatman Filter Paper.

From this solution, 10 ml of sample was withdrawn and diluted to 100 ml with 0.1 N

HCl. Contents of extract (SRME & PGME) was determined spectrophotmeterically at

236 nm(SRME) & 224nm (PGME) respectively using double beam UV-Visible

spectrophotometer.

104

7. In vitro drug release studies147

:

For the determination of in vitro drug release, USP XXIV dissolution testing

apparatus I (basket covered with muslin cloth) was used. Rotation speed was maintained

at 50 rpm. Dilution method was employed to maintain different pH conditions in the

dissolution studies. 5 ml of the solution was added to 750 ml of buffer solution of pH 1.2

contained in the dissolution flask and the temperature was maintained at 37°C with 50

rpm. At the end of 2 hrs, the medium was filled by 150 ml of pH 6.8 Phosphate buffer.

Aliquots of 1 ml were withdrawn at frequent intervals and equal amount of

fresh medium was replaced after each sampling. The dissolution was continued in this

medium up to 8 hrs. For floating system whole dissolution carried out in 900 ml of 1.2

pH buffer. The collected samples were analyzed for the drug content at 236 nm(SRME)

& 224nm (PGME) respectively using double beam UV-Visible spectrophotometer.

8. Measurement of water uptake147

:

The water uptakes by the gels were determined by a simple method. In this

study the in situ gel formed in 40 ml of 0.1 N HCL (pH 1.2) was used. From each

formulation the gel portion from the 0.1 N HCL was separated and the excess HCL

solution was blotted out with a tissue paper. The initial weight of the gel taken was

weighed and to this gel 10 ml of distilled water was added and after every 30 minutes of

the interval water was decanted and the weight of the gel was recorded and the difference

in the weight was calculated and reported.

9. In vitro Bioadhesion test: The in vitro bioadhesion property of in situ gel was

assessed on rat stomach mucosa. The test parameters were pretest speed 0.6mms-1

, test

speed 0.1mms-1

, contact time 3.5 min, preload 1N, load cell500N, diameter of upper

probe 30mm. The rat stomach mucosal tissue was cleaned, washed & stored at -20 C.

Preserved , cleaned & thawed rat stomach mucosa was incised longitudinally just before

the experiment. Rat stomach mucosa was mounted on the plateform below the texture

analyzer probe. A cellophane membrane, equilibrated with simulated gastric fluid at 37 1

c for 24 hrs. was tied the upper probe. Surface of rat stomach mucosa was moisturized

with simulated gastric fluid & gel was applied. The test was run after completing the

pretest requirements.

105

10. Selection of Optimized Batches:

Optimization of preparation of In-Situ Oral Gel was done by Design Expert Software

(Version 7.1.4, Stat-Ease Micromath Inc.). All the above formulations were prepared and

evaluated for various parameters, and the effects of the excipient were studied on the

viscosity, floating time and drug release. The data was input to design expert software and

polynomial equation was obtained.

11. Stability study: 149

Prepared sols were first packed in glass bottles (well stoppered) and then

packing forms were kept for three months and the stability of the gel was monitored up to

3 months at accelerated stability conditions (45 ºC temperature and 75 ± 5% RH).

Periodically (initial, 1, 2 and 3 months interval) samples were removed and characterized

by drug release, drug content, floating time and in-vitro gelation. The results of the

stability study for the selected batch of sodium alginate based in-situ formulation were

recorded.

5.3.22 Evaluation of anti-ulcer activity of formulations (SRME & PGME) 150

I) Animals: Studies were carried out using Wistar albino rats (120–150 gm) of either sex.

They were obtained from the animal house, National Institute of Biological Sciences,

Pune India. All the animals were housed in polypropylene cages maintained in controlled

temperature (27 ± 2°C) and light cycle (12 h light and 12 h dark). They were provided

with standard rat pellet diet and water adlibitum. All the animals were given a week time

to get acclimatized with the laboratory conditions. The experiments were carried out

according to guidelines of Committee for Prevention and Control of Scientific

Experimentation on Animals (CPCSEA).

II) Drugs and Chemicals Omeprazole (Dr.Reddy’s Lab, India) and Topfers reagent

(Nice Chemicals, India) were used in this study. All other chemicals used in present study

were of analytical grade.

III) Methods:

1. Pylorus ligation ulcer model:151

The animals were divided into following groups of six animals each.

Group 1: Served as control and was Animals were treated vehicle only

Group 2: Animals were treated with Fomulation 1 p.o (SRME)

106

Group 3: Animals were treated with Formulation 2 p.o (PGME)

Group 4: Animals were treated with Formulation 3 p.o (NO extract)

Group 5: Animals were treated with standard drug Omeprazole (20mg/kg, p.o)

Overnight fasted rats were anaesthetized with anaesthetic ether. Then an incision of 1cm

long was given in the abdomen just below the sternum. The stomach was exposed and a

thread was passed around the pyloric sphincter and a tight knot was applied. Abdomen

wall was closed by putting the sutures. After 45 minutes of extracts (SRME & PGME)

treatments pyloric ligation was performed. After 4 hr of pyloric ligation animals were

sacrificed by decapitation. Abdomen was opened and the oesophagus was tied at the end

of the stomach. A small cut to the pyloric region just above the knot was given and

contents of the stomach were collected in a centrifuge tube. The following parameters

were analyzed:

1. Volume of gastric juice (in ml): Gastric content was centrifuged at 1000 rpm for

10min and measured the volume.

2. Determination of free and total acidity: Pipetted out 1ml of supernatant liquid and

diluted it to 10ml with distilled water. The PH of this solution was noted with the help of

pHmeter.

Acidity was calculated by using the formula: Acidity = meter. The solution was titrated

against 0.01N NaOH using topfers reagent (Dimethyl-amino-azo-benzene with

phenolptheline) as indicator. The end point was noted when the solution turns to orange

color; this corresponds to the free acidity. Titration was continued further till the solution

regained pink color. This volume corresponds total acidity.

volume of NaOH x Normality x 100 mEq/lt/100g 0.1

3. Ulcer Scoring & Ulcer Index Determination

0 - Normal Mucosa

0.5 - Red coloration

1.0 - Spot ulcers

1.5 - Hemorrhagic streaks

2.0 - Ulcers >3 but <5

2.5 - Ulcer >5.

107

Mean ulcer score of each group were calculated, which was designated as the ulcer index

and percentage of protection was calculated as C – T / C x 100

(C = ulcer index in control group; T = ulcer index in test group)

4. Ethanol induced Ulcer model 152,153

: Administration of Formulations and control

drugs was done for 10 days after which 90% ethanol was administered to the overnight

fasted rats of all groups the next day; at a dose of 1 mL per animal, irrespective of the

weight of the animal through oral route. One hour after ethanol administration, all rats

were sacrificed by an overdose of chloroform and the stomachs were rapidly removed,

opened along their greater curvature and gently rinsed under running tap water. They

were then spread on a paraffin plate and the inner surface was examined with a 6x hand

held magnifier. The scores for each single lesion were then totalled .Mean ulcerative

index was calculated as follows:

Presence of oedema, hyperaemia and single submucosal punctiform

haemorrhages.

Presence of submucosal haemorrhagic lesions with small erosions.

Presence of deep ulcer with erosions and invasive lesions:

Ulcer index = (number of lesion. I) + (number of lesion. II) × 2 + (number of lesion. III)

×3

The percentage inhibition was determined as follows:

= (Control mean lesion index-Test mean lesion index) /X 100Control mean lesion index

3. Histopathology Study 154

: At the end of the study, all the rats were sacrificed by

cervical decapitation and the stomach were isolated, washed in ice cold saline. Then the

tissue was immediately fixed in 10% buffered neutral formalin solution. After fixation,

tissues were embedded in paraffin and serial sections were taken and each section is

stained with hematoxylin and eosin. The slides were then examined under light

microscope and photographs were taken..

4. Statistical analysis 153

: The data were represented as Mean± SEM. The data on

antiulcer activity of formulations were analyzed by one way Analysis of Variance

(ANOVA), ‘P’ value less than 0.05 was considered as statistically significant.

108

CHAPTER 6: RESULT & DISCUSSION:

6.1 Plant Description

6.1.1 Pharmacognostic evaluation

Psidium guajava (Family: Myrtaceae)

Authentication No: BSI/WRC/Tech/2010, dated (22/11/2010)

I) Macroscopic characters of mature leaf

Figure 3. : Mature leaf of Psidium guajava

Morphological Characters

Colour: Green

Odour: Aromatic

Taste: Astringent

Size: 15-20 cm long& 3-6 cm width

Shape: Ovate

II) Microscopic evaluation:

A) Microscopic study of Psidium Guajava L. leaf :

(T.S.) showed presence of upper and Lower Epidermis, Collenchymas, Parenchyma,

Xylem and Phloem

109

Figure 4. T.S. of Psidium guajava ( showing various characters)

B) Microscopic characteristic of powder:

1. Calcium Oxalate crystals:

Calcium oxalate crystals are abundant in powder. The crystals are in the form of thin

pointed needles, which are originally in the form of thick bundles called raphides.

110

2. Starch grains:

The starch grains are not abundant as the needles crystals, but are frequently seen in the

powder. The starch grains are circular to ovoid.

3. Xylem Vessels:

Another characteristic feature of the powder is xylem vessels which are seen in the

powder as short, thick or thin bundles. These bundles consist of broken pieces of xylem

elements, especially vessels. The Spiral xylem vessel is observed.

4. Surface preparation: Surface preparation fallowing character observed

Stomata: Paracytic stomata. Parallel cell with two subsidiary cell arrange guard cell.

Trichomes: Thick wall unicellular covering trichomes.

Epidermal Cell: Polygonal, thin wall Parenchymatus cells present in Upper Epidermis.

Figure 5. A : Calcium oxalate crystal & Starch grains

Figure 5. B : Lignified Xylem vessels &Epidermis & Spiral lignified Xylem

vessel

111

Microscopic study of Psidium Guajava L. showed presence of upper and Lower

Epidermis, Collenchymas, Parenchyma, Xylem and Phloem. T.S. showed, xylem,

phloem, covering trichomes , upper epidermal cell, and parenchyma. Histological findings

in case of leaves powder showed the presence of calcium oxalate crystals in the form of

thin pointed needles, which are originally in the form of thick bundles called raphides .

Broken needles are also seen in the powder. Starch grains, xylem fiber; xylem vessel were

seen in the powder.

Symplocos racemosa (Family: Symplocaceae)

Authentication No: 12-124, dated: 01/08/2012

I) Macroscopic characters of mature stem bark

Shape: Channelled or curved pieces, few fiat pieces

Thickness: Upto 1cm,

Surface: Outer surface uneven and rough due to fissures and cracks

Colour: Grayish brown to grey externally pale to whitish-brown internally

Taste : Astringent and feebly bitter

Fracture: Short and granular in cortical region and somewhat fibrous in inner region

Figure 6. : Barks of Symplocos racemosa

112

II) Microscopic characters:

A) Transverse section of mature bark shows following characters:

1. Cork: A wide cork of thin-walled, rectangular cells arranged in radial rows, cork

cambium 1-3 layered.

2. Secondary cortex: Consists of thin-walled, oval and tangentially elongated

parenchymatous cells towards outer side and rounded cells towards inner side, a number

of stone cells, in singles or in groups present, scattered throughout the region having

highly thickened walls with distinct pits

3 Calcium oxalate crystals: Prismatic and cluster crystals of calcium oxalate, and starch

grains, mostly simple present in a number of cortical cells,

4. Secondary Phloem: Wide consisting of sieve elements, phloem parenchyma, phloem

fibres and stone cells, phloem parenchyma thin walled, oval to rectangular, containing

prismatic crystals of calcium oxalate scattered in phloem parenchyma, phloem fibres

lignified and present in singles or in groups, crystals not present in fibres, isolated fibres

spindle shaped with pointed ends, groups of stone cells as rounded patches distributed

throughout phloem region

5.Medullary rays: Uniseriate to multiseriate consisting of rectangular cells having brown

colouring matter in some cells, broader medullary rays dialating towards outer phloem

region, a number of phloem cells also contain starch grains, mostly arranged in groups,

rarely solitary, simple and rounded.

Microscopic characters of bark

Figure 7. A):T.S. showing cortex B) Sec. phloem & Phloem fibers

113

C): T.S. showing Stone cells & medullary rays (Size of stone cells-65.1µm)

D) T.S. showing presence of cork layer E) Medullary rays & Phloem fiber

B) Microscopic characters of Symplocos racemosa bark powder: Microscopic

characters of bark powder showed presence of shows fragments of cork, stone cells,

fibres, prismatic and cluster crystals of calcium oxalate and starch grains. (Shown in

diagrams)

Figure 8. A): Cork cells B)Unlignified phloem fibers

114

C) Lignified phloem fibers D) Scleride

E)Prism shape Calcium oxalate crystals F)Phloem fibers of various lengths

6.1.2 Evaluation of Physical Constants:

1. Psidium guajava

Table 4. A) : Physical constants of Psidium guajava

Sr. no. Evaluation Parameter Value (%) St. dev.

1 Foreign Matter 1.09 0.176

2 Moisture Content 10.52 0.15

3 Total Ash Value 7.4 0.264

4 Water Soluble Ash Value 3.633 0.152

5 Acid Insoluble Ash Value 1.063 0.118

6 Water Soluble Extractive Value 7.266 0.115

7 Chloroform Soluble Extractive Value 0.366 0.152

8 Methanol Soluble Extractive Value 9.466 0.098

9 Ethanol Soluble Extractive Value 5.333 0.251

115

The Methanol soluble extractive value found to be the highest (9.4%) where water soluble

(7.2%), Chloroform (0.36 %) and Ethanol (5.3%) respectively. The proximate analysis

showed satisfactory result with respect to foreign matter, moisture content, Ash value and

Extractive values.

2. Symplocos racemosa

Table 4 B): Physical constants of Symplocos racemosa

Sr. no. Evalution Parameter Value (%) St. dev.

1 Foreign Matter 1.17 0.1571

2 Moisture Content 10.633 0.1527

3 Total Ash Value 12.5 0.3

4 Water Soluble Ash Value 3.5 0.2

5 Acid Insoluble Ash Value 8 0.2

6 Water Soluble Extractive Value 3.37 0.120

7 Chloroform Soluble Extractive Value 0.36 0.03

8 Methanol Soluble Extractive Value 6.68 0.0253

9 Ethanol Soluble Extractive Value 4.066 0.3055

The Methanol soluble extractive value found to be the highest (6.68%) whereas water

soluble (3.37%), Chloroform (0.36%) and Ethanol (4.06%) respectively. The proximate

analysis showed satisfactory result with respect to foreign matter, moisture content, Ash

value and Extractive values.

116

6.1.3 Extraction of plant constituents(Physical appearence):

Table 5. A) : Characteristics of extracts of Psidium guajava

Sr.No. Type Colour of extract Appearance % Yield pH

1. Aqueous Dark brown Sticky 7.08 5

2. Ethanolic Dark brown Sticky 13.906 5

3. Methanolic Dark brown Sticky 11.7 5

Table 5 B): Characteristics of extracts of Symplocos racemosa

Sr.No. Type Colour of extract Appearence % Yield pH

1. Chloroform Dark brown Sticky 0.36 5

2. Ethanolic Dark brown Sticky 13.906 5

3. Methanolic Dark brown Sticky 11.7 5

4. Aqueous Dark brown Sticky 17.4 6

117

6.1.4 Preliminary phytochemical screening:

Table 6. A) : Phytochemical analysis of Psidium guajava

Sr.no. Chemical test AqExt. Chloroform MeOH EtOH

1 Test for Alkaloids :

a)Dragendorff’s test

b) Mayer’s test

c) Hagers’s test

+

-

-

+

-

-

+

-

-

+

-

-

2 Test for Tannins :

a)Ferric chloride test

b)Lead acetate test

c)Potassium Dichromate test

d) Dilute HNO3

+

+

+

+

+

-

-

-

+

+

+

+

+

+

+

+

3. Test for flavonoids:

a)Lead acetate test

b)Ferric chloride test

c) Sodium Hydroxide test

d) Shinoda test

+

-

+

+

+

-

+

+

+

+

+

+

+

+

+

+

4. Test for Steroids:

a)Salkowski test

b)Liebermann – Burchard test

+

+

+

+

+

+

+

5. Saponification :Foam test + - + +

6. Test for Cardiac Glycosides:

a) Keller-Killiani test

b) Legal’s test

-

+

-

+

+

+

+

-

7. Test for Anthraquinone

Glycosides:

Borntrager’s test

-

-

+

+

8. Test for Saponin Glycosides:

a) Foam Test

b) Heamolytic test

+

+

-

-

+

+

+

+

9. Test for Carbohydrates

a) Molisch’s test

b) Fehlings test

c) Benedict test

+

+

-

-

+

-

+

+

+

+

+

-

10. Test for Proteins:

a) Biuret test

b) Millions test

-

+

-

-

+

-

-

-

11. Test for amino acids -

-

-

-

+ indicates presence of phytoconstituents - Indicates absence of phytoconstituents

118

Table 6 B): Phytochemical analysis of Symplocos racemosa

Sr.no.

Chemical test Aqueous

Extract

Pet. Ether

Extract

Methanol

Extract

Ethanol

Extract

1 Test for Alkaloids :

a)Dragendorff’s test

b) Mayer’s test

c) Hagers’s test

d) wagner’s test

+

-

-

+

--

-

-

-

-

+

-

+

+

-

-

+

2 Test for Tannins :

a)Ferric chloride test

b)Lead acetate test

c)Potassium Dichromate test

d) Dilute Kmno4

+

+

+

+

+

-

-

-

+

+

+

+

+

+

+

+

3. Test for flavonoids:

a)Lead acetate test

b)Ferric chloride test

c) Sodium Hydroxide test

d) Shinoda test

+

+

+

+

+

+

+

+

+

+

+

+

-

-

+

-

4. Test for Steroids:

a)Salkowski test

b)Liebermann – Burchard Reaction

+

+

+

+

+

+

+

+

5. Saponification test:

Foam test

+

-

+

+

6. Test for Cardiac Glycosides:

a) a) Keller-Killiani test

b) b) Legal’s test

-

+

-

+

+

+

+

-

7. Test for Anthraquinone Glycosides:

Borntrager’s test

-

-

+

+

8. Test for Saponin Glycosides:

a) Foam Test

b) Heamolytic test

+

+

-

-

+

+

+

+

9. Test for Carbohydrates

a) Molisch’s test

b) Fehlings test

c) Benedict test

+

+

+

-

+

-

+

+

+

+

+

+

10. Test for Proteins:

a) Biuret test

b) Millions test

-

+

-

-

+

-

-

-

11. Test for amino acids:

Ninhydrin test

-

-

-

-

+ indicates presence of phytoconstituents - Indicates absence of phytoconstituents

119

6.1.5 T.L.C of Extracts:

Thin layer chromatography technique was carried out for characterization of methanolic

extracts. Ethyl acetate: formic acid: glacial acetic acid: water (100:11:11:26) was used as

solvent system for characterization of flavonoids.

Table 7. A) TLC of metahnolic extract of plant Psidium Guajava L.

Sr.

No.

Chemical

constituents

Mobile phase Color of

spot

Rf value

1 Flavonoid Ethyl acetate: Formic acid: Glacial

acetic acid: water (100:11:11:26)

Yellowish

brown

0.9

2 Flavonoid Ethyl Acetate: Methanol

(90:10)

Greenish

brawn

0.9; 0.8

Figure 9. A) : T.L.C. of flavonoid (Methanolic extract of plant Psidium

Guajava)

Table 7 B): Rf values of Metahnolic extract of Symplocos racemosa

Constituent Mobile Phase Detection Rf values

Flavonoid Ethyl acetate: Formic

acid: Glacial acetic acid

:Water(100:11:11:26)

Spraying with

Anisaldehyde

sulpuuric acid

0.09(Blue),0,14(Brown),

0.50(Brown),).0.57(Dark

Pink)

-------,,----------- UV 254 nm 0.05(brown)

Steroid Ethyl acetate:

Benzene(95:5)

Spraying

withVanillin

Sulphuric Acid

Reagent

0.037(darkbrown)

0.075(lightgreen),

0.10(pink)

0.12(purple)

0.65 (violet)

0.72(pink)

120

TLC Profile for Methanolic Extract of Symplocos racemosa: 154,155

a) Flavonoid b) Steroid

Figure 9 B): TLC plates of methanolic extract

Thin layer chromatography of methanolic extract of S.racemosa showed 4 spots with

0.09, 0.14, 0.50 & 0.75 Rf value confirms the presence of flavonoid. Similarly 6 spots in

TLC of steroid with Rf value 0.037, 0.075, 0.10, 0.12, 0.65, 0.72 & confirms the presence

of steroids in extract.

Table 7 C): Rf values of aqueous extract of Symplocos racemosa

Constituent

Mobile Phase

Detection

Rf values

Flavonoid Ethyl acetate: Formic

acid: Glacial acetic acid

:Water (100:11:11:26)

Spraying with

Anisaldehyde

sulpuuric acid

0.025(Redish

brown),0.18(Dark

brown),0.62(brown)

Steroid Ethylacetate:

benzene(95:5)

Spraying

withVanillin

Sulphuric Acid

Reagent

0.062(Redishbrown)

0.10(Lightbrown)

0.125(Lightblue)

0.19,0.25,0.5(Light

brown)

0.75 (pink violet)

121

TLC Profile for Aqueous Extract Extract

a) Flavonoid b)Steroid

Figure 9 C): TLC plates of aqueous extract

Thin layer chromatography of aqueous extract of S.racemosa showed 3 spots with 0.025,

0.18 & 0.62 Rf value confirms the presence of flavonoid. Similarly 5 spots in TLC of

steroid with Rf value 0.0.062, 0.10, 0.125, 0.25& 0.75 confirms the presence of steroids

in extract.155, 156

122

6.1.5) HPTLC: 159

a) HPTLC of MeOH. extract of P. guavaja

Resolution at 220 nm; vol-20µl,

Mobile phase-n-hexane-ethyl acetate (7:3)

Figure 10. A) : showed the presence of total three components with their Rf value Rf

– 0.95,1.11, 1.41, Component number 3 at 1.41at Rf showed maximum

concentration.

HPTLC finger printing of methanol extract of leaf powder revealed presence of three

polyvalent phytoconstituents with their Rf value 0.95, 1.11, 1.41, Component number 3 at

1.41at Rf showed maximum concentration.

b) HPTLC of MeOH extract of P. guavaja

Resolution at 450 nm; vol-10µl,

Mobile phase-n-hexane: ethyl acetate (7:3)

B): showed the presence of total five components with their Rf value Rf – 0.18, 0.91,

1.21, 1.42,1.52 Component number 4 at 1.41at Rf showed maximum concentration.

123

HPTLC finger printing of methanol extract of leaf powder revealed presence of three

polyvalent phytoconstituents with their Rf value 0.95, 1.11, 1.41 at 220nm. Component

number 3 at Rf 1.41 showed maximum concentration and presence of total five

components with their Rf value 0.18, 0.91, 1.21, 1.42, 1.52 at 450nm.Component number

4 at Rf 1.41 showed maximum concentration.

c) HPTLC of Aqueous extract of P. guajava

Resolution at 220 nm; vol-20µl,

Mobile phase Methanol: water (7:3)

C) : showed the presence of total six components with their Rf value Rf –0.29, 0.74,

0.85, 0.96, 1.31.Component number 4 at 0.96 at Rf showed maximum

concentration.

124

HPTLC finger printing of methanol extract of leaf powder revealed presence of six

polyvalent phytoconstituents with their Rf value 0.29, 0.74, 0.85, 0.96, 1.31 at

220nm.Component number 4 at Rf 0.96 showed maximum concentration.

d) HPTLC of MeOH extract of Sy. Racemosa

Resolution at 200 nm; vol-20µl.

Mobilephase: Ethylacetate:methanol (8:2)

D) : showed the presence of total eight components with their Rf value Rf - 0.23,

0.44, 0.57, 0.68 0.97,1.17, 1.35, 1.43 Component number 6 at 1.17 Rf showed

maximum concentration.

HPTLC finger printing of methanol extract of bark powder revealed presence of eight

components 0.23, 0.44, 0.57, 0.68 0.97, 1.17, 1.35, 1.43 at 200 nm (volume 20µl).

Component number 6 at Rf 1.17 showed maximum concentration.

125

e) HPTLC of aq.extract of Sy. Racemosa

Resolution at 224 nm; vol-20µl

Mobile phase-Ethyl acetate:n-butanol(6:4)

E) : showed the presence of total seven components with their Rf value Rf - 0.23,

0.27 0.3, 0.38 0.54, 0.75 Component number 5 at 0.0.54 Rf showed maximum

concentration.2

The results from HPTLC finger print scanned at wavelength 224 nm (volume 20 µl) for

aqueous extract of Symplocos racemosa bark powder showed six polyvalent

phytoconstituents and corresponding ascending order of Rf values 0.23, 0.27, 0.32, 0.38,

0.54, 0.75 Component number 5 at 0.0.54 Rf showed maximum concentration.

126

f) HPTLC of aq.extract of Sy. Racemosa

Resolution at 224 nm; vol-20µl,

Mobile phase-Ethyl acetate: methanol (7:3)

F) : showed the presence of total five components with their Rf value Rf - 0.25, 0.31,

0.48, 0.90, 1.22 Component number 1 at 0.0.25 Rf showed maximum

concentration.

The results from HPTLC finger print scanned at wavelength 224 nm (volume 20 µl) for

aqueous extract of Symplocos racemosa bark powder showed three polyvalent

phytoconstituents and corresponding ascending order of Rf values 0.25, 0.31, 0.48, 0.90,

1.22 Component number 1 at 0.0.25 Rf showed maximum concentration.

127

6.1.7 Characterization of plant extracts:

A) UV Spectra of PGME

Figure 11. A): UV spectra of PGME

B) Caliberation curve:

B) Graph (Calibration curve of PGME)

C) IR Spectra (PGME)

Figure 12. : IR Spectra of PGME

y = 0.0128x + 0.0707 R² = 0.9869

0.000

0.500

1.000

1.500

0 20 40 60 80 100

Ab

sorb

an

ce

conc mcg/ml

Caliberation curve of PGME at 224 nm

Series1

Linear (Series1)

128

Table 8. :Frequencies in IR Spectra of PGME

Sr.No. Frequency(cm-1

) Assignment

1 3520.42 OH- Bond

2 2358.52 -CH Stretch

3 1624.73 -C=O

4 1667.16 -C=C-

5 1512.81 -C=O Aromatic

D) Analytical characterization of Marker of Psidium guajava

Name of Marker compound: Quercetin

Synonym: 2-(3, 4-Dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-

one, 3,3′,4′,5,6-Pentahydroxyflavone, Quercetin-3-O-rhamnoside

CAS Number 117-39-5

Empirical Formula (Hill Notation) C15H10O7

Molecular Weight 302.24

1) Physical Characterstics:

a) Colour : Yellow

b) Odour : Characteristic

c) Taste : Tasteless

d) Melting Point : 310C Dissociates

Solubility : Soluable in Ethanol, Methanol and Alkaline solvent

2) UV of Quercetin:

Figure 13. A) UV Spectrum of Quercetin

ƛ max were observed as 212, 256, & 372nm

129

3) Calibration curve of of Quercetin:

B) Graph (Calibration curve of of Quercetin)

4) IR of Quercetin

Figure 14. : IR Spectrum of Quercetin

Table 9. :Frequencies in IR Spectra of Quercetin

Sr.No. Frequency(cm-1

) Assignment

1 3413.39-3132.79 OH- Bonded

2 2900.41-2355.62 -CH Strech

3 1767.44 -C=O

4 1667.16 -C=C-

5 1611.23 -C=O Aromatic

6 1520.6 -C=C- Aromatic

7 1382.71-1264.11 -C-O-C

8 1201.43 -C=O Strech

9 1168.65-1094.4-1013.41 -C-O-C

10 727.032-823.455 -C-H-bending

y = 0.0103x + 0.0123 R² = 0.9985

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

10 20 40 50 60 80

Ab

sorb

an

ce

Conc in µg

Calibration curve of Quercetin

Series1

Linear (Series1)

130

The IR spectrum of Quercetin is highly specific for each chemical structure with a small

structure difference resulting in significant spectral changes. FTIR Spectrum is

characteristic of entire molecule and it helps structural information by referring to

generalized chart of characteristic group Frequencies.

E) UV Spectra of SRME

Figure 15. A): UV Spectra of SRME

F)Caliberation curve:

B) Graph (Calibration curve of of SRME)

y = 0.0976x + 0.1847 R² = 0.9945

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

0 2 4 6 8 10

ab

sorb

an

ce

concentration in mcg/ml

Calibration curve of SRME at 235nm

Series1

Linear (Series1)

131

G) IR Spectra (SRME)

Figure 16. : IR Spectra of SRME

Table 10. : Frequencies of SRME

Sr.No. Frequency(cm-1

) Assignment

1 3624.55 Primary OH- Bond

2 2848.35 C-O-C

3 2338.27 CH Stretch

4 1514.81 C=O Aromatic

5 1452.14 Phenolic-OH

H) Analytical characterization of Marker of Symplocos racemosa

Name of Marker compound: Gallic Acid

CAS Number 149-91-7

Linear Formula (HO)3C6H2CO2H

Molecular Weight 170.12

Physical Characterstics:

a) Colour : White

b) Odour : Characteristic

c) Taste : Tasteless

d) Melting Point : 2510C Dissociates

e) Solubility :Soluable in Water, Ethanol, Methanol and Alkaline solvent.

132

I) UV of Gallic acid:

Figure 17. A):UV Spectrum of Gallic acid

J) Calibration curve of Gallic acid

B) Graph: Calibration curve of Gallic acid

K) IR Spectrum of Gallic acid:

Figure 18. IR Spectrum of Gallic acid

y = 0.0102x + 0.0132 R² = 0.9969

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

10 20 40 50 60 80

A

b

s

o

r

b

a

n

c

e

Concentration on µg

Calibration curve of Gallic acid

Series1

Linear (Series1)

133

Table 11. :Frequencies of Gallic acid

Sr.No. Frequency(cm-1

) Assignment

1. 3399.13cm‐1 Polymeric O‐H stretching

2. 2922.7 cm‐1 aliphatic C‐Hstretching

3. 1651.6cm‐1 C=C absorption peak

4. 1529.25 cm‐1 C=O stretching

5. 1417.3cm‐1 CH2

6. 1372.6cm‐1 Phenol or Tertiary alcohol(OHbond)

7. 1247.40cm-1 C-O stretch

8. 1078.7cm‐1 C-C stretch

9. 881.6 cm‐1. CH out of plane bending

6.1.8 Determination of total phenolic content: 157, 158

By Folin-ciocalteau calorimetric reaction:

Table 12. A): Absorbance for Total Phenolic Content

Conc. in µg/ml Absorbance 1 Absorbance 2 Average + SDEV

10 0.033 0.0364 0.0347 ± 0.024

20 0.038 0.1033 0.07065±0.046

40 0.1301 0.1711 0.1506±0.028

60 0.196 0.1965 0.19625±0.003

80 0.1985 0.2245 0.2115±0.018

100 0.4089 0.4063 0.4076±0.0018

200 0.5635 0.5635 0.5635±0

Figure 19. : Caliberation curve for Total Phenolic Content

y = 0.0028x + 0.0266 R² = 0.94

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 50 100 150 200 250

abso

rban

ce a

t n

m

Concentration in µg/ml

Absorbance of Gallic acid (at nm)

Absorbance (nm)

Linear (Absorbance (nm))

134

Table 12 B): Total Phenolic Content of extracts

Extract Absorbance (nm) Total Phenolic content

MeOHSy. racemosa 0.2440 77.00µg/ml

Aq. Sy. racemosa 0.1461 42.35µ/ml

MeOH P.guavaja 0.2035 63.00µg/ml

Aq. P. guavaja 0.1270 35.85µg/ml

6.1.9) Determination of total flavonoid content: 157, 158

By Aluminium Chloride colorimetric assay:

Table 13. A):Absorbance for Total Flavonoid Content

Conc (μg/ml) Absorbance 1 Absorbance 2 Average Std Dev

100 0.026 0.023 0.025 ±0.002

200 0.042 0.044 0.043 ±0.002

300 0.056 0.058 0.057 ±0.002

400 0.069 0.071 0.070 ±0.002

800 0.087 0.091 0.089 ±0.003

1200 0.104 0.109 0.107 ±0.003

1600 0.129 0.131 0.130 ±0.002

2000 0.182 0.184 0.183 ±0.001

2400 0.214 0.219 0.217 ±0.003

2800 0.273 0.278 0.275 ±0.004

3200 0.306 0.311 0.308 ±0.004

3600 0.365 0.369 0.367 ±0.003

Figure 20. : Caliberation curve for Total Flavonoid Content

y = 0.0001x + 0.023 R² = 0.9977

0.000

0.100

0.200

0.300

0.400

0.500

0 1000 2000 3000 4000

ab

sorb

an

ce

concentration (μg/ml)

Total Flavonoid content

Series1

Linear (Series1)

135

Table 13 B): Total Flavonoid Content of extracts

Extract Absorbance (nm) Total Flavonoid content

MeOHSy. racemosa 0.1641 1.4mg/ml

Aq. Sy. racemosa 0.0891 0.6mg/ml

MeOHP.guavaja 0.4045 3.8mg/ml

Aq. P. guavaja 0.2350 2.1mg/ml

The results presented in table no. 13 indicate that the methanolic extract of Psidium

guajava (PGME) & Symplocos racemosa (SRME) contain high concentration of

phenolic compounds. Psidium guajava extract contains 63µg/ml & Symplocos

racemosa extract contains 77µg/ml equivalent to gallic acid as standard.

The total flavonoid content of both extracts was assessed using aluminium chloride

(AlCl3) according to a known method, using catechin as a standard reagent. The results

presented in table no.14 indicate that the methanolic extract of Psidium guajava &

Symplocos racemosa contain high concentration of flavonoid. The total flavonoid

contents were calculated using the linear equation based on the calibration curve of

catechin. Psidium guajava extract contains 3.8mg/ml & Symplocos racemosa extract

contains 1.4mg/ml equivalent to catechin as standard.

6.1.10 Antimicrobial study:

Determination of Minimum Inhibitory Concentration for antibacterial activity

Broth Dilution technique:

A) Preparation of extracts of Psidium Guajava l. leaves. 0.1 gram (100mg) of dried

evaporated extract was dissolved in 100ml of water giving final concentration of 1 mg/ml.

Broth culture : Mullar Hinton Broth

Sample : Psidium Guajava Metanolic Extract

Standard Drug :Stryptomycin sulphate

Bacteria : a) Escherichia coli , b)Pseudomonas aureginosa,

c) Staphylococcus aureus d) Bacillus subtilis.

Fungi : Candida albcans

Standard :Miconazole

Growth Temprature: 370C for 24 hours

136

B) Antimicrobial activity of methanolic extract: [1gm/100ml]

Table 14. A):Composition for determination of MIC

Sr.No. Amount of

Broth (ml)

Amount of

Extract (ml)

Sterile water

(ml)

Concentration of

extract in final

Turbidity

1 5 0.5 4.5 0.5 +-

2 5 1 4 1 +-

3 5 1.5 3.5 1.5 ++

4 5 2 3 2 ++

5 5 2.5 2.5 2.5 ++

6 5 3 2 3 ++

7 5 3.5 1.5 3.5 ++

8 5 4 1 4 ++

9 5 4.5 0.5 4.5 ++

10 5 5 0 5 ++

11 5 Control 0 5 --

12

Standard

5 Std dose 0 5 --

The tubes were incubated at 370C for 24 hours. The tubes having concentration of

extract in final solution from 0.5 mg/ml to 5mg/ml showed the presence of turbidity

when incubated at 370C for 24 hours. Turbidity produced in test tubes were

observed. Following data represents the minimum inhibitory concentration produced

by extracts for different microorganisms.

Table No14 B): Minimum Inhibitory Concentration (mg/ml)

Sr. no. Bacteria/Fungi Test Tube No. M.I.C

(mg/ml)

1 Escherichia coli 1 0.5

2 Pseudomonas aureginosa 2 1

3 Staphylococcus aureus 1 0.5

4 Bacillus subtilis 1 0.5

5 Candida albicans 2 1

137

Figure 21. : A) MIC of methanolic extract Escherichia coli.

B) MIC of methanolic extract Pseudomonas aureginosa

C) MIC of Methanolic Extract Staphylococcus aureus.

D) MIC of Methanolic Extract Bacillus subtilis

138

E) MIC of Methanolic Extract Candida albicans

6.1.11) Antimicrobial activity (in vitro): By Cup plate diffusion method (Zone of

Inhibition):

Table 15. : Zone of Inhibition by extracts (mm)

1.PGMEConc.(mg/ml) E. Coli S.Aureus P.auregenosa C.Albicans

5mg/ml 15.6±0.11 16.8±0.30 18.2±0.11 16.4±0.30

10mg/ml 16.2±0.20 17.4±0.22 18.8±0.20 17.2±0.11

15mg/ml 16.8±0.30 17.8±0.11 19.4±0.30 18.4±0.30

20mg/ml 17.2±0.22 18.6±0.11 19.8±0.30 19.0±0.42

25mg/ml 17.8±0.11 19.2±0.30 20.0±0.11 20.4±0.33

30mg/ml 18.2±0.32 19.4±0.20 20.6±0.05 22.2±0.22

2. PGAE Conc.(mg/ml) E. Coli S.Aureus P.auregenosa C.Albicans

5mg/ml 10.2±0.20 10.4±0.05 9.6±0.20 -

10mg/ml 10.8±0.11 11.2±0.23 10.4±0.33 -

15mg/ml 11.4±0.22 11.8±0.30 10.8±0.22 -

20mg/ml 11.8±0.30 12.6±0.30 11.6±0.05 -

25mg/ml 12.4±0.20 13.0±0.22 11.4±0.42 -

30mg/ml 12.6±0.11 13.4±0.11 10.8±0.33 -

3.PGMEConc.(µg/ml) E. Coli S.Aureus P.auregenosa C.Albicans

50µg/ml 8.4±0.24 6.4±0.30 10.6±0.20 11.4±0.11

100µg/ml 8.2±0.30 6.8±0.20 11.4±0.11 12.6±0.05

200µg/ml - 6.8±0.11 12.2±0.30 12.8±0.30

300µg/ml 8.4±0.11 7.6±0.22 12.6±0.33 13.6±0.20

400µg/ml 9.8±0.11 8.2±0.20 13.2±0.11 12.4±0.11

500µg/ml 8.6±0.20 8.8±0.30 13.4±0.30 11.6±0.22

4. PGAE Conc.(µg/ml) E. Coli S.Aureus P.auregenosa C.Albicans

50µg/ml - - - -

100µg/ml - - - -

200µg/ml - - - -

300µg/ml - - - -

139

Ampicillin was used as a standard antibiotic (10µg/ml). The Zone of inhibition producd by

Ampicillin was observed as follows:

1. Ecoli : 35±0.32mm, 2. S.Aureus:32±0.20mm

3. P.auregenosa: 28± 0. 12mm , 4. C.Albicans: 22±0.14mm

The results of preliminary antibacterial evaluation showed that methanolic extract of

Symplocos Racemosa Roxb &Psidium guavaja possess good antibacterial activity as

compare to aqueous extract. However Symplocos Racemosa Roxb has poor antibacterial

activity against gram negative

Micro-organism like P. aeruginosa and E. coli. In the investigations, results revealed their

antimicrobial activity using the agar diffusion technique, against bacteria such as S.

400µg/ml - - - -

500µg/ml - - - -

5.SRMEConc.(µg/ml) E. Coli S.Aureus P.auregenosa C.Albicans

5mg/ml 16.8±0.11 15.7±0.20 18.2±0.30 10.6±0.30

10mg/ml 15.4±0.20 16.4±0.33 17.6±0.22 17.4±0.11

15mg/ml 15.6±0.11 18.8±0.33 19.2±0.22 20.2±0.20

20mg/ml 17.4±0.05 18.2±0.11 24.6±0.30 14.4±0.30

25mg/ml 17.8±0.11 16.2±0.30 24.6±0.11 22.4±0.22

30mg/ml 18.2±0.30 15.4±0.20 21.0±0.33 20.2±0.16

6.SRAEConc.(µg/ml) E. Coli S.Aureus P.auregenosa C.Albicans

5mg/ml 13.8±0.03 12.6±0.11 19.4±0.32 -

10mg/ml 14.6±0.11 13.4±0.20 19.6±0.22 -

15mg/ml 15.4±0.05 14.2±0.11 21.2±0.30 -

20mg/ml 16.2±0.20 14.6±0.30 23.4±0.11 -

25mg/ml 16.8±0.11 15.0±0.11 23.8±0.30 -

30mg/ml 16.6±0.11 15.4±0.20 24.0±0.11 -

7.SRMEConc.(µg/ml) E. Coli S.Aureus P.auregenosa C.Albicans

50µg/ml 10.2±0.30 16.6±0.22 10.6±0.22 8.0±0.11

100µg/ml 10.6±0.11 14.2±0.30 11.4±0.30 8.4±0.05

200µg/ml 10.8±0.30 14.0±0.11 12.2±0.11 8.8±0.34

300µg/ml 10.6±0.20 15.0±0.32 12.6±0.05 9.6±0.22

400µg/ml 10.4±0.11 15.8±0.40 13.2±0.33 9.8±0.11

500µg/ml - 16.4±0.05 13.4±0.22 10.2±0.30

8.SRAEConc.(µg/ml) E. Coli S.Aureus P.auregenosa C.Albicans

50µg/ml - - 8.2±0.20 3.6±0.11

100µg/ml - - 8.4±0.11 4.2±0.05

200µg/ml - - 8.8±0.33 4.4±0.22

300µg/ml - - 9.6±0.14 5.2±0.30

400µg/ml - - 9.4±0.111 5.6±0.20

500µg/ml - - 9.2±0.05 6.2±0.11

140

aureus, E. coli, P. aeruginosa, and the fungus C. albicans; while our results showed that

the methanolic extract of P. guajava can inhibit the growth of S. aureus, E. coli, P.

aeruginosa, and the fungus C. albicans by disc diffusion method.

The mode of action of antibacterial effects of saponins seems to involve membranolytic

properties, rather than simply altering the surface tension of the extracellular medium.The

present study indicated that strong antibacterial activity exhibited by leaf extracts of

P.guajava & S.racemosa was possibly due to protein degrading activity of extracts.

Tannins known to be present in aqueous & methanolic extracts reportedly have protein

binding activities & can interfere with many substances.

6.1.12) Acute toxicity study:

SRME & PGME administered at a dose of 2000 mg/kg did not show any signs or

symptoms of toxicity or mortality during the observation period. The starting dose was

selected as 1/10th and 1/5th of 2 000 mg/kg. SRME & PGME administered at a dose of

2000 mg/kg did not show any signs or symptoms of toxicity or mortality during the

observation period. The starting dose was selected as 1/10th and 1/5th of 2000 mg/kg.

6.1.13) Determination of h. Pylori activity (in vitro): 92, 93

Graph representing zone of Inhibition (mm)/Drug concentration (mg/disc)

1. PGME

Figure 22. A): Zone of Inhibition (mm) by PGME

141

B) Zone of Inhibition (mm) by PGAE

3. SRME

C): Zone of Inhibition (mm) by SRME

4.SRAE

D): Zone of Inhibition (mm) by SRAE

142

Results revealed that 10% metahnolic extract of S. racemosa & P.guajava had significant

antimicrobial effect. Disc-diffusion method was used to determine the susceptibility of

H. pylori isolates to methanol extracts of both plant extracts. Extracts of Psidium guajava

& Symplocos racemosa showed dose dependent activity from 0.01mg/disc to 5.0mg/disc

against H.pylori in vitro. Among them, the metahnolic extracts of S.racemosa & P.

guajava had remarkable anti-H. pylori activity with a mean inhibition zone diameter of

22-70mm for 0.01-5.0mg/disc respectively. The bioflavonoids and phenolic compounds

present in the methanolic extracts may be responsible for such an activity.

6.1.14) Determination of h. Pylori activity (in vivo)

I) PCR Amplification of virulent and non-virulent genes of H. pylori: 161

Figure 23. A):Amplification of 16S rRNA Gene of H.Pylori

Figure showing the successful amplification of 534 base pair amplicon corresponding to

16S rRNA gene of H. pylori. Lane 1: 100 base pair ladder, Lane 2: Vehicle Control, Lane

3: PGME 100 mg/kg p.o. treated group, Lane 4: PGME 200 mg/kg p.o. treated group,

Lane 5: PGME 400 mg/kg p.o. treated group, Lane 6: CAO treated group, Lane 7: CAO +

PGME 400 mg/kg p.o. treated group Lane 8: Healthy control.

b) Gel image showing amplification of 16S rRNA gene of H. pylori

Figure 23 B): Amplification of 16S rRNA gene of H. pylori

143

Figure showing the successful amplification of 534 base pair amplicon corresponding to

16S rRNA gene of H. pylori. Lane 1: 100 base pair ladder, Lane 2: Vehicle Control, Lane

3: SRME 50 mg/kg p.o. treated group, Lane 4: SRME100 mg/kg p.o. treated group, Lane

5: SRME 200 mg/kg p.o. treated group, Lane 6: CAO treated group, Lane 7: CAO +

SRME 200 mg/kg p.o. treated group Lane 8: Healthy control.

II) The infection status in the CAO and SRME treated animals by PCR and Rapid

urease test (RUT)

Table 16. A): Infection status(SRME)

Sr.

No.

Week 16S rRNA status (PCR)

Vehicle

Control

SRME

(50mg/kg)

SRME

(100mg/kg)

SRME

(200

mg/kg)

SRME (200

mg/kg) +

CAO

CAO Healthy

Control

1 4 6/6 5/6 2/6 1/6 0/6 0/6 0/6

Sr.

No.

Week Infection status (RUT)

Vehicle

Control

SRME

(50mg/kg)

SRME

(100mg/kg)

SRME

(200

mg/kg)

SRME (200

mg/kg) +

CAO

CAO Healthy

Control

1 4 6/6 5/6 2/6 1/6 0/6 0/6 0/6

Sr.

No.

Week 16S rRNA status (PCR)

Vehicle

Control

SRME

(50mg/kg)

SRME

(100mg/kg)

SRME

(200

mg/kg)

SRME (200

mg/kg) +

CAO

CAO Healthy

Control

1 4 6/6 5/6 2/6 1/6 0/6 0/6 0/6

Sr.

No.

Week Infection status (RUT)

Vehicle

Control

SRME

(50mg/kg)

SRME

(100mg/kg)

SRME

(200

mg/kg)

SRME (200

mg/kg) +

CAO

CAO Healthy

Control

1 4 6/6 5/6 2/6 1/6 0/6 0/6 0/6

144

III) Determination of H. pylori infection status determination using polymerase chain

reaction 161

The gastric tissue of animals in the vehicle treated group six out of six (100%)

animals were detected positive for 16S rRNA gene of H. pylori at the end of treatment

period of 4 weeks.

In the SRME 50 mg/kg treated group having 6 rats (83.33%) were detected

positive for 16S rRNA gene at the end of treatment period of 4 weeks. In the SRME 100

mg/kg treated group, group two out of six (33.33%) animals were detected positive for all

the H. pylori genes at the end of treatment period of 4 weeks. In the SRME 200 mg/kg

treated group, group one out of six (16.66%) animals was detected positive 16 sRNA gene

of H. pylori at the end of treatment period of 4 weeks. In the animals treated with piperine

(SRME 200 mg/kg +CAO), (CAO) and healthy control group, group none of the animals

were detected positive for 16S rRNA gene of H. pylori at the end of treatment period of 4

weeks.

IV) Infection Status determination using rapid urease test

The gastric tissue of animals in the vehicle treated group six out of six (100%)

animals were detected positive for 16S rRNA gene of H. pylori at the end of treatment

period of 4 weeks.

In the SRME 50 mg/kg treated group having 6 rats (83.33%) were detected

positive for 16S rRNA gene at the end of treatment period of 4 weeks. In the SRME 100

mg/kg treated group, group two out of six (33.33%) animals were detected positive for all

the H. pylori genes at the end of treatment period of 4 weeks. In the SRME 200 mg/kg

treated group, group one out of six (16.66%) animals was detected positive 16 sRNA gene

of H. pylori at the end of treatment period of 4 weeks. In the animals treated with piperine

(SRME 200 mg/kg +CAO), (CAO) and healthy control group, group none of the animals

were detected positive for 16S rRNA gene of H. pylori at the end of treatment period of 4

weeks.

145

V) The infection status in the CAO and PGME treated animals by PCR and Rapid

urease test (RUT)

Table 16 B) : Infection status by PGME

Sr.

No.

Week 16S rRNA status (PCR)

Vehicle

Control

PGME

(100mg/kg)

PGME

(200mg/kg)

PGME

(400

mg/kg)

PGME (400

mg/kg) +

CAO

CAO Healthy

Control

1 4 6/6 5/6 2/6 1/6 0/6 0/6 0/6

Sr.

No.

Week Infection status (RUT)

Vehicle

Control

PGME

(100mg/kg)

PGME

(200mg/kg)

PGME

(400

mg/kg)

PGME (400

mg/kg) +

CAO

CAO Healthy

Control

1 4 6/6 5/6 2/6 1/6 0/6 0/6 0/6

Sr.

No.

Week 16S rRNA status (PCR)

Vehicle

Control

PGME

(100mg/kg)

PGME

(200mg/kg)

PGME

(400

mg/kg)

PGME (400

mg/kg) +

CAO

CAO Healthy

Control

1 4 6/6 5/6 2/6 1/6 0/6 0/6 0/6

Sr.

No.

Week Infection status (RUT)

Vehicle

Control

PGME

(100mg/kg)

PGME

(200mg/kg)

PGME

(400

mg/kg)

PGME (400

mg/kg) +

CAO

CAO Healthy

Control

1 4 6/6 5/6 2/6 1/6 0/6 0/6 0/6

VI) Determination of H. pylori infection status determination using polymerase chain

reaction

The gastric tissue of animals in the vehicle treated group six out of six (100%)

animals were detected positive for 16S rRNA gene of H. pylori at the end of treatment

period of 4 weeks.

146

In the PGME 100 mg/kg treated group having 6 rats (83.33%) were detected

positive for 16S rRNA gene at the end of treatment period of 4 weeks. In the PGME 200

mg/kg treated group, group two out of six (33.33%) animals were detected positive for all

the H. pylori genes at the end of treatment period of 4 weeks. In the PGME 400 mg/kg

treated group, group one out of six (16.66%) animals was detected positive 16 sRNA gene

of H. pylori at the end of treatment period of 4 weeks. In the animals treated with piperine

(PGME 400 mg/kg +CAO), (CAO) and healthy control group, group none of the animals

were detected positive for 16S rRNA gene of H. pylori at the end of treatment period of 4

weeks.

VII) Infection Status determination using rapid urease test

The gastric tissue of animals in the vehicle treated group six out of six (100%)

animals were detected positive for 16S rRNA gene of H. pylori at the end of treatment

period of 4 weeks.

In the PGME 100 mg/kg treated group having 6 rats (83.33%) were detected

positive for 16S rRNA gene at the end of treatment period of 4 weeks. In the PGME 200

mg/kg treated group, group two out of six (33.33%) animals were detected positive for all

the H. pylori genes at the end of treatment period of 4 weeks. In the PGME 400 mg/kg

treated group, group one out of six (16.66%) animals was detected positive 16 sRNA gene

of H. pylori at the end of treatment period of 4 weeks. In the animals treated with piperine

(PGME 400 mg/kg +CAO), (CAO) and healthy control group, group none of the animals

were detected positive for 16S rRNA gene of H. pylori at the end of treatment period of 4

weeks.

VIII) Rapid Urease test: Weighed quantity of the excised pyloric antrum tissue (50 mg)

was immersed in 5 mL RUT solution. The colour change was recorded. The colour

changed from yellow to pink in one hour if H. pylori was present in the pyloric antrum

tissue. Change in color of RUT solution from yellow to pink showing the presence of H.

pylori in the vehicle control group of animals.

147

Figure 24. : Rapid Urease Test

IX) Biochemical Estimations: Mitochondrial Estimations

A) Complex-I (NADH dehydrogenase activity)

Figure 25. A): Complex I estimation

The respiratory chain of Helicobacter pylori has been investigated. The total insensitivity

of activities of NADH dehydrogenase to rotenone and of NADH-cytochrome c reductase

to antimycin is indicative of the absence of the classical complex I of the electron transfer

chain in this bacterium. NADPH-dependent respiration was significantly stronger than

NADH-dependent respiration, indicating that this is a major respiratory electron donor in

H. pylori. PGME & SRME extracts exhibited a concentration-dependent inhibitory effect

148

on the activity of succinate dehydrogenase. The activity of succinate-cytochrome c

reductase was inhibited by antimycin, implying the presence of a classical pathway from

complex II to complex III in this bacterium. The presence of NADH-fumarate reductase

(FRD) was demonstrated in H. pylori and fumarate could reduce H2O2 production from

NADH, indicating fumarate to be an endogenous substrate for accepting electrons from

NADH.

B) Complex-II (succinate dehydrogenase (SDH) activity)

Figure 25 B): Complex II estimation

This enzyme is a key enzyme in the alternative respiratory chain under anaerobic

conditions. No other significant effects on the enzymes of the respiratory chain were

found. The synergistic effect of on COA & extracts showed more resistance against H.

pylori strains could be explained by the effect on fumarate reductase, whereas the effect

of omeprazole is different and could be an inhibition of a proton pump in H. pylori.

149

C) Complex-III (MTT ability)

Figure 25 C): Complex III estimation

D)Complex-IV(cytochromeoxidaseassay):

Figure 25 D): Complex IV estimation

The value of the mitochondrial complex III (No. of viable cells /well) was calculated for

each sample using the standard curve by extrapolation of the optical density values. H.

pylori is oxidase positive and has been reported to have menaquinone-6 and an

unidentified quinone as respiratory quinones. H.pylori lives in the mucous layer

overlying the gastric epithelium of humans, where the micro- environment is low in

150

oxygen because of rapid growth of the epithelium cells. H. pylori is an obligate aerobe

which does not grow in the presence of normal air-oxygen pressure nor anaerobically in

the absence of oxygen. H. pylori does not catabolize saccharides but derives energy from

in aerobic eubacteria, the cytochrome aa, or caa,-type cytochrome-c oxidase is very

similar to the mitochondria1 enzyme with respect to its chromophores. The results of

MTT ability revealed that extracts have shown dose dependent activity. 400mg/kg has

shown maximum response which is comparable to COA.There are a cytochrome be

oxidase & cytochrome c peroxide in the respiratory chain of bacteria, which was

inhibited by COA & extract combination.

E) Determination of DNA content 161

Figure 25 E): Estimation of DNA Content

Molecular methods such as PCR has the capability to sensitively and accurately

determine both the presence of infection and the genotype of bacteria. These techniques

have been used successfully to detect H. pylori DNA in gastric tissue by amplifying

genes such as the adhesin gene, the urease gene, and the 16S rRNA gene. The 16S rRNA

gene of H. pylori is a highly specific target for amplification and has been used

previously to help reclassify the organism. This study investigated the direct effects of

an H.pylori extract on gastric epithelial cell DNA synthesis, cell proliferation, ROS

151

formation and apoptotic DNA fragmentation. The effect of study reveals that extract

exhibited activity on DNA content (mU/mg).DNA content was found to be decreased as

dose of extract was increased. Maximum effect was observed in PGME (400mg/kg)

which is better than COA.

F) Calculation of ulcer area

The images were processed using Image J and Adobe photoshop softwares to determine

the ulcer area of the stomach. The software was calibrated at 95 pixels = 1 mm

Figure 25 F): Estimation of Ulcer area

152

6.1.15 Development of formulation: buccal patch

6.1.15.1 Preformulation studies:

A) Polymer characterization:

Name of Polymers: HPMC K15 & Carbapol

1. HPMC K15:

Description: Hypromellose is an odorless and tasteless, white or creamy-white

fibrous or granular powder

Physical Properties:

pH : 5.5.8.0 for a 1% w/w aqueous solution.

Ash value : 1.5.3.0%, depending upon the grade and viscosity.

Density (bulk) : 0.341 g/cm3

Density (tapped) : 0.557 g/cm3

Density (true) : 1.326 g/cm3

Melting point : Browns at 190.200°C; chars at 225.230°C.

Glass transition temperature is 170.180°C.

Solubility: Soluble in cold water, forming a viscous colloidal solution, practically

insoluble in chloroform, ethanol (95%), and ether, but soluble in mixtures of

ethanol / methanol and dichloromethane, and mixtures of water and alcohol.

Specific gravity: 1.26

3. Carbopol :

Description: Carbomers are white-colored, .fluffy. Acidic, hygroscopic powders

with a slight characteristic odor.

Physical Properties:

Acidity/alkalinity : pH = 2.7.3.5 for a 0.5% w/v aqueous dispersion; pH =

2.5.3.0 for a 1% w/v aqueous Dispersion.

Density (bulk) : 1.76.2.08 g/cm3

Density (tapped) : 1.4 g/cm3

De Glass transition temperature: 100.105°C

Melting point : Decomposition occurs within 30 minutes at 260°C.

Specific gravity : 1.41

153

Moisture content: Normal water content is up to 2% w/w. However, carbomers

are hygroscopic and typical equilibrium moisture content at 25°C and 50%

relative humidity is 8.10% w/w.

Solubility: Soluble in water and, after neutralization, in ethanol (95%) and

glycerin. Although they are described as soluble.Carbomers do not dissolve but

merely swell to a remarkable extent, since they are three-dimensionally

crosslinked microgels.

B) DSC Studies of Extracts with polymers:

Figure 26. A):Graph(Thermogram of pure PGME)

Figure 26 B): Graph (Thermogram of pure PGME, HPMC & carbopol)

154

Pure extract of PGME showed sharp peak at 150 C. Thermogram of PGME, HPMC &

carbopol did not show any changes in peak so both polymers can be considered to be

compatible with extract & can be used for making formulations

Figure 26C): Graph(Thermogram of pure SRME)

Figure 26 D): Graph (Thermogram of pure SRME, HPMC & carbopol)

Pure extract of SRME showed sharp peak at 140ο

C. Thermogram of SRME, HPMC &

carbopol did not show any changes in peak so both polymers can be considered to be

compatible with extract & can be used for making formulations

155

6.1.15.2 Preliminary formulation

A) Result of buccal patches containing PGME & SRME (Trial batches)

Table 17. :Evaluation of trial batches

Patch

code

Thickness Weight

uniformity

Surface

pH

Mucoadhesive

Strength

Mucoadhesion

Time (in min.)

Invitro

release (in

8 hrs)

B1 0.3±0.02 0.0156±0.23 6.85±0.01 9.35±0.35 178±0.24 81%

B2 0.4±0.01 0.0135±0.5 6.7±0.01 10.08±0.46 210±0.35 83%

B3 0.4±0.03 0.0143±0.34 6.6±0.02 11.35±0.23 223±0.46 84%

B4 0.26±0.02 0.0132±0.30 6.8±0.02 15.62±0.43 278±0.38 86%

B5 0.3±0.02 0.0075±0.55 6.7±0.01 23.4±0.50 310±0.25 89%

B6 0.28±0.03 0.0069±0.2 7.0±0.01 19.2±0.41 276±0.53 82%

1. Thickness uniformity & diameter: As the total amount of polymer increases the

thickness of the film were found to be increased. The thickness for formulation B-1 to B-

6 varied from to 0.28±0.02 to 0.4±mm

2. Swelling Index: The swelling of the patches were observed in phosphate buffer

solution (pH 6.8) and shown in table. Swelling was more pronounced in patches B4 and

B5 which contain HPMC and Carbopol in a ratio of (1.5:1) and (2:0.5) respectively.

Patches B2, and B6 showed less swelling (weight basis), may be due to the presence of

Eudragit RL 100 and ethyl cellulose, respectively. These results were in agreement with

the increase in area due to swelling. The results revealed that all the formulations provide

an acceptable swelling index in the range of formulation B-1 to B-6.

3. Mucoadhesion strength: As the amount of mucoadhesive polymer increases the

mucoadhesion was found to be increase. In formulation B-1 to B-6 four different polymer

was used in which Carbopol 934P have better mucoadhesion property than other so B-4

shows greater mucoadhesion strength (10.37 gm). Mucoadhesion strength of formulation

B-1 to B-6 varied from 9.35 gm to 23.4 gm

4. Mucoadhesive Time: In formulation B-1 to B-6 four different polymer was used in

which Carbopol 934P have better mucoadhesion property than other so B5 shows greater

mucoadhesion time 310 min than the formulation containing Eudragit RL-100 and ethyl

cellulose. Mucoadhesion time of formulation BP-1 to BP-6 varied from 178 minute to

310 minute.

156

5. In-vitro drug release study: The release data of Extracts from all the patches are

given (table). In case of formulation B-1 to B-6 the release data of Extract from all the

patches indicated that the drug release was higher in HPMC (patch B1) and HPMC-

Carbopol combinations (patches B4 and B5) at pH 6.8,An increase in the polymer

content was associated with a corresponding decrease in the drug-release rate B-5 shows

good in-vitro drug release.

The patches were found to be smooth in appearance, uniform in thickness, weight

uniformity, drug content, swelling behaviour, and surface pH. The B5 formulation

containing extract, HPMC: Carbopol (4:1), Glycerine, Acetone and Tween 80 showed a

release of 89% after 8hours in phosphate buffer (pH, 6.8). This formulation was further

optimized by varying % of HPMC k15 and carbopol and other variables and 9 new

formulations F1 to F9 were prepared & evaluated.

B) Optimization of Batch containing Extracts:

Table 18. :Optimization of formulation of buccal patch

Patch

code

Amount of

Drug (mg)

(SRME &

PGME)

Total

amount

of po-

lymer

(mg)

Amount of

HPMC

Amount of

carbopol

Amou

nt of

Tween

80(ml)

Amount

of gly-

cerin(ml)

Solvents

% Mg % mg Wat

er

Alcoh

ol

Acet

one

F1 100mg 150 100% 150mg 0% 0mg 0.1 0.1 1 10 0

F2 100mg 150 90% 135mg 10% 15mg 0.2 0.3 1.5 15 0

F3 100mg 150 80% 120mg 20% 30mg 0.3 0.5 2 20 0

F4 100mg 150 70% 105mg 30% 45mg 0.1 0.1 1 8 2

F5 100mg 150 60% 90mg 40% 60mg 0.2 0.3 1.5 11 4

F6 100mg 150 50% 75mg 50% 75mg 0.3 0.5 2 14 6

F7 100mg 150 40% 60mg 60% 90mg 0.1 0.1 1 7 3

F8 100mg 150 30% 45mg 70% 105mg 0.2 0.3 1.5 9 6

F9 100mg 150 20% 30mg 80% 120mg 0.3 0.5 2 11 9

157

6.1.16 Evaluation of mucoadesive herbal buccal patchs:

1. Physical properties: Physical appearance and surface texture of PGME & SRME

Buccal Patch:

Table 19. : Physical appearance and surface texture

Sr.No. Formulation Colour

PGME & SRME

Flexibility Surface

Texture

1 F1 Light green/Brown + smooth

2 F2 Light green/Brown + Smooth

3 F3 Light green/Brown + Smooth

4 F4 Light green/ Brown + Smooth

5 F5 Light green/Brown + Smooth

6 F6 Light green/Brown + Smooth

7 F7 Light green/Brown + Smooth

8 F8 Light green/Brown + Smooth

9 F9 Light green/Brown + Smooth

Figure 27. : Images of Buccal Patch(PGME & SRME)

158

2. Thickness uniformity and Diameter of PGME & SRME Buccal Patch:PGME

Table 20. :Thickness uniformity and Diameter

Formulation

Batch

(PGME)

Thickness

(mm)

Diameter

(cm)

Formulation

Bathch

(SRME)

Thickness

(mm)

Diameter

(cm)

F1 0.21±0.03 3.8±0.41 F1 0.24±0.02 3.9±0.43

F2 0.25±0.0143 3.5±0.13 F2 0.22±0.0152 3.8±0.11

F3 0.23±0.029 3.6±0.18 F3 0.26±0.026 3.5±0.15

F4 0.24±0.007 3.53±0.16 F4 0.253±0.009 3.6±0.09

F5 0.22±0.007 3.46±0.124 F5 0.25±0.007 3.6±0.124

F6 0.236±0.009 3.36±0.04 F6 0.24±0.009 3.6±0.163

F7 0.226±0.004 3.6±0.08 F7 0.23±0.011 3.5±0.124

F8 0.24±0.014 3.4±0.04 F8 0.25±0.002 3.60.081

F9 0.226±0.011 3.5±0.08 F9 0.233±0.002 3.4±0.169

Thickness of Buccal Patch (PGME) F1 to F9 varied from to 0.21±0.01 mm to

0.25±0.01mm whereas thickness of Buccal patch (SRME) F1 to F9 varied from

0.22±0.01mm to 0.26±0.02mm. Diameter of Buccal patch (PGME) F1 to F9 varied

from 3.36±0.04 cm to 3.8±0.41cm and diameter of buccal patch (SRME) F1 to F9

varied from 3.4±0.16 to 3.9±0.43.

159

3. Swelling Index of PGME & SRME Buccal Patch:

Table 21. A): Swelling Index in PGME Buccal patch

Formulation

(PGME)

Initial wt.(gm) Final wt.(gm) Avarage

Result%

F1 0.250±0.013 0.3375±0.02 35.00

F2 0.251±0.02 0.349±0.04 39.83

F3 0.252±0.002 0.377±0.034 49.71

F4 0.251±0.004 0.390±0.001 55.5

F5 0.251±0.002 0.423±0.023 68.81

F6 0.252±0.03 0.417±0.014 65.86

F7 0.252±0.008 0.372±0.03 47.72

F8 0.250±0.005 0.354±0.04 41.75

F9 0.250±0.045 0.341±0.03 36.72

Table 21 B): Swelling Index in SRME Buccal patch

Formulation

(SRME)

Initial wt.(gm) Final wt.(gm) Avarage

Result%

F1 0.250±0.21 0.339±0.032 35.77

F2 0.250±.44 0.349±0.002 39.74

F3 0.250±0.007 0.365±0.003 46.11

F4 0.251±0.21 0.385±0.001 53.21

F5 0.250±0.014 0.414±0.001 65.26

F6 0.251±0.11 0.394±0.004 56.67

F7 0.252±0.13 0.353±0.001 41.37

F8 0.250±0.20 0.347±0.002 39.0

F9 0.251±0.07 0.332±0.0009 32.48

Swelling index in Buccal patch (PGME) in F1 to F9 varied from 35.0% to 68.81%. The

best swelling index was found in Batch F5. Swelling index in Buccal patch (SRME) in F1

to F9 varied from 32.48% to 65.26%. The best result of swelling index was found in

Batch F5 where ratio of HPMC & Carbapol was kept 60%:40% (90mg & 60mg

respectively)

160

4. Surface pH of PGME & SRME Buccal Patch:

Table 22. :Surfae pH of SRME & PGME

Formulation Surface

pH

Average

pH(PGME)

Surface

pH

Avarage

pH(SRME)

F1 6.5 6.46±0.04 6.4 6.53±0.12

6.4 6.7

6.5 6.6

F2 6.4 6.63±0.20

6.9 6.7±0.16

6.9 6.7

6.6 6.5

F3 6.8 6.76±0.04

6.9 6.76±0.18

7.0 6.9

6.8 6.5

F4 6.5 6.66±0.16 6.5 6.63±0.12

6.9

6.6

6.8

6.6

F5 6.8 6.53±0.24

6.6 6.66±0.09

6.2 6.8

6.6 6.6

F6 7.0 6.96±0.04

6.7 6.73±0.04

7.0 6.8

6.9 6.7

F7 6.4 6.73±0.24 6.5 6.63±0.09

7.0 6.7

6.8 6.5

F8 7.0 7.0±0.08

7.0 6.9±0.08

6.9 6.9

7.1 6.8

F9 6.8 6.6±0.21

6.5 6.5±0.08

6.7 6.6

6.3 6.4

The results revealed that all the formulations provide an acceptable swelling index.An

acidic or alkaline formulation is bound to cause irritation on the mucosal membrane.

Surface pH of Buccal Patch (PGME) F1 to F9 varied from 6.46 ± 0.04 to 7.00 ± 0.08 &

buccal patch (SRME) F1to F9 varied from 6.5±0.08 to 6.9±0.08 (Table 26). Each sample

is analyzed in triplicate (n=3). The surface pH of all formulations was within ± 0.5 units

of the neutral pH and hence no mucosal irritation was expected and ultimately achieves

patient compliance.

161

5. Moisture uptake by PGME & SRME Buccal Patch:

Table 23. A): Moisture uptake by PGME Buccal patch

Formulation

(PGME)

Batch Initial

wt.(gm)

Final wt.(gm) Result% Average%

F1

a) 0.0164 0.0204 24.39 22.26

b) 0.0149 0.0179 20.13

F2 a) 0.0153 0.0186 21.56 22.31

b) 0.0156 0.0192 23.07

F3

a) 0.0128 0.0163 27.34 25.11

b) 0.0118 0.0145 22.88

F4 a) 0.0137 0.0176 28.46 28.57

b) 0.0122 0.0157 28.68

F5

a) 0.0148 0.0198 33.40 35.41

b) 0.0163 0.0224 37.42

F6 a) 0.0168 0.0237 41.07 39.22

b) 0.0154 0.0209 37.71

F7

a) 0.0108 0.0129 19.45 19.00

b) 0.0097 0.0115 18.55

F8 a) 0.0083 0.0097 16.86 17.2

b) 0.0098 0.0115 17.34

F9 a) 0.0083 0.0097 16.86 17.1

b) 0.0096 0.0114 18.75

Maximum moisture uptake was shown by F5 & F6 batch. Moisture uptake polymer ratio

of HPMC: Carbopol was kept 60%:40%.

162

Table 23 B): Moisture uptake by SRME Buccal patch

Formulation

(SRME)

Batch Initial

wt.(gm)

Final

wt.(gm)

Result% Average%

F1 a) 0.01668 0.0204 22.302 20.85

b) 0.0146 0.0174 19.17

F2 a) 0.0148 0.0182 22.97 20.975

b) 0.0158 0.0188 18.98

F3 a) 0.0132 0.0166 25.76 26.105

b) 0.0119 0.0147 23.52

F4 a) 0.0136 0.0174 27.94 27.57

b) 0.0125 0.0159 27.20

F5 a) 0.0150 0.0194 29.33 35.35

b) 0.0167 0.0221 41.37

F6 a) 0.0169 0.0238 40.82 38.055

b) 0.0156 0.0208 35.29

F7 a) 0.0106 0.0128 20.75 10.97

b) 0.0099 0.0118 19.19

F8 a) 0.0087 0.0099 13.89 15.61

b) 0.0098 0.0115 17.34

F9 a) 0.0091 0.0108 18.68 18.01

b) 0.0098 0.0115 17.34

Maximum moisture uptake was shown by F6 batch Moisture uptake polymer ratio of

HPMC: Carbopol was kept 50%:50%.

163

6. Folding endurance of PGME & SRME Buccal Patch:

Table 24. : Folding endurance of Buccal Patches

Formulation Batch

(PGME)

Folding Pass/Fail Batch

(SRME)

Folding

F1 a) 280 Fail 290 Fail

b) 303 Pass 304 Pass

F2 a) 249 Fail 248 Fail

b) 312 Pass 316 Pass

F3 a) 337 Pass 338 Pass

b) 315 Pass 319 Pass

F4 a) 328 Pass 325 Pass

b) 325 Pass 327 Pass

F5 a) 312 Pass 316 Pass

b) 324 Pass 326 Pass

F6 a) 315 Pass 316 Pass

b) 316 Pass 318 Pass

F7 a) 313 Pass 310 Pass

b) 290 Fail 288 Fail

F8 a) 304 Pass 308 Pass

b) 313 Pass 312 Pass

F9 a) 312 Pass 313 Pass

b) 310 Pass 315 Pass

As the amount of glycerin increases the folding endurance was found to be increases. The

folding endurance for all the formulation was found more than 300 times which was

satisfactory to reveal good film properties for all the formulation except F1, F2 &F7.

164

7. Uniformity weight of Patch of PGME & SRME Buccal Patch:

Table 25. : Uniformity weight of Buccal patches

Formulation

Batch

Uniformity weight(gm)

PGME

Uniformity weight(gm)

SRME

F1 0.250±0.013 0.250±.21

F2 0.251±0.02 0.250±.44

F3 0.252±0.002 0.250±0.007

F4 0.251±0.004 0.251±0.21

F5 0.251±0.002 0.250±0.014

F6 0.252±0.03 0.251±0.11

F7 0.252±0.008 0.252±0.13

F8 0.250±0.005 0.250±0.20

F9 0.250±0.045 0.251.4±0.07

Weight uniformity in buccal patch (PGME) F1 to F9 varied from 0.250±0.013 gm to

0.252±0.03gm. Similarly weight uniformity in buccal patch (SRME) F1 to F9 varied

from 0.250±0.014gm to 0.252±0.13gm.The patches were found uniform.

MECHANICAL PROPERTIES:

1) In-vitro Bioadhesion Studies of PGME & SRME Buccal Patch:

Table 26. : In-vitro Bioadhesion Studies

Formulation

Patch

PGME

Mucoadhesion

strength

weight(gm)

Formulation

Patch

SRME

Mucoadhesion

strength

weight(gm)

F1 9.981±0.58 F1 9.55±0.150

F2 9.65±0.22 F2 9.82±0.29

F3 20.22±0.35 F3 20.35±0.285

F4 16.51±0.822 F4 16.20±0.26

F5 22.63±0.61 F5 23.63±0.64

F6 19.60±0.35 F6 19.87±0.47

F7 8.79±0.11 F7 7.85±0.81

F8 8.09±0.266 F8 10.26±0.55

F9 6.76±0.134 F9 7.84±0.05

In formulation F1 to F9 mucoadhesion strength increases with increase in the amount of

HPMC so F5 shows greater mucoadhesion strength. Mucoadhesion strength of

165

formulation F1 to F9 varied from 6.76±0.134 gm to 22.63±0.61gm &from 7.84±0.05gm to

23.63±0.64gm for PGME & SRME respectively.

2) Drug Content of PGME & SRME Buccal Patch:

Table 27. A): max for Drug Content

Sr.

no.

Formulati

on

Batch

(PGME)

Observe

max

Average Batch

(SRME)

Observe

max

Average

1.

F1 a) 0.2381 0.2396 a) 0.1964 0.1966

b) 0.2412 b) 0.1968

F2 a) 0.1956 0.2143 a) 0.2004 0.1974

b) 0.2331 b) 0.1945

2.

F3 a) 0.2331 0.2360 a) 0.1898 0.1923

b) 0.2389 b) 0.1948

F4 a) 0.2429 0.2409 a) 0.1962 0.1951

b) 0.2474 b) 0.1940

3.

F5 a) 0.2287 0.2300 a) 0.1796 0.1895

b) 0.2314 b) 0.1995

F6 a) 0.2334 0.2346 a) 0.1896 0.1917

b) 0.2358 b) 0.1938

4.

F7 a) 0.2291 0.2318 a) 0.1689 0.1738

b) 0.2346 b) 0.1788

F8 a) 0.2301 0.2299 a) 0.1978 0.1968

b) 0.2297 b) 0.1958

5. F9 a) 0.2343 0.2323 a) 0.1962 0.1873

b) 0.2303 b) 0.1785

Table 27 B): Drug content in Buccal patches

:

The drug content varied from 89.60% to 94.70% in batches F1 to F9.

Patchcode % Drug content (SRME) %Drug content( PGME)

F1 90.32±2.18 92.13±2.45

F2 90.76±2.25 90.56±2.65

F3 90.71±1.57 89.60±1.70

F4 92.48±2.71 92.52±2.42

F5 93.08±2.25 93.50±2.35

F6 94.34±2.12 94.26±2.87

F7 94.48±1.35 94.70±3.35

F8 93.78±2.79 93.75±2.93

F9 93.07±2.40 93.61±2.20

166

OTHER PROPERTIES: In vitro release study of PGME & SRME Buccal Patch:

1) Drug release in percentage of Buccal patch SRME (F1-F9)

Table 28. A): % Invitro release in SRME Buccal Patch

Batch 0min 60min 120min 180min 240min 300min 360min 420min 480min

F1 0.00 20.33±

0.13

30.71±

0.530

44.28±0

.41

55.68±0.

14

66.46±0.

082

73.60±0.

11

75.71±

0.13

75.90±0.

42

F2 0.00 20.69±

0.291

32.34±

0.093

43.80±0

.12

54.80±

0.41

62.55±

0.166

74.82±

0.10

78.57±

0.18

78.55±0.

34

F3 0.00 18.49±

0.064

32.74±

0.038

45.68±0

.226

55.74±

0.19

66.72±

0.138

76.26±

0.07

84.51±

0.14

84.33±0.

23

F4 0.00 23.22±

0.39

36.63±

0.143

45.45±0

.20

55.38±

0.20

64.57±

0.188

75.54±

0.08

84.56±

2.22

84.23±1.

02

F5 0.00 22.545

±

0.102

34.34±

0.036

46.66±0

.244

59.31±

0.198

72.60±

0.35

85.18±

0.48

90.44±

0.12

92.12±0.

48

F6 0.00 20.53±

0.052

32.65±

0.053

44.44±0

.188

55.39±

0.11

68.56±

0.014

79.43±

0.15

86.53±

0.18

88.39±0.

26

F7 0.00 19.96±

0.22

32.45±

0.075

42.42±0

.19

54.86±

0.130

62.75±

0.14

72.48±0.

10

78.77±

0.15

81.45±0.

42

F8 0.00 18.75±

0.15

30.66±

0.143

40.62±

0.20

48.75±

0.098

60.46±

0.06

69.60±0.

08

76.45±

0.25

78.57±0.

38

F9 0.00 17.69±

0.23

25.67±

0.0949

35.90±

0.30

45.75±

0.09

57.45±

0.026

66.78±

0.05

75.55±

0.14

77.63±0.

70

In formulation F1 to F9 in-vitro drug release varied from 77.63±0.70% to 92.12±0.48% in

8 hours. In formulation F1-F3 which was made of HPMC alone gave faster drug release as

compared to which have HPMC in combination with Carbopol. Formulation F1-F3 release

167

of drug within 6 hours, while formulation F4-F9 showed uniform and sustain drug releases

drug in 8 hours as represented in graph.

2) Drug release in percentage of Buccal patch PGME(F1-F9)

Table 28 B): % In vitro release in PGME buccal patch

Batch 0

min

60

min

120

min

180

min

240

min

300

min

360

min

420

min

480

min

F1 0.00 18.4

1±

0.09

29.90

±

0.29

41.33

±

0.90

53.44

±

0.26

62.61

±

0.25

73.11±

0.2309

88

76.78±

0.26

76.81±

0.39

F2 0.00 18.7

±

0.31

29.99

±

0.36

41.92

±

0.67

54.66

±

0.31

64.34

±

0.47

75.74±

0.3987

76

80.27±

0.53

78.55±

0.27

F3 0.00 16.7

8±

0.33

30.86

±

0.39

42.15

±

0.50

55.53

±

0.39

66.44

±

0.37

76.42±

0.2212

59

81.35±

0.09

80.96±

0.52

F4 0.00 21.1

9±

0.54

34.96

±

0.50

42.26

±

0.87

55.26

±

0.16

68.27

±

0.21

77.71±

1.6186

48

83.31±

0.39

84.23±

0.43

F5 0.00 20.9

±

0.48

32.78

±

0.47

46.14

±

0.42

59.08

±

0.13

72.19

±

0.24

85.01±

0.4359

66

90.84±

0.30

92.09±

0.32

F6 0.00 22.5

9±

0.14

30.88

±

0.25

42.19

±

0.57

55.55

±

0.25

66.79

±

0.37

76.01±

0.5681

74

85.9±

0.40

84.23±

0.19

F7 0.00 18.8

8±

0.49

29.75

±

0.36

41.77

±

0.38

54.67

±

0.26

62.71

±

0.31

74.75±

0.1461

35

82.4±

0.24

82.27±

0.26

F8 0.00 18.3

±

0.31

29.99

±

0.38

41.95

±

0.65

55.59

±

0.18

60.41

±

0.81

73.45±

0.1798

77

79.41±

0.45

80.44±

0.36

F9 0.00 16.2

0±

0.69

27.32

±

0.58

40.47

±

0.37

54.05

±

0.34

58.66

±

0.44

71.28±

0.4450

47

75.87±

0.26

79.28±

0.23

168

In formulation F1 to F9 in-vitro drug release varied from76.81±0.39 to 92.09±0.32% in 8

hours. In formulation F1-F3 which was made of HPMC alone gave faster drug release as

compared to which have HPMC in combination with Caropol. Formulation F1-F3 release

of drug was found 76.81±0.39, 78.55±0.27 & 81.35±0.09% respectively within 6 hours,

while formulation F4-F9 showed uniform and sustain drug releases drug in 8 hours as

represented in graph.

Figure 28. A): Cumulative % Drug release in SRME(F1-F5)

Figure 28 B): Cumulative % Drug release in SRME(F6-F9)

-20

0

20

40

60

80

100

-100 0 100 200 300 400 500 600

%

R

e

l

e

a

s

e

Time in Minutes

% In vitro release in SRME Buccal Patch

F1

F2

F3

F4

F5

-20

0

20

40

60

80

100

-100 0 100 200 300 400 500 600

%

R

e

l

e

a

s

e

Time in MInutes

% In Vitro release in SRME buccal Patch

F6

F7

F8

F9

169

Figure 28 C): Cumulative % Drug release in PGME(F1-F5)

Figure 28 D): Cumulative % Drug release in PGME (F6-F9)

-20

0

20

40

60

80

100

-100 0 100 200 300 400 500 600

%

R

e

l

e

a

s

e

Time in Minutes

% in-vitro release in PGME Buccal patch

F1

F2

F3

F4

F5

-10

0

10

20

30

40

50

60

70

80

90

100

-100 0 100 200 300 400 500 600

%

R

e

l

e

a

s

e

Time in Minutes

% In-vitro release in PGME Buccal Patch

F6

F7

F8

F9

170

4. In-vitro Residence time (mucoadhesive time) of PGME & SRME Buccal

Patch:

Table 29. :Mucoadhesive time

Formulation PGME Patch(Time in

Minutes)

SRME Patch(Time in

Minutes)

F1 177.33±2.05

178±1.63

F2 208.66±2.86

206±1.33

F3 231.66±4.78

225.33±2.05

F4 252±3.26

247.33±2.49

F5 307.33±4.10

305±2.44

F6 299±2.94

301±2.94

F7

275.66±1.69

274±3.26

F8 236.66±2.49

242±1.63

F9 212.66±2.05

213.33±2.49

Mucoadhesion time of formulation F1 to F9 varied from 177.33±2.05min to

307.33±4.10min &178±1.63min to 305±2.44 min. (Table No.33A & B).

5.Short term stability studies : PGME & SRME optimized buccal patches showed no

significant change in the physical appearance, mucoadhesion time, mucoadhesive

strength, was determined at 0, 15, 30 and 45 days. Also showed no significant change at

room temperature and in stability chamber at 40±1ºC and 75 % relative humidity this

indicates that optimized formulations were stable.

171

6.1.17 Antimicrobial activity of buccal patches:

1. Zone of Inhibition: The glass petri plates were incubated 370C for 24 hours. The plates

were observed for the antibacterial activity zone of inhibition was measured.

Table 30. : Zone of Inhibition (mm)

Sr.No Bacteria/Fungi F1 F4 F5 F6

1 Escherichia coli 36 42 39 38

2 Pseudomonas aureginosa 35 36 33 35

3 Staphylococcus aureus 31 32 30 27

4 Bacillus subtilis 32 35 33 30

5 Candida albicans 28 34 34 33

6. Aspergillus niger 30 28 36 32

Figure 29. : A) Zone of inhibition of Formulation F3, F4, F 5&F 6; E.coli,

P.aureginosa.

Figure 29 B): Zone of inhibition of F3, F4, F 5&F 6 C.albicans and S.aureus.

172

Figure 29 C) : Zone of inhibition of Formulation F3, F4, F 5&F 6; Bacillus

subtilis.

2) Release of Extracts from the Patch

Release of extracts from the patches was evaluated by in vitro antimicrobial

pharmacodynamic protocol. An optimized patch was kept in flask containing 25ml of

sterile saline saliva solution and shaken throughout the experiment with a mechanical

shaker saliva solution and shaken throughout the experiment with a mechanical shaker.

About 1 ml of the solution was withdrawn at time intervals of 5, 10, 20, 30, 60 and 120

min. This was introduced into pre-bored holes or cups in brain heart infusion agar for

bacteria plates previously seeded with standardized inoculums of bacteria (S. aureus or P.

aeruginosa). This was left in the cup for 15 min at room temperature (to allow for pre

diffusion of extracts) and later incubated for 24 h at 37˚C. A fresh normal saline saliva

mixture was similarly introduced into appropriate cup as a negative control. The zone of

inhibition were later observed visually and measured.

3) Pre-extinction Time studies:

The optimized patch was introduced into a flat-bottomed flask containing 25 ml of sterile

saline-saliva solution and shaken for 30 min in a mechanical shaker. Using a sterile 2 ml

needle/syringe, 1 ml was sampled out in duplicate. While 1 ml was mixed with a reaction

mixture containing S. aureus. The same procedure has been carried out for P. aeruginosa.

A loop-full was taken from each of these mixtures and transferred to 5 ml of appropriate

recovery medium. The recovery media was incubated for 24 hr after which the tubes were

observed for signs and degree of microbial growth.

173

Significant kill, evidenced by growth reduction was witnessed with the test patch against

Staphylococcus aureus at the 15th

min, respectively against Pseudomonas aeruginosa.

The extinction times of test drug against Staphylococcus aureus and Pseudomonas

aeruginosa were predictably above 30 min. Antimicrobial studies showed that the drug

had good activity against Gram positive and Gram negative micro-organism.

Table 31. : Pre-extinction time

Time (min) of contact organism with drug

Microorganism 0 2 4 6 8 10 15 20 25 30

Staph aureus +++ +++ +++ +++ +++ +++ ++ ++ ++ +

P. aeruginosa +++ +++ +++ +++ +++ ++ ++ ++ + +

C.albicans +++ +++ +++ +++ +++ ++ ++ ++ + +

B.Subtillis +++ +++ +++ +++ +++ ++ ++ ++ + +

A.niger +++ +++ +++ +++ +++ ++ ++ ++ + +

+++: Highly turbid (showing growth), ++: Moderately turbid, +: Turbid

174

6.1.18.:In-situ gel formulation:

As mentioned in the experimental section, various procedural steps were followed to

formulate and develop the formulation. The In-Situ Oral Gel from polymer i.e; sodium

alginate (Floating System).The results of the various methodological steps are

summarized below.

6.1.18.1 Preformulation (Polymer Characterization): The formulation of In-situ Oral

Gel polymers used Sodium alginate and. The characterization of polymer is follows:

Sodium Alginate (Floating system):

1) Organoleptic properties: The sodium alginate was subjected to organoleptic

evaluation.

No. Parameters Observation 1. Colour: Yellowish 2. Odour: Gummy

3. Taste: Slightly bitter 4. Nature: Crystalline

2) Melting point: The melting point of the sodium alginate was found to be 310 0C. In

this step the polymer did not melted but its colour turned brown to black at this

temperature.

3) Solubility: The solubility of sodium alginate in different solvents was studied and is

depicted

Sr.No. Solvent Observation 1. Distilled water : Soluble 2. Methanol : Freely Soluble 3. Phosphate Buffer 6.8 pH: Freely soluble

4. Chloroform: Insoluble

4) Swelling index: Swelling index of sodium alginate was found to be 6.5

175

5) IR Spectra of sodium alginate

Figure 30. : IR Spectra of sodium alginate

Table 32. : IR Interpretation of Sodium Alginate

Wave number (cm-1

) Stretching

1020.16 C-H

1317.14 -C-O-C-

1598.7 -C=O

3206.2 -OH

Above interpretation of IR spectra confirms structure of sodium alginate.

6) Moisture Content: The moisture content of sodium alginate was found to be 2.04

7) Determination of Viscosity:

The viscosity of sodium alginate was found to be 15.33 m Pa.s. Higher viscosity denotes

greater swelling capacity with stronger mucaoadhesion property.

8) Determination of total Ash value: Total ash value of the sodium alginate was found

to be 8.4 %.

9) Loss on drying: Loss on drying of the sodium alginate was found to be 2.33 %.

176

10) DSC Studies:

Figure 31. A): Graph: Thermogram of pure PGME

Figure 31 B) Graph: Thermo gram of pure PGME, Sodium alginate, Calcium

chloride, Sod.citrate

Pure extract showed a single peak at 110 c which was unultered in the thermogram of

above polymer combination. Extract & polymers showed no interaction hence can be

considered compatible for preparation formulations

177

Figure 31 C): Graph: Thermogram of pure SRME

Figure 31 D): Thermo gram of pure SRME, Sodium alginate & Calcium

chloride, Sodium citrate

Pure extract showed a single peak at 150 c which was unaltered in the thermo gram of

above polymer combination. Extract was found to be compatible with polymer

combination.

178

6.1.18.2 Preliminary formulation:

Before actual formulation the preliminary study gives the correct selection of excipient

with their concentration. From the literature survey many trials are done but results of

trial are not enough to get the desired in-situ oral gel.

1)Trials for Floating System: The sodium alginate is used as gelling agent 0.25, 0.5, 1,

and 1.5 %with sodium citrate 0.25, and calcium chloride 0.05, 0.075 & 0.1%.

Table 33. A): Preliminary values of sodium alginate Floating system

Sr.

No. Viscosity

(Poise) Speed

(RPM) FSR

(%) Sheer

Stress(N/m2)

Shear

rate(1/sec) Temp

(C) Time

interval(mm:ss.t)

1. 2.063 5 5.5 137.49 66.66 31.4 00:10.2

2. 2.250 5 6.0 149.98 66.66 35.4 00:10.2

3. 0.713 5 1.9 47.50 66.66 36.8 00:10.2

4. 0.600 5 1.6 40.00 66.66 37.4 00:10.2

5. 0.600 5 1.6 40.00 66.66 37.7 00:10.2

Floating time of the formulation is instant in the 1.2pH buffer and viscosity is enough to

administer orally.

2)Preliminary trail batches

Table 33 B): Preliminary evaluation trail batches

Batch

No.

Conc. of

Sodium

alginate

pH Viscosity(cp)* Floating

time

Characteristic of

In situ Gel

J1 0.25 7.4 90±1 7hr 50min Gel is not formed

properly J2 0.25 7.4 92±2 7hr 30min

J3 0.25 7.3 91±3.2 8hr

J4 0.5 7.1 150±2.5 9hr 15min Gel formation

J5 0.5 7.1 153±4.6 9hr 15min

J6 0.5 7.2 155±1.72 8hr 45min

J7 1.0 7.0 238±2.5 8hr 35min Gel formation

J8 1.0 6.0 236±2.0 8hr 35min

J9 1.0 7.0 235±2.3 8hr 30min

J10 1.5 6.8 331±0.86 8hr 25min Gel formation

J11 1.5 6.7 332±1.2 8hr 30min

J12 1.5 6.8 331±1.0 8hr 45min

All batches prepared using 0.075% (w/v) Calcium Chloride & 0.25%(w/v) Sodium

citrate.* Mean± S.D.(n=3)

179

In preliminary trial batches of J1to J12 were prepared using different concentration of

sodium alginateto see the effect on viscosity of solution, drug content , pH & physical

properties of gel in simulated gastric fluid(pH1.2). The concentration of sodium alginate

was varied from 0.25%w/v to 1.5%w/v. In the batches J1 to J13 (0.25%w/v) improper

gelation was observed which leads the rapid flow of formulation. In addition, the time

required for gelation was very low. Batches J4 to J6 preparded using 0.5% w/v of sodium

alginate, the time required for gelation aws slightly better than batches J1 to J3, while in

batches J 7-J12 all the characterstics were good but, in the batches of J10 to J12 the

viscosity of the solutions were very high because of higher concentration of sodium

alginate which was difficult to pour while it was not observed in batches J7to J19. Thus it

was concluded thah 1% w/v sodium alginate was optimum concentration.The

concentration of Sodium citrate was constant in all the batches (0.25%) & observed no

significant effect. The floating ability of prepared formulation was evaluated in SGF pH

2.0. The time the formulation took to emerge on the medium surface (Floating lag time)

& time the formulation constantly floated on dissolution medium surface (duration of

floating) were evaluated. All the batches showed the floating lag time is less than 15 sec.

& duration of floating was more than 8 hrs. Formulation containing Calcium chloride

demonstrated excellent floating ability. Upon contact with an acid medium, gelation &

crosslinking by Ca++

ions occurred to provide a gel barrier at surface of the formulation.

On the basis of floating time and viscosity of in situ oral gel the concentration of different

polymers and excipients are selected for 32 factorial designs.

180

3) 32

Full factorial design layout:

Table 33 C: Evaluation of 32

Full factorial design layout

Batch

No. Variables levels in coded

form Viscosity*

(cp) Floating time

X1 X2

F1 -1 -1 98±1.8 7hr 34min

F2 -1 0 132±1.34 7hr 02min

F3 -1 +1 154±2.58 8hr

F4 0 -1 192±1.74 9hr 35min

F5 0 0 236±2.64 9hr 30 min

F6 0 +1 266±2.29 8hr 35min

F7 +1 -1 296±2.0 8hr

F8 +1 0 335±±1.0 8hr 45min

F9 +1 +1 365±2.4 8hr50min

Translations of coded levels in actual units

Variable levels Low(-1) Medium(0) High(+1)

Concentration of

Sodium alginate(X1) 0.5% 1% 1.5%

Concentration of

Calcium chloride(X2) 0.05% 0.075% 0.1%

All the batches contain 500mg of SRME & 500mg of PGME, duration of floating was

approximate 9hrs,viscosity was measured at 60rpm *Mean±SD(n=3)

6.1.19) Evaluation of formulations(In-situ gel) : 80,162

1) Physical appearance:

All the prepared sodium alginate based in situ gel of SRME & PGME were checked for

their clarity were found to be satisfactory.

The haziness that was observed after autoclaving (due to precipitation of Polymer

at elevated temperature) was found to disappear and the original clarity was regained

after overnight standing.

2) pH of formulation-

The pH of the formulations was found to be satisfactory and it was in the range of 6.7-

7.4.

181

Table 34. : pH of In-Situ gel

Floating

System

pH

(SRME)

pH

(PGME)

F1 6.9±0.02 6.8±0.04

F2 6.7±0.04 6.8±0.03

F3 7.4±0.03 7.3±0.04

F4 7.1±0.03 7.2±0.02

F5 7.0±0.02 7.1±0.01

F6 7.3±0.03 7.2±0.02

F7 6.8±0.01 6.8±0.02

F8 7.2±0.03 7.2±0.03

F9 7.1±0.05 7.0±0.04

In this range of pH SRME & PGME retains their activity. Therefore, pH adjustment is

not required by any other reagent.

3) Rheological properties: The viscosity two different formulation of in situ oral gel was

determined. It is the main parameter that determines the residence time in the GIT and it

satisfies in-situ formulation.

All formulations showed evidence of shear thinning behavior; with higher viscosities

being observed with the commercial suspension at all shear rates. The in-situ gel is of

lower viscosity than the suspension and should not present difficulties in swallowing.

Rheology of In- situ oral Gel:

The rheological properties of the sols are of importance in view of their proposed oral

administration. Rheology of the two formulations is found to be better as the shear stress

decreases with the steady flow rate. F1 shows that fast decrease in shear stress than the

shear strain, batch F3 and shows that steady decrease in the shear strain than shear strain.

4) In-Vitro Floating Ability:

Time taken by formulation to emerge on the medium surface (floating lag time) and time

for which formulation continuously floated (duration of floating). The released CO2 was

entrapped in gel network producing buoyant formulation and then calcium ion reacted

with Sodium alginate produced a cross linked 3-D gel network and swelled structure that

might further diffusion of CO2 and drug molecule and resulted in extended period of

floating and drug release respectively.

182

Table 35. : Floating time for sodium alginate in situ gel

Floating

System

Floating Lag

Time(Second)SRME

Floating

time(hrs)SRME

Floating Lag

Time(Second)PGME

Floating time

(hrs)PGME

F1 38±0.04 455.6±1.6 40±0.05 447±1

F2 62±0.05 424.5±0.5 65±0.06 433.5±1.5

F3 64±0.03 480±2 64±0.009 463±1

F4 69±0.05 570.5±1.5 70±0.08 572±10.5

F5 42±0.02 573±3 45±0.06 563.5±1.5

F6 48±0.04 512.5±0.5 48±0.08 518.5±1

F7 89±0.06 423.5±1.5 90±0.06 433±1

F8 62±0.02 553±1 65±0.02 535±0.5

F9 60±0.06 542.5±0.5 60±0.008 543.5±1

Floating Lag time for In-Situ gel varied from 38±0.04 to 89±0.06 seconds for SRME gel

& 40±0.05 to 90±0.06 seconds for PGME gel. Floating Time varied from 423±1.4

minutes to 573±3.0 minutes for SRME gel & 433.5±1.5 to 572±10.5 minutes for PGME

gel.

5) In Vitro Gelling Studies:

Gelling studies were carried out using 0.1N HCl, (pH 1.2). In these studies the gelling

capacity (extent and speed of gelation) for all formulations were determined. The in-situ

gel so formed should preserve its integrity without dissolving or eroding so as to localize

the drug at absorption site for extended duration. Gelation characteristic was assessed on

an ordinal scale ranging between - - - to +++. After ingestion, the liquid polymeric

solution should undergo a rapid sol-to-gel transition by means of ionic gelation.

In Vitro Gelling Studies

Table 36. : In-vitro gelation time

Floating

System Geling Studies

(SRME) Duration of Gelation

(SRME) GelingStudies

(PGME) Duration of Gelation

(PGME)

F1 +++ 9hr 32min +++ 9hr 38min

F2 + 8hr + 8hr 10min

F3 ++ 7hr 30min ++ 7hr 10min

F4 +++ 8hr 05min +++ 8hr 25min

F5 +++ 8hr 18min +++ 8hr 8min

F6 +++ 9hr 5min +++ 9hr 15min

F7 + 4hr 38min + 4hr 26min

F8 +++ 8hr 35min +++ 8hr 38min

F9 + 7hr 50 min ++ 8hr 40min

183

6)Drug content: This is one of significant necessity for any type of dosage form that

quantity of the drug present in the formulation should not deviate beyond certain

specified limits from the labelled amount.

Table 37. : Drug content in In-Situ gel

The drug content varied from 91.23% to 96.23% in batches F1 to F9.

7) In-vitro drug release: The effect of polymer concentration on in-vitro drug release

from insitu gels is significant decrease in the rate and extent of drug release was observed

with the increase in polymer concentration and is attributed to increase in the density of

the polymer. It can be judged from various release patterns of formulations. Role of

sodium alginate was primarily in formations of sol-gel phenomenon, but it also did affect

release from formulations to some extent.

Formulation

code

% Drug content

(SRME)

%Drug content

(PGME)

F1 91.23±2.01 92.33±2.45

F2 93.66±2.25 92.76±2.65

F3 94.71±1.97 94.80±1.50

F4 92.88±2.81 92.62±2.61

F5 95.08±2.25 95.50±2.15

F6 96.23±2.55 96.21±2.35

F7 94.28±1.25 94.80±2.25

F8 95.73±2.89 94.93±2.09

F9 95.03±2.50 95.60±2.10

184

In-vitro % drug release (SRME)

Table 38. A): Cumulative % drug release of in situ gel system

Formula

tion

(SRME)

0 hr 1hr 2hr 3hr 4hr 5hr 6hr 7hr 8hr

F1 0% 6.8±0

.32 14.9±0.

34 20.4±0.

41 31.8±

0.32 42.6±0.

12 51.2±0.

28 60.4±0.

36 68.5±0.

43

F2 0 8.2±0

.14

16.4±0.

33

22.6±0.

33

32.5±

0.43

44.8±0.

31

54.5±0.

34

64.2±0.

37

72.2±0.

24

F3 0 10.6±

0.39 18.5±0.

16 29.3±0.

42 40.2±

0.21 51.2±0.

33 60.3±0.

12 71.2±0.

19 76.5±0.

33

F4 0 12.3±

0.23

20.2±0.

26

32.8±0.

36

42.5±

0.23

53.6±0.

21

64.2±0.

23

73.5±0.

36

78.6±0.

24

F5 0 16.2±

0.19

24.8±0.

37

38.2±0.

44

48.0±

0.41

55.6±0.

28

66.8±0.

35

72.8±0.

36

80.2±0.

37

F6 0 14.2±

0.38 23.6±0.

42 35.8±0.

37 46.4±

0.21 53.1±0.

30 62.4±0.

12 70.6±0.

30 77.8±0.

46

F7 0 13.0±

0.35

22.4±0.

27

36.2±0.

51

43.8±

0.23

52.6±0.

42

60.8±0.

23

68.2±0.

27

74.4±0.

38

F8 0 13.4±

0.24 21.6±0.

36 35.3±0.

22 44.7±

0.32 50.9±0.

34 65.8±0.

32 69.6±0.

32 72.4±0.

25

F9 12.6±

0.35

20.2±0.

32

31.8±0.

34

42.8±

0.23

48.2±0.

33

56.4±0.

32

60.5±0.

38

65.4±0.

30

Figure 32. A):Graph representing % release (Series represents: F1-F5 batches)

-20

0

20

40

60

80

100

0hr 1hr 2hr 3hr 4hr 5hr 6hr 7hr 8hr

%

R

e

l

e

a

s

e

Time in hours

% in vitro release in SRME insitu gel

(SRME)

F1

F2

F3

F4

F5

185

Figure 32 B): Graph representing % release (Series represents: F6-F9 batches)

In-vitro % drug release (PGME)

Table 38 B): Cumulative % drug release of in situ gel system

Formula

tion

(PGME)

0hr 1hr 2hr 3hr 4hr 5hr 6hr 7hr 8hr

F1 0 8.8±0

.43 13.6±0.2

2 19.8±0.

24 30.6±0.

12 38.8±0.

50 48.5±0.

26 59.6±0.

29 66.6±0.

43

F2 0 9.4±0

.26 16.2±0.3

1 22.5±0.

21 36.4±0.

32 42.5±0.

26 50.6±0.

31 62.8±0.

37 68.8±0.

11

F3 0 11.5±

0.18

19.2±0.0

9

30.6±0.

34

41.2±0.

22

50.1±0.

31

56.8±0.

12

65.3±0.

42

70.2±0.

27

F4 0 14.6±

0.37 21.0±0.1

2 31.8±0.

23 40.6±0.

43 51.6±0.

47 59.0±0.

18 67.5± 73.8±0.

34

F5 0 16.8±

0.29 23.9±0.2

1 37.6±0.

51 48.2±0.

09 54.5±0.

35 62.8±0.

28 68.5±0.

37 76.0±0.

43

F6 0 15.2±

0.24

22.4±0.1

9

35.8±0.

33

46.2±0.

23

53.2±0.

27

61.0±0.

18

66.5±0.

26

72.6±0.

34

F7 0 13.8±

0.35 22.4±0.3

6 36.8±0.

23 44.8±0.

34 50.0±0.

39 58.8±0.

31 64.8±0.

45 70.2±0.

21

F8 0 12.4±

0.21 21.6±0.3

3 34.2±0.

27 42.6±0.

22 47.6±0.

29 52.6±0.

34 59.2±0.

53 64.3±0.

23

F9 0 10.8±

0.32 20.2±00.

08 32.5±0.

45 38.2±0.

32 43.5±0.

34 48.2±0.

29 56.2±0.

32 62.8±0.

42

-10 0

10 20 30 40 50 60 70 80 90

0hr 1hr 2hr 3hr 4hr 5hr 6hr 7hr 8hr

%

R

e

l

e

a

s

e

Time in Hours

% in vitro release in SRME in situ Gel

(SRME)

F6

F7

F8

F9

186

Figure 32 C): Graph representing % release (Series represents: F1-F5 batches)

Figure 32 D): Graph representing % release (Series represents:F-F9 batches)

The release of SRME & PGME from floating in-situ gel was analyzed by plotting %

cumulative drug relesse against time (in Hours). The effect of Sodium alginate

concentration was shown in graph. The significant variation in the rate & extent of drug

release was observed. From F1to F3 gel could release only 66% to 70% of drug content

where the concentration of sodium alginate was 0.5% concentration of Calcium chloride

was 0.05%. F7 to F9 also showed less release with concentration of sodium alginate 1.5%

& concentration of calcium chloride was 0.1%. F4 to F6 showed better release with

-10

0

10

20

30

40

50

60

70

80

0 hr 1hr 2hr 3hr 4hr 5hr 6hr 7hr 8hr

%

R

e

l

e

a

s

e

Time in Hours

% in Vitro release in PGME in situ Gel

(PGME)

F1

F2

F3

F4

F5

-10

0

10

20

30

40

50

60

70

80

0 hr 1hr 2hr 3hr 4hr 5hr 6hr 7hr 8hr

%

R

e

l

e

a

s

e

Time in Hours

% in vitro release in PGME Insitu Gel

(PGME)

F6

F7

F8

F9

187

concentration of sodium alginate 1% & concentration of Calcium chloride was 0.075%.

Batch F5 showed maximum release in both Formulations (SRME 80.2% & PGME 76%

respectively)

8) Water Uptake Studies: Release of the drug from the polymer matrix depends on the

amount of water associated with the system. The release of the drug may involve the

penetration of water into the matrix and simultaneously release of the drug via dissolution

(Table 39 A and 39 B).

188

Table 39. A) : Water uptake studies in SRME In-Situ gel

Formulation

Code(SRME)

Initial

Weight

(gm)

Time

(Hours) % Water

Gain

F1 48.4 0 0

0.5 5.893

1 5.965

1.5 6.230

F2 41.7 0 0

0.5 4.484

1 4.698

1.5 4.963

F3 53.8 0 0

0.5 6.183

1 6.234

1.5 6.576

F4 48.2 0 0

0.5 5.846

1 5.974

1.5 6.306

F5 42.8 0 0

0.5 4.907

1 4.946

1.5 5.201

F6 44.9 0 0

0.5 5.173

1 5.367

1.5 6.783

F7 50.2 0 0

0.5 5.587

1 5.897

1.5 6.297

F8 47.8 0 0

0.5 4.820

1 4.925

1.5 5.298

F9 46.4 0 0

0.5 4.832

1 4.984

1.5 5.310

System shows first increase in the water gain then it is maintained throughout system

which is good to release the drug after penetration in to the gel.

189

Table 39 B) : Water uptake studies in PGME In-Situ gel

Formulation

Code(PGME)

Initial

Weight

(gm)

Time

(Hours) % Water

Gain

F1 48.7 0 0

0.5 4.658

1 4.865

1.5 5.206

F2 41.8 0 0

0.5 3.978

1 4.260

1.5 4.371

F3 54.2 0 0

0.5 5.801

1 6.288

1.5 6.484

F4 43.8 0 0

0.5 4.198

1 4.274

1.5 4.573

F5 44.4 0 0

0.5 4.251

1 4.431

1.5 4.788

F6 46.3 0 0

0.5 3.891

1 4.277

1.5 4.581

F7 51.4 0 0

0.5 5.478

1 5.843

1.5 6.405

F8 49.2 0 0

0.5 5.430

1 5.501

1.5 5.691

F9 50.5 0 0

0.5 5.440

1 5.568

1.5 5.741

System shows first increase in the water gain then it is maintained throughout system

which is good to release the drug after penetration in to the gel.

190

9) In vitro Bioadhesion test: The force required to detach the formulations from the

surface of tissue was determined. The bioadhesive strength of F5 formulation was found

to be 0.5N, pointing to good bioadhesion. In-vitro bioadhesion test showed that SRME &

PGME In situ gel adhered more strongly to gastric mucous layer & could retain in GIT

for an extended period.

6.1.20 Optimization by design expert software: The previously evaluated batches

optimized with the help of Design Expert software the floating system optimized as

following. The concentration of sodium alginate and other excipient in In-Situ Oral Gel

and its response was analyzed by optimization.

Factorial design & responses (SRME)

Table 40. A) : Factorial design & responses(SRME)

Sr.no Formulation

code(SRME)

Actual Units

Response 1

Floating

Time

Response 2

%drug

Release X1 X2 X1(gm)

w/v

X2

(gm)w/v

1 F1 -1 -1 0.5 0.05 454 68.5

2 F2 -1 0 0.5 0.075 422 72.2

3 F3 -1 +1 0.5 0.1 480 76.2

4 F4 0 -1 1 0.05 575 78.6

5 F5 0 0 1 0.075 570 80.2

6 F6 0 +1 1 0.1 510 77.8

7 F7 +1 -1 1.5 0.05 420 74.4

8 F8 +1 0 1.5 0.075 550 72.4

9 F9 +1 +1 1.5 0.1 540 65.4

191

A) Floating Time: SRME (3D graph)

Figure 33. A): 3D graph for Floating time (SRME)

B) Floating time (Predicted vs. Actual)

Figure 33 B): Predicted Vs Actual graph for floating time (SRME)

192

C) In-vitro % release (SRME)

Figure 33 C): 3D graph for % Invitro release for SRME

D) In vitro % release (Predicted vs. Actual)

Figure 33 D): Predicted Vs Actual graph In vitro % release (SRME)

193

From the 3D graph it can be interpreted that % in vitro release & floating time responses

were found to be different for different formulations. The range for responses for % in

vitro release was obtained 65.4% (lowest) & 80.2% (Highest). Floating time is varying

for all formulations. Maximum floating time was observed as 540 for F9, 550 for F8, 570

for F5, & highest 575 mins for F4 respectively.

From the predicted vs. actual graph for floating time various points were observed near

line .The nearest point(red) showing response at 575 minutes for F4 batch.% in-vitro

release from predicted vs. actual graph nearest point (red) showing 80.2% release. From

both graphs revealed that batch no. F4 & F5 have shown better response for both factors.

But % in vitro release was found to be best for F5 batch hence batch F5 (SRME) having

concentration 1% sodium alginate & 0.075% calcium chloride can be considered for

optimization for further evaluation.

Factorial design & responses (PGME)

Table 40 B): Factorial design & responses (PGME)

Sr.no Formulation

code(PGME)

Actual Units

Response 1

Floating Time

Response 2

%drug

Release X1 X2 X1(gm)

w/v

X2

(gm)w/v

1 F1 -1 -1 0.5 0.05 455 66.6

2 F2 -1 0 0.5 0.075 422 70.2

3 F3 -1 +1 0.5 0.1 445 66.6

4 F4 0 -1 1 0.05 570 73.2

5 F5 0 0 1 0.075 560 76.0

6 F6 0 +1 1 0.1 515 72.6

7 F7 +1 -1 1.5 0.05 430 70.2

8 F8 +1 0 1.5 0.075 535 64.3

9 F9 +1 +1 1.5 0.1 540 73.8

194

A) Floating time 3D graph (PGME)

Figure 34. A) : 3D graph for floating time ( PGME)

B) Floating time (Predicted vs. Actual)

Figure 34 B): predicted Vs actual graph for floating time(PGME)

195

C) In-vitro % Drug release (PGME)

Figure 34C): 3D graph for % in vitro drug release (PGME)

D) In-vitro % Drug release (Predicted vs. actual)

Figure 34 D): Predicted Vs actual graph for % in vitro drug release (PGME)

196

From the 3D graph it can be interpreted that % in vitro release & floating time responses

were found to be different for different formulations. The range for responses for % in

vitro release was obtained 64.3% (lowest) 76% & (Highest). Floating time is varying for

all formulations. Maximum floating time was observed as 535 for F8, 540 for F9, 560 for

F5 highest 570 minutes for F4 respectively.

From the predicted vs. actual graph for floating time various points were observed near

line .The nearest point(red) showing response at 570 minutes for F4 batch.% in-vitro

release from predicted vs. actual graph nearest point (red) showing 76% release. From

both graphs revealed that batch no. F4 & F5 have shown better response for both factors.

But % in vitro release was found to be best for F5 batch hence batch F5 (PGME) having

concentration 1% sodium alginate & 0.075% calcium chloride can be considered for

optimization for further evaluation.

Optimized response from floating system:

Table 41. : Optimized Response of Floating system

Formulation Response Experimental value Predicted value

SRME(F5) Floating time 570min 563min

PGME(F5) 560min 558min

SRME(F5) % in vitro-release 80.2% 79.8%

PGME(F5) 76% 74.35%

6.1.21 Stability studies:

The stability studies were carried out on the optimized formulation as per ICH guidelines.

The accelerated stability studies were performed on Optimized formulations i.e. F5. The

results indicated that these formulations remained stable for a period of 6 months.

I) Appearance: The In-Situ Oral Gel was evaluated for appearance and no significant

changes were observed during the stability testing period.

197

II) % Drug Release: The optimized batch was evaluated for in vitro dissolution study up

to 8 hrs and showed slight decrease in drug release after 3 months of period.

Table 42. A): Drug Release (%)

Formulation

Code

Drug Release (%) after 8 hrs.

Day 1 2 Months 4Months 6Months

F5(SRME) 80.2±0.22 80 ±0.12 79.58±0.18 78.21±0.19

F5(PGME) 76.2±0.17 71.08±0.15 71.92±0.13 70.21±0.19

III) Floating time of F5 (SRME) and F5 (PGME) batch during stability

Table 42 B) : In-Vitro Gelation during stability

Formulation

Code

In-Vitro gelation (in Minutes)

Day 1 2Month 4Months 6Months

F5(SRME) 570min 570 min 568min 567min

F5(PGME) 560min 558min 555min 552min

The floating time of F5 (SRME & PGME) batch from floating system is found to be

initially maintained with respect to the duration.

After the series of experimental steps, it is finally concluded that;

The floating system in-situ formulation exhibited well viscosity and in-vitro

gelation.

The results of a 32

factorial design found that the concentration of sodium alginate

significantly affected the dependent variables in-vitro gelation, floating time and

drug release.

Floating system of sodium alginate and cross-linking agent calcium chloride shows

the enough viscosity of sols which are easy for oral administration.

Floating system easily float on the gastric medium that ensures retention of gel in

the acidic medium stomach.

Gel formed by the floating system totally release drug in the acidic medium and it

is also observed that gel float on the medium through the drug release study.

198

6.1.22 Determination of antiulcer activity

6.1.22.1 Pyloric Ligation Method:

I) Observations: The number of ulcers was counted by using magnifying glass.Severity

score was observed as under:

Normal ulceration : 0

Red ulceration : 0.5

Spot ulceration : 1.0

Hemorrhagic stress : 1.5

Deep Ulcer : 2.0

Perforation : 3.0

Table 43. A) : Observations of pyloric ligation methods

Sr.

no.

Treatment Dose

mg/kg

Normal

stomach

Red

Coloration

Spot

ulcer

Hemorrhagic

streak

Ulcer Perforation

1 Normal

contorl

1ml/an

imal

------- +++ +++ ++++ +++ ++

2 Formulation

I(SRME)

300mg -- + + ---- ---- ---

3 Formulation

II(PGME)

300mg -- + + ------ ----- ----

4 Standard

(OMP)

20mg +++ ---- --- ----- ----- ------

Ulcer index = (UN+US+UP) * 10 -1

UN: Avarage no. of ulcer per animal

US: Average no. of severity score

UP: % of animal with ulcer

Observations from above table revealed that formulations made of SRME & PGME

showed good response against all symptoms. Only red colouration & spot ulcers were

observed but the intensity was very less. Other symptoms wers not observed. All

observations were comparable with standard drug.

199

II) Effect of Formulations (SRME & PGME) on various parameters in pyloric

ligation induced gastric ulcers: 163

Table 43 B): Evaluation of parameters in pyloric ligation method

Group Ulcer

index

%Protection pH of gastric

fluid

Gastric juice in

ml

Normal control 4.5±1.5 0.00 2.45±0.02 2.2±0.1

Formulation 1(SRME) 1.9±0.09 65 3.15±0.04** 3.05±0.06**

Formulation 2(PGME) 1.5±0.08 60 2.8±12** 2.82±.08*

Formulation 3(No extract) 4.3±1.05 25 2.45±0.05 2.3±.05

Standard(Omeprazole) 1.2±0.08 75 3.35±0.05** 3.45±.04**

Values are mean ± SEM for six animals in each group.

*P< 0.05 considered statistically significant as compared with control group.

** P< 0.01 considered statistically significant as compared with control group.

In pyloric ligation model, the Formulation 1 and Formulation 2 showed significant

(p<0.01) rise in pH as compared to control. The free acidity gastric content is increased in

control animals. The Formulation 1 & 2 both showed significant (p<0.01) decrease in free

acidity as compared to control. Both Formulations significantly reduced the total acidity

and ulcer index (p<0.001) as compared to control (Table 1).The percentage protection of

Formulation 1 & 2 was found to be 65% and 60% respectively whereas the percentage

protection of omeprazole was found to be 75%.

6.1.22.2 Ethanol induced Ulcer model:

The administration of Formulation 1SRME, Formulation 2 PGME (500 mg/kg body

weight) causes a decrease in ulcer index of ethanolic induced ulceration in the stomach of

Wistar rats. Ethanol induced gastric ulcer was employed to study the ulceroprotective

effect of both formulations.

Ethanol induced gastric lesion formation may be due to stasis in gastric blood flow which

contributes to the development of the haemorrhage and necrotic aspects of tissue injury.

Alcohol rapidly penetrates the gastric mucosa apparently causing cell and plasma

200

membrane damage leading to increased intracellular membrane permeability to sodium

and water. The massive

Intra cellular accumulation of calcium represents a major step in the pathogenesis of

gastric mucosal injury. This leads to cell death and exfoliation in the surface epithelium.

In ethanol induced gastric ulcer, on induction of gastric ulceration by using ethanol (96%,

v/v), the pretreatment with both formulations showed a reduction in the severity of the

lesions.

III) Effect of Formulations (SRME & PGME) on ethanol induced gastric ulcers: 152,163

Table 44. A) : Observationsof different parameters in ethanol induced method

Sr. no Hyperemia

and

hemorrhages

Edema Necrosis Leucocytic

infiltration

Ulceration

Protcetion

Normal

control

00 00 00 00 00

Formulation

1

+++ ++ ++ ++ +++

Formulation

2

++++ ++++ +++ +++ ++++

Formulation

3

+ + + + +

Standard ++ +++ ++ +++ ++

0: no changes detected

+: active changes up to less than 25 %

++: active changes up to less than 50 %

+++: active changes up to less 75 %

++++: active changes up to more than 75 %

201

A) Morphological investigation

Ethanol (50%) induced gastric damage showed marked gross mucosal lesion, including

long hemorrhage bands and petechial lesion. On gross examination these hemorrhagic

bands were characterized by different sizes along the longitudinal axis of the glandular

part of stomach (Fig 1A). Animals pretreated with formulations 1 & showed very mild

lesions with interstitial hemorrhage and sometimes no lesion at all.

B) Investigation of gastric lesions

Both formulations significantly increased macroscopic curative ratio compared with

control groups (Table 3). Morphometric evaluation was also carried out to evaluate the

extent of ulcer. The ulcer index was significantly (P<0.01) reduced in animals pretreated

with Formulatios compared to distilled water and Omaprazole treated rats.

C) Biochemical investigation

Moreover, SRME & PGME significantly reduced the gastric ulcer in ethanol induced

gastric ulcer model by confirmation of significant decreases (P<0.05) in acid volume and

increases (P<0.05) in pH when compared with (P<0.05) (Table-2) ethanol treated group.

D) Histopathological investigation

On microscopic examination, ethanol treated rats showed mucosal hemorrhage,

segmental mucosal necrosis of gastric epithelium, edema and ample infiltration of

leukocytes in submucosa .Only patchy mucosal epithelial loss was seen in pre-treated

rats.

Table 44 B): Evaluation parameters for ethanol induced method

Group Ulcer index % Protection

Normal control 76±1.08 0.00

Formulation 1 19.50±.05** 68

Formulation 2 15.75±.02** 63

Formulation 3 70.45±03 10.00

Standard(Omeprazole) 22.70±.09** 75

Values are mean ±SEM for six animals in each group. **

202

From the selected formulation (F5) of sodium alginate based insitu gel of SRME &

PGME “Pyrolus legation method in rats”was used for in vivo study and gel formation

was also checked in collected gastric juice from the rats.

In the present study, the control group treated orally with ethanol produced the expected

ulceration. Pretreatment at the dose of 500mg/kg, p.o and 500mg/kg, p.o with

formulation containing methanolic extracts of significantly (p<0.01) decreased the ulcer

index when compared with control rats. These results indicate that PGME & SRME

extract displays an antiulcerogenic effect related to cytoprotective activity, since it

significantly reduced the ethanol induced ulcer. In histopathological examination of

stomach mucosa ethanol treated group shows the ulcerated mucosa with haemorrhage

and discontinuity of lining of epithelium.Pretreatment with SRME & PGME (500mg/kg,

p.o. and 500mg/kg, p.o.) protected the mucosal epithelial from the damage caused by

ethanol.The antioxidant activity of flavonoids has been well documented in the literature.

Moreover, flavonoids have been reported for their antiulcerogenic activity and gastro

protection already.It has been also reported that flavonoids like Quercetin seem to play a

very important role in the prevention and treatment of peptic ulcer. It acts by promoting

mucus secretion, thereby serves as gastroprotective agent.

Histopathological investigation

Figure 35. A): Normal stomach 10X

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Figure 35 B): Normal stomach 40X

Figure 35 C): Standard 10X : Photograph showing necrosis (black arrow),

hyperemia and hemorrhages (red arrow), leucocytic infiltration (blue arrow),

ulceration (orange arrow) and edema (yellow arrow)H&E stain 100X

Figure 35 D): Standard 40X : Photograph showing necrosis (black arrow),

hyperemia and hemorrhages (red arrow), leucocytic infiltration (blue arrow),

ulceration (orange arrow) and edema (yellow arrow)H&E stain 400X

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Figure 35E):Formulation 1(SRME) 10X : Photograph showing necrosis (black

arrow), hyperemia and hemorrhages (red arrow), leucocytic infiltration (blue

arrow), ulceration (orange arrow) and edema (yellow arrow) H&E stain 100X

Figure 35 F): Formulation 1(SRME) 40X : Photograph showing necrosis (black

arrow), hyperemia and hemorrhages (red arrow), leucocytic infiltration (blue

arrow), ulceration (orange arrow) and edema (yellow arrow) H&E stain 400X

Figure 35G): Formulation 2(PGME) 10X : Photograph showing necrosis (black

arrow), hyperemia and hemorrhages (red arrow), leucocytic infiltration (blue

arrow), ulceration (orange arrow) and edema (yellow arrow) H&E stain 100X

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Figure 35 H): Formulation 2(PGME) 40X : Photograph showing necrosis (black

arrow), hyperemia and hemorrhages (red arrow), leucocytic infiltration (blue

arrow), ulceration (orange arrow) and edema (yellow arrow) H&E stain 100X

Figure 35 I): (Normal Control) 10X : Photograph showing necrosis (black

arrow), hyperemia and hemorrhages (red arrow), leucocytic infiltration (blue

arrow), ulceration (orange arrow) and edema (yellow arrow) H&E stain 100X

Figure 35J): Normal Control 40X: Photograph showing necrosis (black arrow),

hyperemia and hemorrhages (red arrow), leucocytic infiltration (blue arrow),

ulceration (orange arrow) and edema (yellow arrow) H&E stain 100X

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Figure 35 K): ControlFormulation 3 10X: Photograph showing necrosis (black

arrow), hyperemia and hemorrhages (red arrow), leucocytic infiltration (blue

arrow), ulceration (orange arrow) and edema (yellow arrow) H&E stain 100X

Figure 35 L): Control Formulation 3 40X: Photograph showing necrosis (black

arrow), hyperemia and hemorrhages (red arrow), leucocytic infiltration (blue arrow),

ulceration (orange arrow) and edema (yellow arrow) H&E stain 100X

Toxicity studies of SRME & PGME carried out in rats indicate no lethal effect at

least up to an oral dose of 4.0 g/kg for 14 days indicating that LD50 of PGME &

SRME will be higher than that dose. The studies of extracts on hematological

parameters are close to or within the normal range suggested that there are no

adverse effects. Induced gastric ulcers and was comparable to the reference drug

OMP.

The finding of present study demonstrated that Formulations prepared by

methanolic extract of Symplocos racemosa & Psidium guavaza significantly

protected against mucosal damage induced by ethanol (50%) and were found 68%

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& 63%respectively. It is remarkable that these doses produced a greater protection

than omeprazole (20 mg/kg) against the ethanol.

Narcotizing agents such as ethanol, when given intragastrically to rats produce

severe gastric hemorrhagic erosions. Ethanol induced both long ulcers and

petechial lesions with-in a short time, which makes this technique suitable for

screening experiments for investigation of antiulcer drugs. The genesis of ethanol-

induced gastric lesion is of multifactorial origin with the decrease in gastric mucus

amount also it is associated with significant production of free radicals leading to

increased lipid peroxidation which inturn causes damage to cell and cell

membranes .

There was decrease in gastric volume and reduction in free and total acidity in the

animals treated with both formulations and was found to be devoid of ulcerogenic

potential. The above discussion shows that, the herbal formulation is said to

produce beneficial antiulcer activity

In the microscopic observation of EtOH-induced rat, damaged mucosal epithelium,

glands, inflammatory exudates, proliferated fibroblasts, mixed leucocytic infiltrate

and cellular debris was found in ulcerated wall of the stomach. Protection against

these histopathological changes was observed by apparent epithelializations,

glandular organization, regeneration of mucosa and reduced size of ulcer crater in

PGME & SRME pretreated rats .In the present study we have checked the

protective effects on gastric ulcer models, ulcer healing and antisecretory property

of Psidium guajava & Symplocos racemosa, which is very important plant in

herbal medicine practice.

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CHAPTER 7: SUMMARY & CONCLUSION:

Mouth ulcers are small, painful sores on the inside lining of the mouth. They usually

develop on the inside of the lips and cheeks and on the underneath and edge of the

tongue. Mouth ulcers include lesions, sores, laceration, abrasions, or any open break in

the mucosa of the mouth, lips or tongue. This condition may also be called stomatitis and

could be a symptom of a variety of mild to serious diseases, disorders and conditions.

Mouth ulcers can result from vitamin deficiencies, infection, inflammation, trauma,

malignancy and other diseases and abnormal process.

Stomach infection with pathogenic strains of H. pylori causes severe gastro duodenal

diseases in a large number of patients worldwide. The H. pylori infection breaks up in

early childhood, persists lifelong if not eradicated, and is associated with chronic gastritis

and an increased risk of peptic ulcer and gastric cancer. Across population of children, H.

pylori prevalence ranges from under 10 to over 80% in developed and developing

countries, respectively.

Killing H. pylori with antibiotics can cure most patients specifically with duodenal ulcer.

Many antibiotic-linked treatments have been recommended for eradication of H. pylori

infection but the appearance of antibiotic resistance makes the treatment more

complicated and the infection is persistent at higher levels when the drug treatment is

stopped. It is also reported that above 15% of the patients undergoing drug treatment

experience therapeutic failure. Recently, screening of natural products has gained much

interest for the safe inhibition of urease as potential new antiulcer drugs. Numerous

studies have concentrated on the eradication of H. pylori infection using herbal

medicines.

Psidium guajava (Myrtaceae) is a widely cultivated shrub in India and neighboring Asian

countries for its edible fruits. The plant is indigenously known as Amrud in Hind,

Perukah in Sanskrit, Peyara in Bengali and Perala-hannu in Kannada. The various parts

of the tree are widely used in Ayurveda, Siddha and Unani systems of medicine for a

variety of diseases like diarrhoea, dysentery, ulcers, cholera, haemorrhages, gingivitis,

vomiting etc. Leaf and unripe fruit extracts of P. guajava has been reported to possess

antidiarrhoeal activity. The plant is reported to contain catechol, tannins, wax, resins,

quercetin, β-sitosterol, sugars, carotene, vitamins B1, B2, B6 and niacin. However, the

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literature survey afforded no scientific claim on antiulcer activity of P. guajava leaf

extract. In view of this, the present study was taken up.

The plant Sympolocos racemosa Roxb belongs to the family Symplocaceae. Drug

yielding plant is an evergreen tree found in plain and lower hills throughout India. It

contains three alkaloids Loturine, Colloturine, Loturidine and a large quantity of red

coloured matter. The Bark is used by Unani Physicians as analgesic, antidysentric, anti-

inflammatory. Besides these, it is also useful in curing eye diseases as eye tonic and also

used in treating urinary disorders.

Thus keeping in mind the medicinal importance of the both medicinal plants it was felt

desirable to formulate & evaluate the extract formulation for antibacterial & antiulcer

activity.

Therefore the aim of this research project is to develop a formulation easily palatable,

mucoadhesive buccal patch using transmucosal drug delivery system rendering significant

oral antiseptic activity & to develope In-Situ Gel using Floating Drug Delivery system

that can be effectively used in Peptic ulcers.

1. Psidium guajava leaf

The leaf of the plant Psidium Guajava was taken for its morphological,

pharmacognostical, phytochemical and antibacterial evaluation. Morphological

characteristics were studied and found similar as reported in the official compendia. The

important histological findings in case of leaves powder suggest that there is presence of

calcium oxalate crystals in the form of thin pointed needles, which are originally in the

form of thick bundles called raphides. Broken needles are also seen in the powder.It also

shows frequent presence of starch grains, xylem fiber; xylem vessel leaf powder.

The proximate Analysis showed satisfactory result with respect to foreign matter,

moisture content and ash values. The total ash, acid-insoluble ash, and water soluble ash

were found to be slightly higher as specified in literature. Likely reason for this may be

due to contamination or sometimes due to unwanted parts of the drug. The methanol

soluble extractive was found to be more as compared to water soluble, ethanol soluable,

and chloroform soluble extractive values.

In preliminary phytochemical tests of the methanolic extract presence of flavonoid,

saponins, tannins, carbohydrates, glycoside (Cardic and Anthraquinone) steroids and

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Saponin, was confirmed.Where as the test for alkaloid, proteins, amino acids, and fats

were observed as negative. The significant data pertaining to morphological,

microscopical and phytochemical nature of the plant could be obtained which can be

significant to establish a monograph of Psidium guajava l. of the plant successfully.

The TLC of methanolic extract for confirmation of flavonoid developed systems was

done and Rf value of it was found to be same as falvonoids 0.9; 0.94 Rf value of

flavonoid was found same as standard Quercetin.

HPTLC finger printing of methanol extract of leaf powder revealed presence of three

polyvalent phytoconstituents with their Rf value 0.95, 1.11, 1.41 at 220nm. Component

number 3 at Rf 1.41 showed maximum concentration and presence of total five

components with their Rf value 0.18, 0.91, 1.21, 1.42, 1.52 at 450nm.Component number

4 at Rf 1.41 showed maximum concentration. Aqueous extract of leaf powder showed

total six components with their Rf value 0.29, 0.74, 0.85, 0.96, 1.31 at 220nm.Component

number 4 at Rf 0.96 showed maximum concentration.

UV spectrum of the methanolic extract of the drug Psidium guajava was plotted for light

absorbed versus wavelength, and the drug showed maximum absorption (λmax), which is

characteristic of a particular drug and helps in standardization of herbal drug. Infrared

spectrum of methanolic extract was also recorded and major characteristic bands were

noted. Charactrization of marker compound of Psidium guajava i.e.Quercetin was done

by UV Spectrscopy & IR Spectroscopy.

2. Symplocos racemosa Bark

The bark was in slightly curved pieces of approximately 1cm thickness, outersurface

uneven and rough due to fissures and cracks, grayish brown woody externally while inner

surface was pinkish brown, feebly bitter in taste and odourless. Microscopic Characters

(T.S.) of bark revealed presence of Cork, Secondary cortex, Secondary Phloem,

Medullary rays and other characters of bark. Bark powder showed presence of fragments

of cork layer, lignified & unlignified phloem fibers, sclerides & Calcium oxalate crystals

(Prismatic and cluster crystals of calcium oxalate),and starch grains, mostly simple

present in a number of cortical cells.

Preliminary phytochemical screening of the extract showed the presence of alkaloids,

triterpenes, tannins, saponins, glycosides, phenolic compounds and flavonoids. The

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proximate analysis showed satisfactory result with respect to foreign matter, moisture

content, ash value and extractive values.

Thin Layer Chromatography (TLC): Thin Layer Chromatographic analysis of methaolic

extract was carried out using different solvent systems and visualizing agents, and Rf

values were calculated to standardize the drug for its identity and purity. HPTLC finger

printing of methanol extract of bark powder revealed presence of eight components 0.23

0.44, 0.57, 0.68 0.97,1.17,1.35,1.43 Component number 6 at Rf 1.17 showed maximum

concentration.Aqueous extract of bark powder showed seven peaks with Rf values in the

range with their Rf value Rf - 0.23 0.27 0.32, 0.38 0.54, 0.75 , Component number 5 at

0.54 Rf showed maximum concentration.

UV spectrum of the methanolic extract of the drug Symplcos racemosa was plotted for

light absorbed versus wavelength, and the drug showed maximum absorption (λmax),

which is characteristic of a particular drug and helps in standardization of herbal drug.

Infrared spectrum of methanolic extract was also recorded and major characteristic bands

were noted. Charactrization of marker compound of Symplocos racemosa i.e. Gallic acid

was done by UV Spectrscopy & IR Spectroscopy

Thus significant data pertaining to morphological, microscopical and phytochemical

nature of the plant could be obtained which can be effectively utilized in establishing the

monograph of the plant successfully. Thus the crude extracts or the isolated

phytoconstituents of the herb can be exploited for designing effective herbal formulations

for aforesaid indications.

Staphylococcus, Pseudomonas and Candida species are an important flora of

the Oropharynx. Mouth contains a wide variety of pathogenic microorganism. The

microorganism frequently identified in root caries are Lactobacillus acidophilus,

Actinomyces viscosus, Nocardia spp. and streptococcus mutans, Streptococcus aureus,

Neisseria mucosa, Candida albicans, Pseudomonas aeruginosa. Streptococcus aureus,

Pseudomonas aeruginosa and candida albicans frequently encountered in high

proportions in smooth tooth surfaces and gingiva, in moderate proportions in pits and

fissure of teeth, periodontal ligament, saliva, cheek and tongue In the oral cavity they

have been commonly implicated as a cause of gingivitis further progressing to

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periodontitis Streptococcus aureus was taken as representative for common gram

positive microorganism, Pseudomonas aeruginosa was taken as representative for

common gram negative microorganism and Candida albicans was taken as representative

for fungi inhabiting the oral cavity, to study the effect of herbal buccal patch formulated

by using extract of leaf powder of Psidium guajava & bark powder of

Symplocos racemosa Linn. Antibacterial activity of extract was evaluated against

these microorganisms.

Determination of Minimum Inhibitory Concentration for antibacterial activity was

carried out by using Broth Dilution technique MIC of the extracts of Psidium guajava&

Symplcos racemosa. The lowest concentration that inhibited the growth of

microorganism, was not found in broth dilution technique; therefore MIC of the

extracts of extracts was found with the help of cup plate diffusion method.

In Cup plate diffusion method the petri plates having concentration of extract

5 m g - 3 0 mg/ml & 50µg-500µg/ml incubated at temperature 30˚C for 48 hours

produced zone of inhibition (table no.28-35). Ampicillin was used as positive control

produced zone of inhibition. The test procedure was repeated three times to check the

reproducibility of the results. Then the optimized concentration used to formulate buccal

patch.

Psidium guajava & Symplocos racemosa methanoic extract revealed the presence of

flavonoids, tannins & steroids. The antimicrobial activitiy of the extracts of the bark of

Symplocos racemosa L. and leaves of the Psidium guajava L. and zone of inhibition are

represented in Table no 15 .The antimicrobial studies showed that methanolic extract of

Psidium guajava L & Symplocos racemosa had inhibitory effects on all microorganisms

with zones of inhibition ranging from 16 mm to 25 mm, in the concentration range of

5mg-30mg/ml of extract. Antibacterial effects were also observed in the concentration

range of 50µg-500µg/ml. Maximum effect was observed for S.Aureus & P.aurigenosa.

Zone of inhibition of both extracts were found satisfactory & comparable with

Ampicillin. This shows that the methanolic extracts of these plants could be used to treat

mouth ulcer and other infections caused by these micro-organisms.As the rapid

emergence of drug-resistant organisms necessitates the continuous search of new

antimicrobial substances, natural products may act as alternative for antibiotics and

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chemotherapeutic agents in certain circumstances. The results showed that the methanolic

extract of P. guajava leaf was able to inhibit all of the bacteria and fungi used in this

study with different degree of inhibition. The information obtained may provide

validation for its reported medicinal uses. In conclusion, the guava leaf methanolic

extract & bark of Symplocos racemosa are most effective against the tested bacterial

strains than the fungal strains.

H.Pylori (in vitro):

Evaluation of the inhibitory effect of Psidium guajava & Symplocos racemosa extracts

was determined on Helicobacter pylori growth in vitro. Activity of Methanolic &

aqueous extract of P. guajava against clinical isolate of H. pylori was evaluated by using

the agar-well diffusion method. Amoxycillin and clarithomycin was used as a control.

Mean diameters of H. pylori growth inhibition was determined. The inhibitory

concentrations of Psidium gaujava extract against H. pylori isolates by the agar diffusion

method are presented in bar graphs as shown, at concentrations of 0.01, 0.05, 0.1,1 &

mg/disc. Zone of inhibition of both extracts were found satisfactory & comparable with

Amoxicillin & Clarithromycin. In conclusion, methanolic extracts of Psidium guajava

(PGME) & Symplocos racemosa (SRME) possess considerable antibacterial activity

against H. pylori. The potential of Psidium guajava & Symplocos racemosa in the

prevention or treatment of H. pylori infection is worth further extensive evaluation.

Total Phenolic content: The total phenolic content of both extracts was assessed using

Folin-Ciocalteu reagent. The results indicate that the methanolic extract of Psidium

guajava (PGME) & Symplocos racemosa (SRME) contain high concentration of phenolic

compounds. Psidium guajava extract contains 63µg/ml & Symplocos racemosa extract

contains 77µg/ml equivalent to gallic acid as standard.

Total Flavonoid Content: The total flavonoid content of both extracts was assessed

using aluminium chloride (AlCl3) according to a known method, using catechin as a

standard reagent. The results indicate that the methanolic extract of Psidium guajava &

Symplocos racemosa contain high concentration of flavonoid. The total flavonoid

contents were calculated using the following linear equation based on the calibration

curve of catechin. Psidium guajava extract contains 3.8mg/ml & Symplocos racemosa

extract contains 1.4mg/ml equivalent to catechin as standard.

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Buccal patch:

The purpose of this study was to develop formulations and systematically evaluate in-

vitro & Ex vivo permeation performances of buccoadhesive patches of methanolic extract

of Psidium guajava & Symplocos racemosa using different polymer and chose the

polymer to develope the release of drug in immediate and sustained manner.

The DSC of extracts alone and its combination with polymer shown in graphs.Extracts

peak did not affected and prominently observed in DSC of extracts along with polymer

which shown in graph indicates no interaction.

In trial batches of buccal patches B-1 to B-6 four different polymers was used in which

Carbopol 934P have better mucoadhesion property than other so B-4 shows greater

mucoadhesion time 385 min than the formulation containing Eudragit RL-100 and ethyl

cellulose.Mucoadhesion time of formulation B-1 to B-6 varied from 178minute to 276

minutes.

This formulation was further optimized by varying concentration of HPMC K15 and

Carbopol and other variables and 9 new formulations F1 to F9 were prepared & evaluated.

Color of all formulations (F1 to F9) appeared light green made by PGME & brown made

by SRME extract. Texture of all patches was found to be smooth.

Buccal patches were evaluated for physical properties like diameter, thickness, diameter,

swelling index & pH.

Diameter of F1 to F9 for PGME buccal patches varied from 3.36±0.04 to 3.8± & for

SRME buccal patches varied from 3.4±0.16 to 3.9±0.43.

Thickness of Patches were found uniform.Thickness was observed as 0.21±0.01mm to

0.25±0.01mm.

Any polymer with good swelling property is expected to be a good candidate for

bioadhesive application. When bioadhesive comes in contact with aqueous medium they

swell and form a gel. The faster this phenomenon occurs more rapid will be the polymer

adherence to the buccal mucosa. The swelling of the patches were observed in phosphate

215

buffer solution (pH 6.8) .These results were in agreement with the increase in area due to

swelling.swelling index varied from 35.0% to 68.81%.The results revealed that all the

formulations provide an acceptable swelling index.Best result was shown by Batch F5.

An acidic or alkaline formulation is bound to cause irritation on the mucosal membrane.

Surface pH of formulation F1 to F9 varied from 6.4 ± 0.04 to 7.00 ± 0.08. Each sample is

analyzed in duplicate (n=3). The surface pH of all formulations was within ± 0.5 units of

the neutral pH and hence no mucosal irritation was expected and ultimately achieves

patient compliance.

Moisture uptake was found satisfactory for all batches. It was found maximum in batches

F5 & F6.The concentration of HPMC & carbopol was 60% & 40% respectively.

As the amount of glycerin increases the folding endurance was found to be increases. The

folding endurance for all the formulation was found more than 300 times which was

satisfactory to reveal good film properties for all the formulation except F1, F2 & F7.

Weight of Patches were found uniform.Weights were observed as 0.250±0.013gm to

0.252±0.03gm for PGME patches & 0.250±0.14gm to 0.252±0.13gm for SRME patches

respectively.

In formulation F1 to F9 mucoadhesion strength increases with increase in the amount of

HPMC so F5 shows greater mucoadhesion strength. Mucoadhesion strength of

formulation F1 to F9 varied from 6.76±0.134gm to 22.63±0.61gm & 7.84gm to

22.63±0.64 gm for PGME & SRME respectively.

Drug content varied from 89.60% to 94.70% in F1 to F9 batches.

In patches F1 to F9 in-vitro drug release varied from 767.63±0.07% to 92.12±0.48% in

SRME buccal patch in 8 hours & 76.81±0.39% to 92.09±0.32% in PGME buccal patch in

8 hours.(Table no. 52 & 53). In formulation F1-F3 which was made of HPMC alone gave

faster drug release as compared to which have HPMC in combination with Carbopol.

Formulation F1-F3 release of drug within 6 hours, while formulation F4-F9 showed

uniform and sustain drug releases drug in 8 hours.Best result was obtained in Batch F5 for

SRME & PGME.

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Mucoadhesion time of formulation F1 to F9 varied from 177±2.05 minute to 307minute

for SRME buccal patch & 178±1.63minute to 305±2.44minute for PGME buccal patch.

The best results were obtained in Batch F5 for major factors like In-vitro release &

mucoadhesive time.Therefore batch F5 for PGME & SRME was considered as best for the

formulation of buccal patches where ratio of HPMC K-15 & Carbopol was 60%:40%.

PGME & SRME optimized buccal patches showed no significant change in the physical

appearance, mucoadhesion time, mucoadhesive strength, was determined at 0, 15, 30, 45

& 60 days. Also showed no significant change at room temperature and in stability

chamber at 40±1ºC and 75 % relative humidity this indicates that optimized formulations

were stable.

The main advantage of this formulation is that it contains a lower drug dose, sufficient for

therapeutic effect as it bypass first pass metabolism. The results showed that

mucoadhesive buccal patch containing 90mg HPMC-K15 & 60mg Carbopol produced

buccal patches having good mucoadhesive strength and better drug release in 8 hr .Good

results were obtained for in vitro conditions for bioadhesive buccal patch for PGME &

SRME so it may be concluded that buccal patch can be used to administer the drug or

plant extract for antibacterial activity for microorganisms residing in oral cavity causing

mouth ulcer.

It may be concluded that mucoadhesive patches for oral cavity are a promising drug

delivery system for both drug extract. The combination of polymers HPMC K-15,

Carbopol showed good mucoadhesive and swelling characteristics.Good correlation

observed between the in-vitro and ex-vivo profile. Medicated patches showed good

release of the drug over a relatively long period (7-8 hrs.).Hence the best formulation F5

achieved the objective of present study such as reducing the dose, improving the

bioavailability by avoiding first pass metabolism.

Significant kill, evidenced by growth reduction was witnessed with the test buccal

patch against all microorganisms. Antimicrobial studies showed that the extracts had good

activity against Gram positive and Gram negative microorganism.

The results of this work show that the extract of Psidium guajava & Symplocos

racemosa when formulated into buccal patches for intended antimicrobial and antifungal

effect against susceptible organisms associated with buco-laryngo-oesophagal infections.

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It is therefore recommended that pharmaceutical manufacturers could go into the

commercialization of Psidium guajava & Symplocos racemosa extract as formulated

buccal patch since the plants can easily be sourced and processed.

Gastric hyperacidity and gastro duodenal ulcer is a very common global problem today. It

is now generally agreed that gastric lesions develop when the delicate balance between

some gastro protective and aggressive factors is lost. Major aggressive factors are acid,

pepsin, Helicobacter pylori and bile salts. Defensive factors mainly involve mucus-

bicarbonate secretion and prostaglandins. Hyper secretion of gastric acid is a pathological

condition, which occurs due to uncontrolled secretion of hydrochloric acid from the

parietal cells of the gastric mucosa through the proton pumping H+K+ATPase. Even the

normal rate of acid secretion may cause ulceration in the breached mucosa when some

gastro protective factors are lost. The modern approach to control gastric ulceration is to

inhibit gastric acid secretion, to promote gastro protection, to block apoptosis and to

stimulate epithelial cell proliferation for effective healing. Most of the antisecretory drugs

such as proton pump inhibitors (omeprazole, lansoprazole, etc.) and histamine H2-

receptor blocker (ranitidine, famotidine, etc.) are extensively used to control increased

acid secretion and acid related disorders caused by stress, NSAID’s and H. pylori, but

there are reports of adverse effects and relapse in the long run. On the contrary most of

the herbal drugs reduces the offensive factors and proved to be safe, clinically effective,

better patient tolerance, relatively less expensive and globally competitive. Plant extracts,

however, are some of the most attractive sources of new drugs and have been shown to

produce promising results in the treatment of gastric ulcers.

The gastro retentive drug delivery system can be retained in the stomach & can contribute

in improving the oral sustained delivery of drug that have an absorption window in a

particular region of gastrointestinal tract. These systems help in continuously releasing

the drug before it reaches the absorption window, thus ensuring optimal bioavailability.

In-situ gel, or oral in-vivo gel, environment sensitive gel is a new dosage form. The

alginate based in situ gelling liquid formulation containing calcium ion in complex form

gets converted into gel when reaches to acidic environment of stomach & make

formulation to float for prolonged period of time.

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In preliminary trial batches of J1to J12 were prepared using different concentration of

sodium alginateto see the effect on viscosity of solution, drug content , pH & physical

properties of gel in simulated gastric fluid(pH1.2). On the basis of floating time and

viscosity of in situ oral gel the concentration of different polymers and excipients are

selected for 32 factorial designs.F1-F9 batches were formulated containing SRME &

PGME. Effect of concentration of sodium alginate & calcium chloride were evaluated for

viscosity, drug content, floating time & invitro release.A numerical optimization

technique using the desirability approach was employed to develop a new formulation

with desired responses.

Floating Lag time for In-Situ gel varied from 38±0.04 to 89±0.06 seconds for SRME gel

& 40±0.05 to 90±0.06 seconds for PGME gel. Floating Time varied from 423±1.4

minutes to 573±3.0 minutes for SRME gel & 433.5±1.5 to 572±10.5 minutes for PGME

gel.

Gelation characteristic was assessed on an ordinal scale ranging between - - - to +++.

After ingestion, the liquid polymeric solution should undergo a rapid sol-to-gel transition

by means of ionic gelation.

The drug content varied from 91.23% to 96.23% in batches F1 to F9.

The effect of Sodium alginate concentration was shown in graph. The significant

variation in the rate & extent of drug release was observed. From F1to F3 gel could

release only 66% to 70% of drug content where the concentration of sodium alginate was

0.5% concentration of Calcium chloride was 0.05%. F7 to F9 also showed less release

with concentration of sodium alginate 1.5% & concentration of calcium chloride was

0.1%. F4 to F6 showed better release with concentration of sodium alginate 1% &

concentration of Calcium chloride was 0.075%. Batch F5 showed maximum release in

both Formulations (SRME 80.2% represented in Table no.38 A & Figure No. 32 A,B and

PGME 76% 38 B Fig no.32 C,D respectively)

For water uptake studies system shows first increase in the water gain then it is

maintained throughout system which is good to release the drug after penetration in to the

gel.(Table no.39 A & B)

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The bioadhesive strength of F5 formulation was found to be 0.5N, pointing to good

bioadhesion. In-vitro bioadhesion test showed that SRME & PGME In situ gel adhered

more strongly to gastric mucous layer & could retain in GIT for an extended period.

Constrains like maximizing in vitro release at the end of 8 hrs.Minimizing viscosity,

maximizing in vitro gelation time. The optimized in situ gel formulation (F5) was

developed using 1% of sodium alginate & 0.075 % calcium chloride. The in-vitro

gelation time & in vitro release was found to be good thus batch F5 was selected for

further study, which exhibit gelation time 7-9.5 hrs. & drug release 80-95% low viscosity

which was easy for swallowing & good ability for immediately after oral administration.

Based on visual identification, the in-situ gel has remained as liquid for 6 months without

the occurrence of turbidity or gelation at 40 2 C.The present investigation deals with the

formulation, optimization and evaluation of sodium alginate based In-situ gel of SRME

& PGME. Sodium alginate was used as a polymer and CaCl2 was used as a cross-linking

agent. The insitu forumulation exhibited well, viscosity, drug content and sustained drug

release; this study reports that oral administration of aqueous solutions containing sodium

alginate results in formation of insitu gel, such formulations are homogenous liquid when

administered orally and become gel at the contact site.

The previously evaluated batches optimized with the help of Design Expert software.The

concentration of sodium alginate and other excipient in In-Situ Oral Gel and its response

was analyzed by optimization.From both graphs revealed that batch no. F4 & F5 have

shown better response for both factors. But % in vitro release was found to be best for F5

batch hence batch F5 (SRME) having concentration 1% sodium alginate & 0.075%

calcium chloride can be considered for optimization for further evaluation.

The study reports that oral administration of aqueous solutions containing sodium

alginate results in formation of In-situ gel at stomach site. The results of 32 full factorial

design revealed that concentration of sodium alginate & concentration of calcium

chloride significantly affected on the dependent variables like in-vitro gelation time & in-

vitro drug release.

220

The in vivo study demonstrated the excellent gel formation in the stomach of rat &

significant anti-ulcer effect of alginate based in-situ of extracts (PGME & SRME) over a

longer period.

In pyloric ligation model, the Formulation 1 and Formulation 2 showed significant

(p<0.01) rise in pH as compared to control. The free acidity gastric content is increased in

control animals. The Formulation 1 & 2 both showed significant (p<0.01) decrease in free

acidity as compared to control. Both Formulations significantly reduced the total acidity

and ulcer index (p<0.001) as compared to control .The percentage protection of

Formulation 1 & 2 was found to be 65% and 60% respectively whereas the percentage

protection of omeprazole was found to be 75%.Our results are in corroboration with the

antigastric ulcer activity of the extract observed under the studies on pharmacological and

biochemical evaluation.

Based on data & previous findings, a tentative scheme has been proposed for

electron transferrin H.Pylori. Several dehydrogenases are present, depending on [H]

donar. It seems that NADH dehydrogenase, a classic Complex I, is absent in respiratory

chain of H.Pylori.NADPH might be a major respiratory donar in bacterium. There are

succinate dehydrogenase, a classic complex-II & succinate cytochrome C reductase, a

classic pathway from complex II to complex-III, in the respiratory chain of H.Pylori. The

complex-II could be inhibited by malonate & pathway from complex-II to complex-III

could be inhibited by antimycin. There are a cytochrome bc oxidase & cytochrome c

peroxide in respiratory chain of bacteria, which was inhibited by COA & other non-

antibiotics. This preliminary study helps to understand the respiratory chain of H.pylori &

to classify pathogenesis. It is likely that detailed characterization of special features of

electron transport pathway or target enzyme complexes may be helpful in opening new

lines for chemotherapy against H.Pylori.

In conclusion, the oral administration of plant extracts displayed a significant antiulcer

activity without any apparent toxicological effects, which supports the use of Psidium

guajava & Symplocos racemosa in herbal medicine of India for ulcer therapy.

The gastric retention approaches as well as herbal drugs described here have application

for treatment of H.pylori infections although further development is required for each to

be fully effective especially, in human studies. Overcoming high mucous turnover rate &

221

resulting limited retention times is challenging for bio adhesive systems & swelling

systems must guarantee clearance from the stomach after a certain time to prevent any

obstruction.

Floating systems are available commercially, & combination approaches, using floating

behavior & mucoadhesion, have also shown promise. Exploiting dual mechanism of

retention may provide the strength & reproducibility required to permit successful

advancement in this field. So in future, a combination of herbal drugs with a novel drug

delivery systems mentioned above, may lead to an important breakthrough in

herbal/integrative treatment of H.pylori infections & other types of ulcer.

222

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