Rajiv Gandhi University of Health Sciences Karnataka, …accp.co.in/Thesis-M.Pharm/M Pharm...

ISOLATION AND EVALUATION OF FENUGREEK MUCILAGE AS A

EXCIPIENT IN THE TABLET DOSAGE FORM

By

AKSHATHA R.S. B.Pharm.,

Reg. No: 13PU088

A Dissertation Submitted to the

Rajiv Gandhi University of Health Sciences Karnataka, Bangalore

In partial fulfillment of the requirements for the

MASTER OF PHARMACY

In

PHARMACEUTICS

Under the guidance of,

ABDUL NASIR KURNOOL

M. Pharm,

DEPARTMENT OF PHARMACEUTICS

SAC COLLEGE OF PHARMACY

B.G.NAGARA, KARNATAKA -571448

MAY-2015

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

KARNATAKA, BANGALORE

DECLARATION BY THE CANDIDATE

I hereby declare that this dissertation entitled “ISOLATION AND EVALUATION OF

FENUGREEK MUCILAGE AS A EXCIPIENT IN THE TABLET DOSAGE FORM” is a

bonafide and genuine research work carried out by me under the guidance of ABDUL NASIR

KURNOOL Assistant Professor, Department of Pharmaceutics, S.A.C College of Pharmacy,

B.G.Nagara. This work is original and has not been submitted in part or full to any other

university for the award of any degree or diploma or fellowship.

Date: AKSHATHA R.S B.Pharm.,

Place: B.G.Nagara Department of Pharmaceutics,

SAC College of Pharmacy,

B.G.Nagara,

Mandya-571448

Karnataka

SRI ADICHUNCHANAGIRI COLLEGE OF PHARMACY

B.G.NAGARA-571448

CERTIFICATE BY THE GUIDE

This is to certify that the dissertation entitled “ISOLATION AND EVALUATION OF


bonafide research work done by Ms. AKSHATHA R.S in partial fulfillment of the requirement

for the Degree of MASTER OF PHARMACY IN PHARMACEUTICS. This work was

carried out by her in the laboratory of SAC College of Pharmacy, B.G.Nagara, under my direct

supervision and guidance.

Date: ABDUL NASIR KURNOOL M. Pharm., Place: B.G.Nagara Associate Professor Department of Pharmaceutics,


B.G.Nagara,

Mandya-571448

Karnataka


B.G.NAGARA-571448

ENDORSEMENT BY THE HEAD OF THE DEARTMENT



bonafide research work carried out by Ms. AKSHATHA R.S under the guidance of ABDUL

NASIR KURNOOL, Assistant Professor, Department of Pharmaceutics, SAC College of

Pharmacy, B.G.Nagara.

Date: Dr. MOHAMMED GULZAR AHMED

M.Pharm., Ph.D., Place: B.G.Nagar Professor and Head,

Department of Pharmaceutics,


B.G.Nagara-571448

Karnataka


B.G.NAGARA-571448

ENDORSEMENT BY THE PRINCIPAL/HEAD OF THE INSTITUTION



bonafide research work carried out by Ms. AKSHATHA R.S under the guidance of ABDUL

NASIR KURNOOL Assistant Professor, Department of Pharmaceutics, SAC College of

Pharmacy, B.G.Nagara.

Date: Dr. B. RAMESH

M. Pharm., Ph.D., Place: B.G.Nagara Principal,

Department of Pharmaceutics,


B.G.Nagara-571448

Karnataka

COPYRIGHT

DECLARATION BY THE CANDIDATE

I hereby declare that the Rajiv Gandhi University of Health Sciences, Karnataka shall

have the rights to preserve, use and disseminate this dissertation in print or electronic format for

academic / research purpose.

Date: AKSHATHA R.S B.Pharm.,

Place: B.G.Nagara Department of Pharmaceutics,


B.G.Nagara-571448

Karnataka

ACKNOWLEDGEMENT

After months of preparation and weeks of agony,

my thesis defense is finally over!!!!

It is a pleasure to thank the many people who made this thesis

possible.

First and foremost, I wish to place my soulful thanks to the Paramapoojya

Karnatakarathna Padmabushana Dr. Sree Sree Sree Shivakumar Maha

Swamiji, President, Sree Siddaganga Mutt and Paramapoojya Jagadguru

Sree Sree Sree Nirmalanandanatha Maha Swamiji, President, Sree

Adichunchanagiri Mutt.

My Deepest Gratitude is to my guide, Abdul Nasir Kurnool. He is one of

the best teachers that I had in my life. He sets high standards for his students

and he encourages and guides them to meet those standards. He has given

me enough freedom during my research, and he has always been nice to

me. I will always remember his calm and relaxed nature, and the way he asks

“YES! How can I help you? ”, Whenever I approach him. I am thankful to the

Almighty for giving me a mentor like him. I have been amazingly fortunate to

have a guide who gave me the freedom to explore on my own to think

about the final purpose and applications of my research and at the same

time the guidance to recover when my steps faltered. He taught me how to

question thoughts and express ideas. He has been always there to listen and

give advice. I have learned a lot from his attention to detail, patience and

step-by step approach to any task. I am deeply grateful to him for the long

discussions that helped me sort out the technical details of my work. I am also

thankful to him for encouraging the use of correct grammar and consistent

notation in my writings and for carefully reading and commenting on

countless revisions of this manuscript. His patience and support helped me

overcome many crisis situations and finish this dissertation. Thank you for

giving me an opportunity to attend conference at IPC, SRM and Vijapur.

I express my profound sense of reverence to my principal Dr. B. Ramesh

and HOD Dr. Mohammed Gulzar Ahmed, for their constant guidance,

support and for giving me the opportunity to do my Post graduation through

the Department of Pharmaceutics at SACCP.

I extend my thanks to the teaching faculty of SACCP especially Mr. G.B. Kiran

Kumar, Mr. Senthil Kumar, Dr. N.K. Sathish, Mr. Chandra Prakash.

I am also grateful to the non-teaching staff especially Lokesh,

Poornima, Mahalakshmi, Prashant, and Krishna Gowda deserve special

mention for assisting me in many different ways. I am thankful their help

towards the completion of my work in their various forms of support during my

study.

I acknowledge and express my special thanks to Radiant Research

laboratory, Bangalore, for assisting in extraction of mucilage and for carrying

out In-vitro glucose uptake activity.

Most importantly, none of this would have been possible without the

love and patience of my family. My family has been a constant source of

love, concern, support and strength all these years. I thank my family for

encouraging me in all of my pursuits and inspiring me to follow my dreams. I

deeply thank my parents Kalpana and Shivakumar for their unconditional

trust, timely encouragement, and endless patience. It was their love that

raised me up. I always knew that you believed in me and wanted the best for

me. Thank you for teaching me that my job in life was to learn, to be happy,

and to know and understand myself only then could I know and understand

others. I can’t imagine my current position without the love and support from

my family. I have to give a special mention for the support given by grandpa

(Chandrashekariah), grandma (Gangamma), Dodamma (Shylaja), dodappa

(Parmeshwarappa), maama (Niranjan), athe(Rekha) and my dearest

brothers Siddesh and Akshay. I strongly believe that their prayers played very

important role in my life.

My humble thanks to Dr. S. Badami and K.P. Manohar, Encouraged me

to pursue degree and who mentored me for almost six years. I immensely

appreciate your encouragement during the different stages of my life. I am

grateful to you both for sharing your knowledge and for motivating me to

choose right things at the right time. I am truly honored to have you both as

mentors.

I am grateful to my school and college teachers, each of you have given

your time, energy, and expertise and I am richer for it; Mr.Manjunath,

Prof.Subbaiah,Mr.B.V.Manjunath, Dr.B.S.Thippeswamy, Dr.Veeresh.P.Veerapur,

Dr.R.Nandeesh, Prof.Bhaghavathi, Dr.Manjunath, who laid seeds of

enthusiasm and passion in my pursuit of knowledge.

Many friends have helped me stay sane through these years. I wish to

thank my Besties who have enriched my life sharing part of themselves and

for being the secret of my happiness; Manasa, Ashwin, Kushal, Abhishek, for

helping me get through the difficult times, and for all the emotional support,

camaraderie, entertainment, boosting my confidence and caring they

provided. They are the charming gardeners who make my soul blossom.

A special thanks to Chandra Prakash sir and Mamta madam for your

love and care & for being second parents to me in times of need, your

support and care helped me overcome setbacks and stay focused on my

study.

Many more thanks to my best friends that I met in this Chunchunland,

who never made me feel the difference of staying far away from home. I

greatly value their friendship and I deeply appreciate their belief in me.

Thanks to Ammi Raju, Meenu pandey, Priyanka Biswas, Vishal Mandal and

Abel Abraham for being great friends.

I also convey my gratitude to Harika and Ashwini my dear friends and

roommates, thank you for listening, offering me advice, for being great

company and supporting me through this entire process. I enjoyed our chit-

chats, lab days, tea times, birthday treats, debates and dinners. Harika, I had

a great time working with you as a team. Thank you for always being there to

help me with any issue.

I am indebted to my many student colleagues for providing a

stimulating and fun environment in which to learn and grow, I am grateful to

Vinay anna, Madhu anna, Guru, Kharel, Dachu, Hansi, Sachin, Ankit, Suyash,

Shilpa, Sarala, Poorvini, Anwar. Many thanks especially to Amrit Timalsina &

Ravi chaudhary for a giving me a joyful workplace and for being great

friends and colleagues.

Thank you very much everyone!

And off course, I must not forget to thank God for all of this. Thank you Lord

for all your blessings and for your guidance, especially during the moments I

felt so lost and hopeless.

- AKSHATHA.R.S

ABSTRACT

Department of Pharmaceutics, SACCP

ABSTRACT

The aim of the current research work was to isolate and evaluate Fenugreek mucilage

(FGM) and to develop and evaluate Pioglitazone tablets using FGM and Polyvinylpyrrolidone

(PVP) K30, in varying concentrations of FGM. Tablets of Pioglitazone containing 15mg drug

were prepared by non-aqueous wet granulation technique. Compatibility study was carried out

by using FTIR and confirmed that no chemical interaction took place during entrapment process.

Pre-compressional parameters and post-compression parameters were evaluated and the results

were within the acceptable official limits. In-vitro drug release rate was carried out by USP

dissolution rate apparatus type-II using 2different dissolution media (0.1N HCL and Phosphate

buffer of pH 7.4) and data was subjected to various kinetic models. In-vitro drug release shows

concentration of Fenugreek increases then drug release decreases. The release data was fitted to

various mathematical models to evaluate the kinetics of drug release. The drug release follows

first order kinetics and mechanism was found to be non-Fickian diffusion. The Stability studies

were carried out for 2months. Cytotoxic effect of the formulations was evaluated by MTT assay

and In-vitro anti-diabetic effect was studied using the glucose uptake model in rodent skeletal

muscle cells (L-6) involved in glucose utilization, results exhibited moderate anti-diabetic

activity and merits further investigation in animal models. In conclusion the results suggest that

the formulated delayed release tablets of Pioglitazone could therapeutically better than

conventional dosage form, leading to improved efficacy and better patient compliance.

Keywords: Delayed release tablets, Pioglitazone, Fenugreek, Non-aqueous wet granulation,

Cytotoxicity, In-vitro anti-diabetic effect.

LIST OF ABBREVIATIONS



% Percentage

°C Degree centigrade

µg Microgram

λmax Maximum Wavelength

% CDR

Percentage cumulative drug release

Abs

Absorbance

Conc.

Concentration

cm Centimeter

Cmax Maximum concentration

Hr Hour

RPM Revolution per minute

ICH International conference on harmonization

IP Indian Pharmacopoeia

IR Infra red

Kg Kilogram

Sec Seconds

tmax

Time of peak concentration

AUC Area under the curve

GIT Gastro intestinal tract



FT-IR

Fourier Transform Infrared Spectroscopy

mm Millimeter

gm Gram

mg Milligram

pH Negative logarithm of hydrogen ion concentration

RH Relative humidity

min Minute

ml Milliliter

nm Nanometer

t1/2 Half life

USP United states pharmacopoeia

UV Ultra violet

Vs Versus

w/w Weight by weight

w/v Weight by volume

TABLE OF CONTENTS


TABLE OF CONTENTS

CHAPTER NO CHAPTER NAME PAGE NO

1 INTRODUCTION 1-22

2 OBJECTIVES 23-26

3 REVIEW OF LITERATURE 27-34

4 MATERIALS AND METHODS 35-70

5 RESULTS 71-88

6 DISCUSSION 89-96

7 CONCLUSION 97-98

8 SUMMARY 99

9 BIBLIOGRAPHY 100-106

LIST OF TABLES


LIST OF TABLES

Table

No Title

Pg

No

1 Complications of DM 1

2 Taxonomy of Trigonella foenum-graecum 19

3 Potential Medicinal Values of Trigonella foenum-graecum 20

4 List of chemicals used 35

5 Details of Equipment’s used 36

6 Effect of Carr’s Index and Hausner’s Ratio on flow property 54

7 Effect of Angle of repose (ϴ) on Flow property 55

8 Selected Ingredients for formulation with function 55

9 Formulation developed 56

10 Weight variation tolerances for uncoated tablets 61

11 Mechanism of Drug Release as per Korsmeyer Equation/ Peppa’s Model 65

12 Drug substances intended for normal storage 66

13 Solubility determination 71

14 Spectrophotometric Data for the Estimation of Pioglitazone in 0.1N HCL 74

15 Spectrophotometric Data for the Estimation of PGZ in 7.4pH Phosphate

buffer 75

16 Results of Organoleptic Characters determination 76

17 Results of Solubility determination 76

18 Results of Yield estimation 76

19 Results of Phytochemical tests of isolated mucilage 77

20 Results of micromeritic Evaluation of isolated mucilage 78

21 Organoleptic properties evaluation results 79

22 Pre-compression parameters results 80

23 Post-Compression Parameter results 81

24 In-vitro drug release profile 82

25 Results of Kinetic data of various models for release study 83

26 Results of Stability studies for F5 formulation 86

27 Results of Stability studies for F6 formulation 86

28 Results of %CDR of Formulation of F5 and F6 at 40ºC/75% RH after 10hrs 86

29 Results of %Cytotoxicity of F5 and F6 formulations against L6 cell line 87

30 Result of In vitro glucose uptake studies of F5 and F6 formulations 88

LIST OF FIGURES


LIST OF FIGURES

Sl No Title Page No

1 Main symptoms of diabetes mellitus 8

2 Patho-physiologic defects of type II diabetes 9

3 Causes of type-II diabetes 11

4 Schematic diagram of insulin resistance progresses

towards type-II diabetes

12

5 Relationship of pharmaceutical Delayed Release dosage

forms

17

6 Mechanism of Anti-diabetic Activity of T. foenum-

graecum

22

7 Structure of Pioglitazone 37

LIST OF SPECTRA & GRAPHS


LIST OF SPECTRA

Sl No Title Page No

1 FT-IR spectra of Pioglitazone Drug 71

2 FT-IR spectra of Fenugreek Mucilage 72

3 FT-IR spectra of PVP K30 72

4 FT-IR spectra of PGZ + FGM + PVP K30 73

LIST OF GRAPHS

Sl No Title Page No

1 Maximum wavelength of Pioglitazone 73

2 Calibration Curve of Pioglitazone in 0.1N HCL 74

3 Calibration Curve of Pioglitazone in 7.4 Phosphate buffer 75

4 In-vitro Cumulative percentage drug released V/S Time for Formulations F1 to

F9 83

5 Plot of % Cum. Drug Released Vs. Time 84

6 Plot of Log % Cum. Drug Retained Vs. Time 84

7 Plot of % Cum. Drug Released Vs. √Time 85

8 Plot of Log % Cum. Drug Released Vs. Log Time 85

9 Cytotoxic effect of F5 and F6 on the L6 Cell line 87

10 Glucose uptake assay of the F5 and F6 on L6 cell line 88

INTRODUCTION The key to growth is the introduction higher

dimensions of consciousness into our awareness.

INTRODUCTION

Department of Pharmaceutics, SACCP Page 1

1. INTRODUCTION

1.1 DIABETES MELLITUS

A. Introduction

“Genetics loads the Gun, Life style pulls the trigger”. There are many

diseases that are caused due to genetic disorders, and are one of the causes for Diabetes

mellitus1.

Diabetes mellitus is a major and growing health problem world wise and an important cause

of prolonged ill health and early death (Arunachalam, Gunasekaran, 2002). Diabetes mellitus

is the major cause of the death and disability in the world. Recent estimates indicate there were

171 million people in the world with diabetes in the year 2000 and this is projected to increase

to 366 million by 2030. The American Diabetes Association (ADA) estimated the national

costs of diabetes in the USA for 2002 to be $US 132 billion, increasing to $US 192 billion in

2020. The focus of the medical community is on the prevention and treatment of the disease,

as is evident from the rising number of research papers every year on the subject.2

India has the highest cases of diabetes in the world (32 million expected to increase to

78 million by 2030 according to WHO estimate). According to World Health Organization

estimates, by 2025, over 350 million would be affected and over 75% of these diabetes cases

will be in the developing world3. As India has no subsidized, coordinated diabetes care

programs, reducing treatment costs through raising public awareness, regular monitoring and

earlier diagnosis should be a key objective4. Impaired glucose tolerance and impaired fasting

glycaemia are risk categories for future development of diabetes and cardiovascular diseases.

In some age groups, people with diabetes have a two-fold increase in the risk of stroke.

Diabetes is the leading cause of renal failure in many populations in both developed and

developing countries. Lower limb amputations are at least 10times more common in people

with diabetes than in non-diabetic individuals in developed countries; more than half of all

INTRODUCTION


non-traumatic lower limb amputations are due to diabetes. Diabetes is one of the leading

causes of visual impairment and blindness in developed countries. People with diabetes

require at least two to three times the health-care resources compared to people who do not

have diabetes, and diabetes care may account for up to 15% of national health care budgets.

In addition, the risk of tuberculosis is three times higher among people with diabetes. The

apparent prevalence of hyperglycemia depends on the diagnostic criteria used in

epidemiological surveys. The global prevalence of diabetes in 2008 was estimated to be 10%

in adults aged 25+years. The prevalence of diabetes was highest in the Eastern Mediterranean

Region and the Region of the Americas (11% for both sexes) and lowest in the WHO

European and Western Pacific Regions (9% for both sexes) The magnitude of diabetes and

other abnormalities of glucose tolerance are considerably higher than the above estimates if

the categories of „impaired fasting‟ and „impaired glucose tolerance‟ are also included. The

estimated prevalence of diabetes was relatively consistent across the income groupings of

countries. Low-income countries showed the lowest prevalence (8% for both sexes), and the

upper middle-income countries showed the highest (10% for both sexes) 5

.

B. Definition

Diabetes mellitus is a chronic disease that is characterized by disorders in

carbohydrate, protein and lipid metabolism6. Its central disturbance appears to involve an

abnormality either in the secretion of or effects produced by Insulin although other factors

also may be involved. Diabetes mellitus is a metabolic disorder in which carbohydrate

metabolism is reduced while that of proteins and lipids is increased7. The external secretion

of the pancreas is digestive in function and the intestinal secretions play a major role in the

regulation of metabolism. The hormones which regulate the level of blood sugar are mainly

two; glucagon from the alpha-cells and Insulin from the β-cells of the islets of Langerhans8.

INTRODUCTION


C. Classification9

Diabetes can be classified into two categories:

1. Clinical Classification

Type 1 diabetes (due to b-cell destruction, usually leading to absolute

insulin deficiency)

Type 2 diabetes (due to a progressive insulin secretory defect on the

background of insulin resistance)

Gestational diabetes mellitus (GDM) (diabetes diagnosed during

pregnancy that is not clearly overt diabetes)

Other specific types of diabetes due to other causes, e.g., genetic defects

in b-cell function, genetic defects in insulin action, diseases of the

exocrine pancreas (such as cystic fibrosis), and drug- or chemical-

induced (such as in the treatment of HIV/AIDS or after organ

transplantation)

Type 1 diabetes10

, formerly called juvenile diabetes, is usually first diagnosed in

children, teenagers, and young adults. In this type of diabetes, the beta cells of the pancreas

no longer make insulin because the body‟s immune system has attacked and destroyed them.

Type 2 diabetes10

, formerly called adult-onset diabetes, is the most common type of

diabetes. About 90 to 95 percent of people with diabetes have type 211

. People can develop

type 2 diabetes at any age, even during childhood, but this type of diabetes is most often

associated with older age. Type 2 Diabetes is also associated with excess weight, physical

inactivity, family history of diabetes, previous history of gestational diabetes, and certain

ethnicities. Type II diabetes usually begins with insulin resistance, a condition linked to

excess weight in which muscle, liver, and fat cells do not use insulin properly. As a result, the

body needs more insulin to help glucose enter cells to be used for energy. At first, the

INTRODUCTION


pancreas keeps up with the added demand by producing more insulin. But in time, the

pancreas loses its ability to produce enough insulin in response to meals, and blood glucose

levels rise.

Gestational Diabetes10

, is a type of diabetes that develops only during pregnancy. The

hormones produced during pregnancy increase the amount of insulin needed to control blood

glucose levels. If the body can‟t meet this increased need for insulin, women can develop

gestational diabetes during the late stages of pregnancy. Gestational diabetes usually goes

away after the baby is born. Shortly after pregnancy, 5 to 10 percent of women with

gestational diabetes continue to have high blood glucose levels and are diagnosed as having

diabetes, usually type 211

. Research has shown that lifestyle changes and the diabetes

medication, metformin, can reduce or delay the risk of type 2 Diabetes in women. Babies

born to mothers who had gestational diabetes are also more likely to develop obesity and type

2 Diabetes as they grow up.

2. Etiological Classification: 12

I. Type 1 Diabetes (cell destruction, usually leading to absolute insulin

deficiency)

A. Immune mediated

B. Idiopathic

II. Type 2 diabetes (may range from predominantly insulin resistance with

relative insulin deficiency to a predominantly secretory defect with insulin

resistance)

III. Other specific types

A. Genetic defects of -cell function

1. Chromosome 12, HNF-1 (MODY3)

2. Chromosome 7, Glucokinase (MODY2)

INTRODUCTION



4. Chromosome 13, insulin promoter factor-1 (IPF-1; MODY4)


6. Chromosome 2, NeuroD1 (MODY6)

7. Mitochondrial DNA

8. Others

B. Genetic defects in insulin action

1. Type A insulin resistance

2. Leprechaunism

3. Rabson-Mendenhall syndrome

4. Lipoatrophic diabetes

5. Others

C. Diseases of the exocrine pancreas

1. Pancreatitis

2. Trauma/pancreatectomy

3. Neoplasia

4. Cystic fibrosis

5. Hemochromatosis

6. Fibrocalculous pancreatopathy

7. Others

D. Endocrinopathies

1. Acromegaly

2. Cushing‟s syndrome

3. Glucagonoma

4. Pheochromocytoma

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5. Hyperthyroidism

6. Somatostatinoma

7. Aldosteronoma

8. Others

E. Drug- or chemical-induced

1. Vacor

2. Pentamidine

3. Nicotinic acid

4. Glucocorticoids

5. Thyroid hormone

6. Diazoxide

7. Adrenergic agonists

8. Thiazides

9. Dilantin

10. Interferon

11. Others

F. Infections

1. Congenital rubella

2. Cytomegalovirus

3. Others

G. Uncommon forms of immune-mediated diabetes

1. “Stiff-man” syndrome

2. Anti–insulin receptor antibodies

3. Others

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H. Other genetic syndromes sometimes associated with diabetes

1. Down‟s syndrome

2. Klinefelter‟s syndrome

3. Turner‟s syndrome

4. Wolfram‟s syndrome

5. Friedreich‟s ataxia

6. Huntington‟s chorea

7. Laurence-Moon-Biedl syndrome

8. Myotonic dystrophy

9. Porphyria

10. Prader-Willi syndrome

11. Others

IV. Gestational diabetes mellitus (GDM)

D. Complications of Diabetes Mellitus: 13

Table No.1: Complications of DM

Body Location Description

Eyes Retinopathy, cataract formation, glaucoma and periodic

visual disturbances; leading cause of new blindness.

Mouth Gingivitis, increased incidence of dental cavities and

periodontal disease.

Pregnancy Born of large babies, miscarriages, neonatal deaths and

congenital defects.

Nervous system Motor, sensory and autonomic neuropathy leading to

impotence, neurogenic bladder, parathesias, gangrene.

Vascular system Large vessel disease and micro angiopathy.

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Skin Numerous infections and specific lesions due to small vessel

disease, increased lipids in blood and pruritus.

Kidneys Diabetic glomerulosclerosis causing nephropathy.

Infections Diabetics have a higher incidence of cystitis, tuberculosis and

skin infections; moniliasis is common in diabetic women.

E. Main symptoms of diabetes mellitus

Figure No.1: Main symptoms of diabetes mellitus

F. Pathophysiology14

The islets of the langerhans are the endocrine components of the pancreas. Insulin is

synthesized in the pancreatic β-cells. Glucose is the major stimulant to insulin release. The

response is triggered both by intake of nutrient and the release of gastrointestinal peptide

hormones. The initial response represents the release of stored insulin and second phase

reflects discharge of newly synthesized insulin. Once released from the pancreas, insulin

enters the postal circulation. It is rapidly degraded by the liver and only 50% reaches the

INTRODUCTION


peripheral circulation. Total daily secretion is approximately40 units. Insulin circulates free

as a monomer, has a half life of 4 to 5 minutes and is primarily metabolized by the liver and

kidneys. Many tissues contains receptors that are highly specific for insulin and to which it

binds reversibly. The biological response to insulin can be altered by either a change in the

receptor affinity for insulin or a change in the total number of receptors. Changes in the

number of receptors occur in two important clinical situations, obesity and chronic exposure

to high insulin levels; these both lead to a decrease in the number of receptors. The

interaction of insulin with the receptor on the cell surface sets off a chain of messengers

within the cell. This opens up transport processes for glucose, amino acids and electrolytes.

Acute deficiency of insulin leads to unrestrained hepatic glycogenolysis and gluconeogenesis

with a consequent increase in hepatic glucose output. Glucose uptake is decreased in insulin

sensitive tissues and hyperglycemia occurs, either as a result of the metabolic disturbance

itself or secondary to infection or other acute illness, there is increased secretion of the

counter regulatory hormones Glucagons, catecholamine and growth hormone. All of this will

further increase hepatic glucose production.

Figure No.2: Patho-physiologic defects of type II diabetes

INTRODUCTION


G. Type-II Diabetes Mellitus

i. Synonyms:

1. Non-insulin dependent diabetes mellitus (NIDDM)

2. Maturity onset diabetes mellitus

ii. About:

Type-II diabetes is a metabolic disorder that results from complex interactions

of multiple factors and is characterized by 2 major defects: decreased secretion of

insulin by the pancreas and resistance to the action of insulin in various tissues

(muscle, liver and adipose), which results in impaired glucose uptake. NIDDM is

heterogeneous and the results of an interaction between genetic and environmental

factors. Type II diabetes represents about 90% of all diabetes cases among persons

older than 45 years of age, approximately 18% of persons 65 to 75 years of age and

40% of those older than 80 years of age. It is further of 2 types obese and non-

obese type II diabetes. Generally has a late onset (past middle age). Over90%

cases are type II diabetes mellitus.

iii. Causes:

The causes are abnormality in glucose receptor of β cells so that they respond

at higher glucose concentration, reduced sensitivity of peripheral tissues to insulin

and excess of hyperglycemic hormones (glucagon).

INTRODUCTION


Figure No.3: Causes of type-II diabetes

iv. Characteristics

1) Usually occurs after 30 years of age, but is now occurring in

children and adolescents.

2) Increased prevalence in some ethnic groups.

3) Strong genetic predisposition.

4) Frequently obese.

5) May or may not have symptoms of hyperglycemia.

6) May also have extreme tiredness, blurred vision, delayed healing,

numbness and tingling of hands and feet, recurring yeast infection.

7) Children between the ages of 10-19 yrs that have one or more of the

following are at an increased risk: family history, Member of certain

ethnic populations.

INTRODUCTION


v. Treatment:

1) Diet/weight management

2) Exercise/increase physical activity

3) Oral hypoglycemic/anti hyperglycemic agents

a. Agents which increase the amount of insulin secreted by the pancreas

b. Agents which increase the sensitivity of target organs to insulin and

c. Agents which decrease the rate at which glucose is absorbed from the

GIT

4) Education

5) Monitoring

6) Treatment of co morbid conditions.

E.g. Hypertension, lipid abnormalities.

Figure No.4: Schematic diagram of insulin resistance progresses towards type-II diabetes

INTRODUCTION


vi. Overview of the pathogenesis12

:

Any rise in glycaemia is the net result of glucose influx exceeding glucose outflow

from the plasma compartment. In the fasting state, hyperglycemia is directly related to

increased hepatic glucose production. In the postprandial state, further glucose excursions

result from the combination of insufficient suppression of this glucose output and defective

insulin stimulation of glucose disposal in target tissues, mainly skeletal muscle. Once the

renal tubular transport maximum for glucose is exceeded, glycosuria curbs, though does not

prevent, further hyperglycemia. Abnormal islet cell function is a key and requisite feature of

type 2 diabetes. In early disease stages, insulin production is normal or increased in absolute

terms, but disproportionately low for the degree of insulin sensitivity, which is typically

reduced. However, insulin kinetics, such as the ability of the pancreatic b-cell to release

adequate hormone in phase with rising glycaemia, are profoundly compromised. This

functional islet incompetence is the main quantitative determinant of hyperglycemia and

progresses over time. In addition, in type 2 diabetes, pancreatic a-cells hyper secrete

glucagon, further promoting hepatic glucose production. Importantly, islet dysfunction is not

necessarily irreversible. Enhancing insulin action relieves b-cell secretory burden, and any

intervention that improves glycaemia from energy restriction to, most strikingly, bariatric

surgery can ameliorate b-cell dysfunction to an extent. More recently recognized

abnormalities in the incretin system (represented by the gut hormones, glucagon-like peptide

1 [GLP-1], and glucose-dependent insulin tropic peptide [GIP]) are also found in type 2

diabetes, but it remains unclear whether these constitute primary or secondary defects. In

most patients with type 2 diabetes, especially the obese, insulin resistance in target tissues

(liver, muscle, adipose tissue, myocardium) is a prominent feature. This results in both

glucose overproduction and underutilization. Moreover, an increased delivery of fatty acids

to the liver favors their oxidation, which contributes to increased gluconeogenesis, whereas

INTRODUCTION


the absolute overabundance of lipids promotes hepatosteatosis. Anti-hyperglycemic agents

are directed at one or more of the pathophysiological defects of type 2 diabetes, or modify

physiological processes relating to appetite or to nutrient absorption or excretion. Ultimately,

type 2 diabetes is a disease that is heterogeneous in both pathogenesis and in clinical

manifestation a point to be considered when determining the optimal therapeutic strategy for

individual patients.

1.2. Anti-Diabetic Agents15

Anti-diabetic drugs are medicines that help to control blood sugar levels in people with

diabetes mellitus (sugar diabetes). Anti-diabetic drugs treat diabetes mellitus by lowering

glucose levels in the blood. There are different classes of anti-diabetic drugs, and their

selection depends on the nature of the diabetes, age and situation of the person, as well

as other factors. The treatment protocol needs to be individualized and can be developed only

after the type of diabetes has been categorized.

Classification of anti-diabetic agents 15

These are classified as follows:

I. Sulfonylurea’s

A) First generation analogs

1. Tolbutamide

2. Chlorpropamide

3. Acetohexamide

4. Tolazamide

B) Second generation analogs

1. Glipizide

2. Glibenclamide

3. Gliclazide

INTRODUCTION


4. Glimepiride

II. Biguanides

1. Phenformin

2. Metformin

III. Meglitinide/Phenyl Alanine Analogues

1. Repaglinide

2. Nateglinide

IV. Thiazolidinediones

1. Rosiglitazone

2. Pioglitazone

V. α-Glucosidase Inhibitors

1. Acarbose

2. Miglitol

Advantages of oral therapy in diabetes15

1. Patient acceptability

2. Ease of administration

3. No need of exogenous insulin – hence decreased insulin antigenicity

4. Insulin – being endogenous – physiological major action on liver and less at

periphery

5. Less frequent and less severe hypoglycemia when compared to insulin therapy

Disadvantages of oral therapy in diabetes15

1. Less of medical supervision

2. Disinclination towards potential dangers of diabetes

3. Drug interactions

4. Toxic reactions – though rare could be serious

INTRODUCTION


5. Limitations of dosage and inflexible dosage

6. Increased incidence of therapeutic failure with the passage of time

Mechanism of TGZ16

:

Thiazolidinediones, such as pioglitazone, are synthetic ligands

for peroxisome proliferator-activated receptors (PPARs). They alter the transcription of genes

influencing carbohydrate and lipid metabolism, resulting in changed amounts of protein

synthesis and, therefore, metabolic changes. Pioglitazone improves glycaemic control in

people with Type 2 diabetes by improving insulin sensitivity through its action at PPAR

gamma 1 and PPAR gamma 2, and affects lipid metabolism through action at PPAR alpha.

The results of these interactions include increases in glucose transporters 1 and 4, lowered

free fatty acids, enhanced insulin signalling, reduced tumour necrosisfactor alpha (TNF

alpha) and remodelling of adipose tissue. Together, these can increase glucoseuptake and

utilisation in the peripheral organs and decrease gluconeogenesis in the liver, thereby

reducing insulin resistance.

1.3 Modified release drug delivery system17

:

The term modified – release dosage form is used to describe products that alter the

timing and rate of release of drug substance. A modified-release dosage form is defined “as

one for which the drug release characteristics of time course and/or location are chosen to

accomplish therapeutic or convenience objectives not offered by conventional dosage forms

such as solutions, ointments, or promptly dissolving dosages forms.

The modified drug delivery systems can be divided into the following categories: 18

Delayed released

Controlled released

Sustained released

Extended released

http://europepmc.org/abstract/MED/11594239/?whatizit_url_Chemicals=http://www.ebi.ac.uk/chebi/searchId.do?chebiId=CHEBI%3A50864


http://europepmc.org/abstract/MED/11594239/?whatizit_url_go_term=http://www.ebi.ac.uk/ego/GTerm?id=GO:0005777

http://europepmc.org/abstract/MED/11594239/?whatizit_url_gene_protein=http://www.uniprot.org/uniprot/?query=PPARs&sort=score




http://europepmc.org/abstract/MED/11594239/?whatizit_url=http://europepmc.org/search/?page=1&query=%22Type%202%20diabetes%22

http://europepmc.org/abstract/MED/11594239/?whatizit_url_gene_protein=http://www.uniprot.org/uniprot/?query=insulin&sort=score

http://europepmc.org/abstract/MED/11594239/?whatizit_url_gene_protein=http://www.uniprot.org/uniprot/?query=PPAR%20gamma&sort=score




http://europepmc.org/abstract/MED/11594239/?whatizit_url_gene_protein=http://www.uniprot.org/uniprot/?query=PPAR%20alpha&sort=score

http://europepmc.org/abstract/MED/11594239/?whatizit_url_gene_protein=http://www.uniprot.org/uniprot/?query=glucose%20transporters&sort=score


http://europepmc.org/abstract/MED/11594239/?whatizit_url=http://europepmc.org/search/?page=1&query=%22tumour%20necrosis%22

http://europepmc.org/abstract/MED/11594239/?whatizit_url_gene_protein=http://www.uniprot.org/uniprot/?query=TNF%20alpha&sort=score

http://europepmc.org/abstract/MED/11594239/?whatizit_url_gene_protein=http://www.uniprot.org/uniprot/?query=TNF%20alpha&sort=score




INTRODUCTION


Site specific targeting

Receptor targeting

Delayed release systems19

The design of such systems involves release of drug only at specific site in the GIT.

The drugs contained in such a system are those that are:

• Destroyed in the stomach or by intestinal enzymes

• Known to cause gastric distress

• Absorbed from a specific intestinal site

• Meant to exert local effect at a specific GI site

The two types of delayed release systems are intestinal release systems and colonic release

systems.

Figure No.5: Relationship of pharmaceutical Delayed Release dosage forms18

1.3 Mucilages as Natural Polymer:

Mucilages are naturally occurring, high molecular weight Polyuroides consisting of

sugars and Uronic acid units. They are normal physiological metabolism products formed

within the cell/deposited on it in layers20.

Mucilages are most commonly used adjuvant in

pharmaceutical preparations. They swell in water and form a gel, such phenomenon is often

INTRODUCTION


called as rheology synergism21

. Mucilages found in rhizomes, roots and seed endosperm may

act primarily as energy reserves whereas foliar mucilages appear not to serve as storage

carbohydrates22

. Plant mucilages are well known, since ancient times, for their medicinal use.

In recent years, plant mucilages have evoked tremendous interest due to their diverse

applications in pharmacy, for formulation of both solid and liquid dosage forms. Plant

mucilages are pharmaceutically important polysaccharides with a wide range of applications

such as thickeners, binding agents, water retention agents, emulsion stabilizers, suspending

agents, disintegrants, gelling agents, and film formers23

. Mucilages find applications in tablet

formulation as binders because of their adhesive nature. They impart cohesiveness to the

powder mass and convert them into granules, which are further compressed into tablets. They

can swell upto 5times their original volume and this swelling leads to breakage of tablets into

smaller pieces, which in turn improves the dissolution rate thereby making them a favorable

candidate as disintegrating agents24

. In several cases, the polysaccharides, resins or the

tannins present in the gums are responsible for imparting release retardant properties to the

dosage form25

.

1.4 Fenugreek:

Fenugreek is as one of the oldest cultivated medicinal plants identified in written

history, and many studies showed that the seeds acquire anti-oxidant properties in seeds and

leaves of fenugreek. India indicates its value in commerce as far back as 2000 -1700. B. C.

India is one of the major producers of the fenugreek [Trigonella foenumgraecum L.] in the

world, and the production is about 45,000-55,000 tonnes per annum.3 Fenugreek have

originated in the Mediterranean region of the "Old World" or parts of Asia and recent years,

it was suggested so as to fenugreek originated in Turkey. Fenugreek is all over the world

(Table 1). The most accurate number of species of fenugreek has not been identified till

now. Taxonomists such as Linnaeus noted that 18 species of Trigonella are currently in a

INTRODUCTION


total of 260 species. Most species, including Trigonella foenum graecum L., are diploids

with 2n = 16 chromosomes. However, some species of Trigonella may include 18, 28, 30,

32 or 44 chromosomes.

Table No.2: Taxonomy of Trigonella foenum-graecum

Kingdom Plantae

Superdivision Angiosperms

Division Eudicots

Class Rosids

Order Fabales

Family Fabaceae

Subfamily Faboideae

Tribe Trifolieae

Genus Trigonella

Species Foenum

Pharmacological attributes:

Fenugreek has different pharmacological attributes such as a hypoglycemic,

hypercholesterolemia, gastro protective, chemo-preventive, anti-oxidant, and laxative and

appetite stimulation. The plant contains alkaloids, flavonoids, salicylate, and nicotinic acid.

Fenugreeks are harmless for human consumption. The biological and pharmacological effects

of fenugreek has related to the variety of its components namely, steroids, N-compounds,

polyphenolic substances, volatile constituents, and amino acids etc. Fenugreek 45-60%,

(galactomannans), 20-30% proteins high in lysine tryptophan, 5-10% (lipids), pyridine

alkaloids, Trigonelline (0.2-0.38%), Choline (0.5%), Carpaine gentianine, Flavonoids

luteolin, Apigenin, Quercetin, Orientin, Isovitexin vitexin, amino as 4-hydroxyisoleucine

INTRODUCTION


(0.09%), Histidine, Arginine Lysine, Calcium, Saponins, Glycosides steroidal Sapogenins on

hydrolysis (yamogenin, diosgenin, neotigogenin, tigogenin), Sitosterol cholesterol, vitamin

A, B1, C nicotinic26

.

Table No.3: Potential Medicinal Values of Trigonella foenum-graecum

Traditional Uses Pharmacological Activities Side Effects

To treat arthritis, asthma,

bronchitis, improve digestion,

increase libido

and male potency, to cure

skin

problems (wounds, rashes

and

boils), to treat sore throat,

and cure

acid reflux, treatment of

reproductive disorders, to

induce

labor, to treat hormonal

disorders,

to help with breast

enlargement,

and to reduce menstrual pain,

blood Sugar Regulation

Anti-diabetic

Anti-inflammatory

Anti-toxic

Anti-cancer

Hypoglycemic,

hypercholesterolemia,

gastroprotective,

chemopreventive,

antioxidant, laxative,

appetite stimulation,

Anti-cataract,

Immunomodulatory activity,

Anti-atherogenic

Minor side effects such as

Nausea, Gastrointestinal

discomfort (diarrhea

and/or

gas)

INTRODUCTION


It is shown that T foenum graecum seeds have hypoglycemic property,

hypocholesterolemic and hyperinsulinemic effects in patients with type 1 and 2 diabetes

mellitus and experimental diabetic animals. T foenum graecum seeds lowers blood glucose,

reduces levels of glycated hemoglobin, and reduces lipidemia in streptozotocin(STZ) -

induced diabetic rats in a dose dependent manner. In addition to its hypoglycemic effects, the

T foenum graecum seeds have also been reported to restore the altered antioxidant status in

various tissues due to diabetes-induced oxidative stress. T foenum graecum seed extract has

been reported to prevent both lipid peroxidation and red blood cell oxidative hemolysis. An

ethyl acetate extract of the T foenum graecum seed significantly decreased the content of

catalase and superoxide dismutase (SOD) activities in the liver, heart, and kidneys of rats that

were fed a cholesterol-rich diet27

. It is used with xanthan gum as a viscosity enhancing

agent28

. The leaves, seeds (whole and gum), chemical fractions such as hydroxyisoleucine as

well as its tender shoots have exhibited antioxidant, anti-diabetic and hypocholesterolemic

characteristics. The potential of fenugreek could be significant for the pharmaceutical and

food industry due to its proposed dual positive impact on hyperglycemia and

hypercholesterolemia. The pharmacological characteristics exhibited by fenugreek in diabetes

mellitus have been related to its insulin secretagogue actions, its effects on peripheral glucose

utilization and the action of the gum fiber on the intestines29

.

Anti-Diabetic Activity 30

Preliminary animal and human trials suggest possible hypoglycemic and anti-

hyperlipedemic properties of fenugreek seed powder taken orally. Fenugreek has been well

known to be used as anti-diabetic remedy for both type I and II diabetes and has been

extensively used as a source of anti-diabetic compounds, from its seeds, leaves and extracts in

different model systems. About 25-50 g fenugreek seeds were given to diabetic patients daily

in diet to prevent and manage long term complications of diabetes and studies have been

INTRODUCTION


made about the glycemic index of fenugreek recipes which showed that the soluble fenugreek

fiber has significantly reduced the glycemic index. On the other hand, water extract of

fenugreek seeds has higher hypoglycemic and anti-hyperglycemic potential and for this

reason it may be used as a supplementary medicine to treat the diabetic population by

significantly reducing the dose of standard drugs. Since fenugreek seeds are a source of

protein, they can replace pulses in the diets of diabetics. 25-50 g fenugreek in the diet of

diabetic patients (taken daily) can be an effective supportive therapy in the management of

diabetes. The bioactive compounds with respect to diabetic conditions include the

galactomannan-rich soluble fiber fraction of fenugreek which may be responsible for the anti-

diabetic activity of the seeds.

Figure No.6: Mechanism of Anti-diabetic Activity of T. foenum-graecum31

OBJECTIVES Failure comes only when we forget our ideals &

objectives.

OBJECTIVES


2. OBJECTIVES

The main aim of the present work was to formulate and evaluate Oral, delayed release

dosage form containing anti-diabetic agent by using natural polymer, i.e. to study the effect

of Trigonella foenum graecum mucilage as binder in combination with oral hypoglycemic

agent.

2.1 Need for the Study:

Pioglitazone is an effective oral anti–diabetic agent that belongs to the thiazolidonediones

drug class. Pharmacological studies indicate that pioglitazone improves glycemic control while

reducing circulating insulin level32

. Pioglitazone has short biological half-life of 3-6 hours and is

eliminated rapidly33.

The drug causes gastro intestinal disturbances such gastric pain,

constipation, nausea and vomiting if present in larger concentration in G.I. tract. Therefore

delayed release (DR) products are needed for pioglitazone to prolong its duration of action and to

improve patience compliance. Delayed release formulation is also needed for pioglitazone for

better control of blood glucose levels to prevent hypoglycemia and enhance clinical efficacy, to

reduce G.I. disturbances and to enhance patient compliance. There are few reports34

on the

formulation of pioglitazone employing coated granules and matrix tablets but no delayed release

formulations of pioglitazone are available commercially. Hence delayed release tablets of

Pioglitazone were designed employing natural polymer Fenugreek mucilage.

2.2 Primary Objectives of the Study

To improve therapeutic efficiency.

To reduce adverse side effects and to improve its tolerability.

To promote usage of natural excipients.

OBJECTIVES


To reduce adverse side effects of synthetic excipients.

To improve patient compliance.

Reduction in health care cost.

2.3 Secondary Objectives of the Study:

1. To isolate and investigate the suitability of the Fenugreek seed mucilage as excipient.

2. To carry out the compatibility studies for possible drug and polymer interactions by FT-

IR studies.

3. To prepare Oral, delayed release dosage form containing Anti-diabetic agent.

4. To carry out various in-vitro evaluation parameters.

5. Treatment of dissolution data with various mathematical models.

6. To carry out short-term stability study for best selected formulations.

7. To carry out in-vitro glucose uptake assay.

2.4 Plan of the work:

1. To isolate mucilage from seed of Trigonella foenum graecum.

2. To carry out characterization studies like organoleptic evaluation, Solubility, Percentage

yield, Physiochemical tests, Determination of Swelling index, and Viscosity

determination.

3. To study the factors for mucilage powder blend such as Angle of repose, Bulk density,

tapped density and powder flow property.

4. To carry out the compatibility studies for possible drug and polymer interactions by FTIR

studies.

5. Literature survey

OBJECTIVES


6. Selection of drug candidate for delayed release dosage form based on Physical and

Biopharmaceutical properties.

7. Procurement of drug, polymer and excipients.

8. To carry out Preformulation studies like Drug solubility, Drug excipient compatibility

melting point, angle of repose, bulk density, tapped density, Carr’s Index and Hausner’s

ratio.

9. Determination of λmax for Pioglitazone in 0.1N HCL (pH 1.2).

10. Preparation of standard calibration curve of Pioglitazone.

11. To develop experimental designing for formulation and characterization of Pioglitazone

delayed release tablet.

12. Development of delayed release tablets of Pioglitazone using polymers like FGM, PVPK30

by non-aqueous wet granulation method.

13. Evaluation of the formulations for weight variation, hardness, friability, drug content and

in-vitro disintegration release studies.

14. To study in-vitro dissolution of pioglitazone tablets in 2different mediums 0.1N HCL (pH

1.2) and Phosphate buffer (pH 7.4)

15. To fit the resultant data to various kinetic models by curve fitting analysis.

16. To suggest a suitable mechanism of drug release based on the curve fitting analysis.

17. To carry out short-term stability study for best selected formulation.

18. To carry out cytotoxicity studies

19. To carry out in-vitro glucose uptake assay.

OBJECTIVES


2.5 Brief Outline of the work:

Isolation of mucilage from seeds of Trigonella foenum graecum.

Characterization of mucilage

Literature survey and selection of Drug

Formulation of tablets by non-aqueous wet granulation

Pre-compression Evaluation

Compatibility study of Drug and Polymer interactions by FT-IR studies

Characterization of Drug and Preformulation Studies

Preparation of Standard curve of the Drug

Post-compression Evaluation

Stability Studies

In-vitro Glucose uptake study

Cyto toxicity test

REVIEW OF LITERATURE To steal ideas from one person is called Plagiarism,

To steal ideas from many is called Research.

REVIEW OF LITERACTURE


3. REVIEW OF LITERATURE

Literature survey was carried out on the proposed topic by referring various scientific journals,

online and offline also referred various text books available in college library. This survey

reveals that no such articles were reported on the proposed work and some related articles are

mentioned below.

A study conducted by Nokhodchi A, et al., in which they evaluated fenugreek mucilage as a

potential excipient for oral controlled-release matrix tablet. An increase in concentration of

the mucilage in matrices resulted in a reduction in the release rate of propranolol

hydrochloride comparable to that observed with hypomellose matrices as standard. The rate of

release of Propranolol hydrochloride from fenugreek mucilage matrices was mainly controlled

by the drug: mucilage ratio. The presence of lactose in matrices containing mucilage increased

the release rate of propranolol hydrochloride. This is due to a reduction in tortuoisity and

increased pore size of channels caused by lactose through which propranolol diffuses and

therefore diffusion of water into the tablet is facilitated35

.

Amit Kumar Nayak, et al., have been developed a Calcium pectinate-fenugreek seed

mucilage (FSM) mucoadhesive beads containing metformin HCL through ionic-gelation for

controlled delivery of metformin HCL. Effects of pectin and FSM amounts on drug

encapsulation efficiency (DEE) and cumulative drug release at 10 h (R10 h) were optimized

using 32 factorial designs. The optimized beads also exhibited good mucoadhesivity and

significant hypoglycemic effect in alloxan-induced diabetic rats over prolonged period after

oral administration36

.

http://www.ncbi.nlm.nih.gov/pubmed?term=Nokhodchi%20A%5BAuthor%5D&cauthor=true&cauthor_uid=18363148



Indranil Kumar Yadav, et al., conducted a study in which they investigated fenugreek

mucilage as sustained release matrix forming material in Diclofenac sodium tablet

formulations. In that study sustained release matrix tablets of Diclofenac sodium, were

developed by using different drug polymer ratios. Moreover compressed tablets were

evaluated for uniformity of weight, content of active ingredient, friability, hardness, thickness,

in vitro dissolution using paddle method and swelling behavior. All the formulations showed

compliance with pharmacopoeial standards. In their study the dissolution study proved that

the seeds mucilage of fenugreek can be used as a matrix forming material for making once

daily Sustained release matrix tablets of Diclofenac sodium. Stability studies also have been

done as per the ICH guidelines37

.

V. Senthil, et al., conducted a study in which they formulated and evaluated Paracetamol

Suspension from Trigonella Foenum Graecum Mucilage by involving extraction of

suspending agent from the Trigonella foenum graecum (fenugreek) seeds, solubility testing of

the mucilage obtained, phytochemical testing, determination of swelling index, preparation of

Paracetamol suspension (blank), determination of sedimentation volume, measurement of

viscosity, and determination of flow rate. In their study the swelling index was found to be

150% and sedimentation volume by using fenugreek as a suspending agent shows highest

sedimentation volume than acacia, tragacanth and Paracetamol alone. They concluded that

fenugreek can be employed as a stabilizer of choice and high viscosity is desired especially in

cosmetic, pharmaceutical and food industries38

.

A study conducted by Ajay Kumar Sav, et al., in which they investigated an extended release

formulation of Theophylline using modified fenugreek gum as a hydrophilic polymer. In their

study various formulations were prepared and evaluated for their physical properties such as



tablet hardness, weight variation, drug content and In vitro drug release. The optimized

formulation was found to show stability for two month as per ICH guideline and the study

suggested that modified fenugreek gum can be used as extended release polymer39

.

Olfa Belguith-Hadriche, et al., were investigated the hypocholesterolemic and antioxidant

activities of various extracts (water, methanol, ethyl acetate, hexane, dichloro-methane) of

fenugreek seeds in cholesterol-fed rats in their study. They concluded that only ethyl acetate

extract of the fenugreek seeds had a significant hypocholesterolemic effect and antioxidant

activity in cholesterol-fed rats, whether this is partly due to the presence of flavonoids in the

extract40

.

Manoj M Nitalikar, et al., examined a study technique for division of husk part of T. foenum

graecum (fenugreek). A variety of physicochemical criteria counting angle of repose,

distribution of particle and swelling factor were determined. As a binder husk of fenugreek in

tablets was studied. To optimize the binding potential of dispersion of methi husk in tablets, as

a sculpt drug Ibuprofen was chosen. Assessment of dispersion of husk with paste of starch

was done. The greatest amount needed of the dispersion of husk was 4-6% as a binder, which

is comparatively less in contrast to standard. Dispersion of methi husk was established to be

advanced over paste of starch41

.

Naser Tavakoli, et al., intention of the nearby examination was to assess the binding potential

of fenugreek mucilage in formulation. Mucilagenous part of fenugreek seed was secluded and

employed in a role of fastening agent in all diverse medicines. Model drugs chosen were

Theophylline (TH), Ibuprofen (IB), Calcium acetate (CA). The consequences demonstrated

that a 2.5% amount of the novel binder in contrast to typical binders studied42

.



Waugh J, et al., has reviewed that pioglitazone is an anti hyperglycaemic agent, that in the

presence of insulin resistance, increases hepatic and peripheral insulin sensitivity, thereby

inhibiting hepatic gluconeogenesis and increasing peripheral and splanchnic glucose uptake

and pioglitazone can be used as monotherapy or in combination with metformin, repaglinide,

insulin or a sulfonylurea, thus offers an effective treatment option for management of patients

with type 2 diabetes43

.

Gilles PS, et al., reviewed that pioglitazone is an insulin sensitizing thiazolidinedione agent

that has been developed for the treatment of type 2 diabetes mellitus which activates the

nuclear peroxisome proliferators activated receptor(PPARγ), leading to increased transcription

of various proteins regulating glucose and lipid metabolism. These proteins amplify the post-

receptor actions of insulin in the liver and peripheral tissue, which leads to improved

glycaemic control with no increase in the endogenous secretion of insulin44

.

Singh C, et al., studied on formulation and evaluation of extended release tablet of

Pioglitazone by melt granulation technique and concluded that, Pioglitazone is a potent and

highly selective agonist for peroxyzome proliferators-activated receptor-gamma (PPAR).

Pioglitazone has short biological half-life of 3-5 hrs & is eliminated rapidly. Therefore Matrix

type tablets of Pioglitazone were developed by using polymer such as Precirol ATO5,

Campritol 888 ATO, Carnuba wax & Hydrogenated castor oil by melt Granulation technique.

The tablets were initially placed in phosphate buffer at pH 7.4 at 8 hrs used dissolution

apparatus USP-245

.

Rajendran N.N, et al., The present study was to establish Bi‐layer tablets containing

Metformin HCl as sustained release and Pioglitazone HCl as immediate release layer. The



formulations (P6M7) having immediate release layer produces immediate effect within 54

second followed by sustained release (97.35%) at 8 hrs46

.

Hatorp, et al., conducted an in vitro study to reviewed that all three thiazolidinediones

(Troglitazone, Pioglitazone and Rosiglitazone) have the potential to induce CYP3A447

.

Chowdary K.P.R, et al., they aimed to enhance the solubility and impart a controlled release

pioglitazone -βCD matrix tablets; pioglitazone is an oral hypoglycemic agent which belongs

to Class II of BCS with relatively short elimination half-life. Inclusion complex of

pioglitazone with β-cyclodextrin was prepared by kneading, co-precipitation, physical

mixture and evaluated for its in-vitro release. The dissolution study of kneading complex

shows significant increase in the drug release from kneading complex than pure drug and

physical mixture48

.

Srinivasa rao .Y, et al., prepared floating tablets of pioglitazone hydrochloride with three

different grades of HPMC K100M,K15M,K4M,MCC,sodium bicarbonate ,magnesium

stearate and talc were used as variant along with pioglitazone hydrochloride as active

pharmaceutical ingredient. Sodium bicarbonate (16%) employed as gas generating agent for

twelve formulation enable tablets to float. Among all formulation tablet prepared with

HPMC K100M, MCC and PVP shows slow and spread over 24h. These tablets exhibited a

floating time of 48h after a floating lag time less than 180 sec. Drug release was diffusion

controlled and followed zero-order Kinetics49

.

Rangapriya .M, et al., prepared floating tablets of Pioglitazone Hydrochloride containing

HPMC of different viscosity grades and poly vinyl pyrolidone to achieve a sustained release

for 24 hrs. The tablets of all formulation were subjected to various physico –chemical

evaluation parameters such as thickness, diameter, weight variation, hardness, friability, drug



content, in-vitro buoyancy lag time, total floating time, tablets density, swelling index and in-

vitro dissolution study. The results of all these tests were found to be satisfactory within the

prescribed limits. The formulations showed higher R2 values for zero order plots indicating

that drug release followed zero order kinetics and drug release from these floating tablets

were by both diffusion and erosion50

.

G.Chinna devi, et al., formulated and evaluated floating tablets of pioglitazone employing

olibanum gum, a natural gum resin in comparison to HPMC K15 M, a synthetic cellulose

derivative. Floating tablets of pioglitazone were prepared employing olibanum gum and

HPMC K15 M as matrix formers, sodium bicarbonate as gas generating agent and bees wax

and ethyl cellulose as floating enhancers and the tablets were evaluated for In vitro buoyancy

and drug release characteristics. The floating characteristics of the formulations which

contained sodium bicarbonate (15%) alone were not satisfactory with both the two polymers

and need to be improved. Increasing the strength of sodium bicarbonate from 15% to 20% has

not much improved the floating characteristics. Addition of bees-wax (15%) and ethyl

cellulose (5%) has significantly enhanced the buoyancy of the tablets formulated with both the

two polymers. Pioglitazone release from the floating tablets prepared was slow and spread

over 24 h and depended on the polymer used and composition of the tablets. Drug release was

diffusion controlled and followed zero order kinetics. Non-Fickian diffusion was the drug

release mechanism from all the tablets formulated. Pioglitazone release from the tablets

containing beeswax and ethyl cellulose along with the matrix forming polymers was slow and

spread over more than 24 h. The T90 values were in the range 19-24 h with these tablets.

These tablets exhibited a floating time of 44 h after a floating lag time in the range 2-6 min.

Olibanum is found suitable as matrix former for floating tablets and is comparable to HPMC



K15M, a widely used polymer for floating tablets and for controlled release. Since olibanum

gum is of natural origin, it is non-toxic, biocompatible and cheaper51

.

Sammour OA, et al., reviewed that PVPK30 has the ability to interact with poorly water-

soluble drugs and drug candidates resulting in an increase in their apparent water solubility.

The mechanism for this solubilisation is rooted in the ability of PVPK30 to cause reduction

in interfacial tension between the drug and the dissolving solution. The increase in the

solubility in the presence of PVPK30 can also be explained by increase in wettability of

rofecoxcib52

.

V. Kalvimoorthi, et al., conducted a study in which six formulations of delayed release

tablets were prepared by the direct compression method and simple pan coating using Drug

coat N-100 and Hydroxy propyl methyl cellulose phthalate (HPMCP) as enteric coating

polymers. The in vitro drug release was carried in pH 1.2 HCl and pH 6.8 phosphate buffer

using USP dissolution Apparatus 2 at 100 rpm. They concluded that F4 batch was

considered to be the best enteric formula it shows 84.23% drug release at end of 45 min in

the phosphate buffer53

.

P. Suresh Kumar, et al., attempts were made in the present investigation to prepare a stable

composition of delayed release tablets of rabeprazole sodium. They concluded that the

prepared formulation offers effective resistance in acidic environment and starts its release in

the alkaline environment of small intestine. Thus, Instacoat EN-HPMCP A34G00031 Yellow

can be successfully employed to retard the release pattern of Rabeprazole sodium thereby

enhancing the therapeutic efficacy54

.

Damodharan et al., have formulated and developed a delayed release doxycycline tablets by

enteric coating using pH dependent polymers like Eudragit and HPMC. Six batches (F1 to



F6) were formulated and evaluated for hardness, friability, weight variation, drug content,

disintegration and in-vitro dissolution study. Among them, batch F4 which contains 25 gms

of Eudragit polymer have shown 94% drug release hence this batch was considered to be best

formulation55

.

Kalvimoorthi, et al., have been developed a tablet formulation of aspirin for delayed release

of drug by direct compression method as enteric coated tablets. Six different formulations

(F1 to F6) with various concentrations of polymers were formulated and evaluated the

preformulation studies, the dissolution of F4 showed % drug release of 84.23 at the end of 45

min in phosphate buffer56

.

METHODOLOGY We must revisit that science is a methodology and not

An Ontology.

MATERIALS AND METHODS


4. MATERIALS AND METHODS

4.1 MATERIALS USED:

Table No.4: List of chemicals used

Sl.no Materials Name of the Supplier

1 Pioglitazone Dr.Reddy’s labs, Hyderabad, India

2 PVPK30 Loba chemie pvt.ltd, Mumbai.

3 Magnesium stearate S.D. Fine Chemical Ltd, Mumbai.

4 Talc S.D. Fine Chemical Ltd, Mumbai.

5 Lactose S.D. Fine Chemical Ltd, Mumbai.

6 Ethanol Fine Chemicals Limited, Delhi.

7 3-(4,5–dimethyl thiazol–2–yl)–5–

diphenyl tetrazolium bromide (MTT)

Sigma Aldrich Co, St Louis, USA

10 Dulbecco’s Modified Eagle’s Medium

(DMEM) Sigma Aldrich Co, St Louis, USA

13 Glucose

Hi-Media Laboratories Ltd., Mumbai

15 Dimethyl Sulfoxide (DMSO)

E.Merck Ltd., Mumbai, India.

17 Bovine Serum Albumin (BSA)

Sigma Aldrich Co, St Louis, USA

18 Insulin (40IU/ml) Torrent Pharmaceuticals



4.2 EQUIPMENTS USED:

Table No.5: Details of Equipment’s used

Sl.no Instrument Manufacturer

1 Electronic Balance Acculab

2 Hot Air Oven Kadavil electro mechanical industries, Kerala,

India.

3 UV-Visible spectrophotometer Spectrophotometer UV-1800, Shimadzu,

Japan.

4 FT-IR Spectrophotometer Thermo Nicolet 380, India

5 Tablet Punching Machine LAB PRESS, Cip Machineries Pvt.Ltd.

Ahmadabad, India

6 Roche Friabilator PSM Industries, Bangalore, India

7 Monsanto Hardness Tester Techno scientific products, Bangalore, India.

8 Digital pH meter Techno scientific products.

9 Disintegration test apparatus SiiSerwell Instruments INC, Bangalore, India

10 Dissolution test apparatus Labindia instruments Pvt. Ltd, DS 8000,

Navimumbai, India.

11 Stability chamber (106 Model) LABTOP, SKY Lab Instruments &

Engineering Pvt. Ltd. India

12 CO2 Incubator Naire

13 Cooling centrifuge REMI

15 Inverted tissue culture microscope Motic

16 ELISA Reader Biotek India



4.3. DRUG PROFILE57,58

:

Name: Pioglitazone

Structure:

Figure No.7: Structure of Pioglitazone

PROPERTIES

Synonym

Pioglitazona

Pioglitazone

Pioglitazonum

Chemical Name 5-({4-[2-(5-ethylpyridin-2-yl)ethoxy]phenyl}methyl)-1,3-

thiazolidine-2,4 dione

CAS number 111025-46-8

Category Hypoglycemic Agents

Molecular formula C19H20N2O3S

Molecular weight 392.91

Appearance Amorphous powder

Physical State Solid

Color White

http://www.drugbank.ca/mesh/hypoglycemic-agents

http://en.wikipedia.org/wiki/File:Pioglitazone.svg



Odor Characteristic

Solubility

Soluble in water (<1 mg/ml at 25 °C), DMF, methanol,

ethanol (4 mg/ml at 25 °C), and DMSO (79 mg/ml at 25 °C).

Melting Point 192-195 °C

Refractive Index n20

D 1.61 (Predicted)

pKa (Strongest Acidic) 6.66

pKa (Strongest Basic) 5.6

Salt form Pioglitazone Hydrocholoride

TAXONOMY

Description

This compound belongs to the class of organic compounds

known as phenol ethers. These are aromatic compounds

containing an ether group substituted with a benzene ring.

Kingdom Organic compounds

Super Class Benzenoids

Class Benzene and substituted derivatives

Sub Class Phenol ethers

Direct Parent Phenol ethers

http://classyfire.wishartlab.com/tax_nodes/C0000000







Alternative Parents

Thiazolidinediones

Alkyl aryl ethers

Pyridines and derivatives

Heteroaromatic compounds

Thioethers

Carboxylic acid amides

Azacyclic compounds

Organonitrogen compounds

Hydrocarbon derivatives

Carbonyl compounds

Substituents

Phenol ether

Thiazolidinedione

Alkyl aryl ether

Pyridine

Heteroaromatic compound

Thiazolidine

Carboxamide group

Azacycle

Organoheterocyclic compound

Thioether

Ether

Carboxylic acid derivative

Hydrocarbon derivative













Organooxygen compound

Organonitrogen compound

Carbonyl group

Aromatic heteromonocyclic compound

Molecular Framework Aromatic heteromonocyclic compounds

PHARMACOLOGY

Indication Treatment of Type II diabetes mellitus

Pharmacodynamics

Pioglitazone, a member of the drug group known as the

thiazolidinediones or "insulin sensitizers", is not chemically

or functionally related to the alpha-glucosidase inhibitors, the

biguanides, or the sulfonylureas. Pioglitazone targets insulin

resistance and, hence, is used alone or in combination with

insulin, metformin, or asulfonylurea as an antidiabetic agent.

Mechanism of action

Activation of PPAR-gamma receptors increases the

transcription of insulin-responsive genes involved in the

control of glucose production, transport, and utilization. In

this way, pioglitazone both enhances tissue sensitivity to

insulin and reduces hepatic gluconeogenesis. Thus, insulin

resistance associated with type 2 diabetes mellitus is

improved without an increase in insulin secretion by



pancreatic β cells.

Absorption

Following oral administration, in the fasting state,

pioglitazone is first measurable in serum within 30 minutes,

with peak concentrations observed within 2 hours. Food

slightly delays the time to peak serum concentration to 3 to 4

hours, but does not alter the extent of absorption.

Volume of distribution 0.63 ± 0.41 L/kg

Protein binding > 99%

Metabolism Hepatic

Route of elimination

Following oral administration, approximately 15% to 30% of

the pioglitazone dose is recovered in the urine. Renal

elimination of pioglitazone is negligible, and the drug is

excreted primarily as metabolites and their conjugates. It is

presumed that most of the oral dose is excreted into the bile

either unchanged or as metabolites and eliminated in the

feces.

Half life 3-7 hours

Clearance apparent cl=5 – 7 L/h [oral administration]

Toxicity Hypogycemia; LD50=mg/kg (orally in rat)



PHARMACOKINETICS

Absorption

Rapid. T max is 2 h. Food slightly delays T max3 to 4 h. Steady

state is reached in 7 days.

Distribution

Vd is 0.63 L/kg (single dose). Protein binding is extensive

(more than 99%), mainly to albumin.

Metabolism

Extensively metabolized in the liver by hydroxylation and

oxidation. Metabolites M-III (keto derivative) and M-IV

(hydroxy derivative) are the major circulatory active

metabolites in humans. The major isoforms involved include

CYP2C8, CYP3A4, and CYP1A1.

Elimination

15% to 30% excreted primarily as metabolites in urine.

Excreted into bile (unchanged as metabolites) and then

eliminated in the feces. Serum half-life is 3 to 7 h

(pioglitazone) and 16 to 24 h (metabolites M-III and M-IV).

Apparent Cl is 5 to 7 L/h.

INTERACTIONS

Drug Colesevelam

Bile Acid Sequestrants may decrease the absorption of

Antidiabetic Agents (Thiazolidinedione). Separate the

dosing of bile acid sequestrants and thiazolidinediones

by at least 2 hours. Monitor for reduced effects of the

http://www.drugbank.ca/drugs/DB00930



antidiabetic agents.

Gemfibrozil

Gemfibrozil may increase the effect and toxicity of

pioglitazone.

Glucosamine Possibly hyperglycemia

Ketoconazole Ketoconazole increases the effect of pioglitazone

Mestranol Possible loss of contraceptive effect

Norethindrone Possible loss of contraceptive effect

Somatropin

recombinant

Somatropin may antagonize the hypoglycemic effect of

pioglitazone. Monitor for changes in fasting and

postprandial blood sugars.

Tamoxifen

Pioglitazone may decrease the therapeutic effect of

Tamoxifen by decreasing the production of active

metabolites. Consider alternate therapy.

Tamsulosin

Pioglitazone, a CYP2D6 inhibitor, may decrease the

metabolism and clearance of Tamsulosin, a CYP2D6

substrate. Monitor for changes in therapeutic/adverse

effects of Tamsulosin if Pioglitazone is initiated,

discontinued, or dose changed.

Tramadol Pioglitazone may decrease the effect of Tramadol by













decreasing active metabolite production.

Tretinoin

The moderate CYP2C8 inhibitor, Pioglitazone, may

decrease the metabolism and clearance of oral

Tretinoin. Monitor for changes in Tretinoin

effectiveness and adverse/toxic effects if Pioglitazone is

initiated, discontinued to dose changed.

Food

Take without regard to meals. Food slightly delays absorption rate but

extent of absorption is not affected.

Dose and

Administration

15 or 30 mg/day (start with 15 mg in patients with CHF [NYHA class I

or II]); may titrate in increments of 15 mg daily (max, 45 mg daily).

Storage Store at 59° to 86°F. Protect from moisture and humidity.

ADVERSE REACTIONS

Cardiovascular Congestive heart failure

CNS Headache

ENT Pharyngitis, macular edema

Hepatic Hepatic failure

Musculoskeletal Bone fractures, myalgia

Respiratory Upper respiratory tract infection, sinusitis

Miscellaneous Edema, urinary bladder tumors.




4.4. EXCIPIENTS PROFILE:

4.4.1 POLYVINYLPYRROLIDONE59, 60

Non-proprietary

names

BP: Povidone

USP: Povidone

PhEur: Povidonum

Synonyms Plasdone k-30, luviskol k-30, plasdone, povidone, pvp k-30,

poly(1-vinyl-2-pyrrolidinone

Description

Fine, white to creamy-white colored, odorless, hygroscopic, amorphous

powder.

Structural Formula

Chemical names

CAS number

1-Ethenyl-2-pyrrolidinone homopolymer

9003-39-8

Chemical formula (C6H9NO)n

Melting point 150-1800C

Solubility Soluble in cold water, chloroform, alcohol, chlorinated hydrocarbons,

amines and lower weight fatty acids.

Functional Category Suspending agent, tablet binder

Stability and storage

conditions

It darkens to some extent on heating at 1500C, with a reduction in

aqueous solubility and should be stored in an airtight container in a

cool, dry place.

Incompatibilities Oxidizing agents.

Safety It may be regarded as essentially nontoxic and nonirritant.

Application

PVP k series can be used as film forming agent, viscosity enhancement

agent and adhesive. In tableting, PVP solutions are used as binders in

wet granulation process. PVP solutions may also be used as coating. It

is also used as suspending, stabilizing-increasing agents in topical and

oral suspensions and solutions.

http://en.wikipedia.org/wiki/File:Polyvinylpyrrolidon.svg



4.4.2 MAGNESIUM STEARATE61,62

Non-proprietary

names

BP: Magnesium stearate

USP/NF: Magnesium stearate

PhEur: Magnesii stearas

Synonyms Magnesium octadecanoate, octadecanoic acid, magnesium salt

Description Very fine, light white, precipitated or milled, impalpable powder of low

bulk density, having a faint odor of stearic acid and a characteristic taste.

The powder is greasy to touch and readily adhere to skin.

Chemical names

CAS Number

Octadecanoic acid magnesium salt

557-04-0

Empirical formula

Molecular weight

C36H70MgO4

591.34

Melting point 117-150

0C (commercial samples)

126-130 0

C (high purity magnesium stearate)

Solubility Practically insoluble in ethanol, ethanol (95%), ether and water, slightly

soluble in warm benzene and warm ethanol (95%).

Functional Category Tablet and capsule lubricant


conditions

It is stable and should be stored in a well-closed container in a cool, dry

place.

Incompatibilities Strong acids, alkalis and iron salts.

Safety It is widely used as pharmaceutical excipient and is generally regarded

as being nontoxic.

Application

It is widely used in cosmetic, foods, and pharmaceutical formulations. It

is primarily used as a lubricant in capsule and tablet manufacture at

concentrations between 0.25% and 5.0% w/w. it is also used in barrier

creams.



4.4.3 TALC63, 64

Non-proprietary

names

BP: Purified talc

USP: Talc

PhEur: Talcum

Synonyms Altalc, E553b, hydrous magnesium calcium silicate, Luzenac Pharma,

Purtalc, steatite, purified French chalk.

Description Very fine, white to grayish-white, odorless, impalpable, unctuous,

crystalline powder.

Structural Formula

Chemical names

CAS Number

Talc

14807-96-6

Empirical formula Mg6(Si2O5)4(OH)4

Functional Category Anticaking agent, glidant, tablet and capsule diluent, tablet and capsule

lubricant.


conditions

It is stable material and may be sterilized by heating at 1600C for not less

than 1 hour. It should be stored in a well-closed container in a cool, dry

place.

Incompatibilities Quaternary ammonium compounds.

Safety

Application

It is widely used oral solid dosage formulations as a lubricant and

diluent. It is also used as lubricant in tablet formulation, in a novel

powder coating for extended-release pellets and as an adsorbant.



4.4.4 LACTOSE65

Non-proprietary

names

BP: Lactose monohydrate

USP/NF: Lactose monohydrate

PhEur: Lactosum monohydricum

JP: Lactose

Synonyms Lactochem Coarse Crystals, Lactochem Crystals, Lactochem Powder,

Pharmatose 50M, NF Lactose 310.

Description White to off-white crystalline particles or powder. Lactose is odorless

and slightly sweet-tasting.

Chemical names

CAS Number

O- -D-Galactopyranosyl-(1 4)- -D-glucopyranose

64044-51-5

Empirical formula

Molecular weight

C12H22O11.H2O

360.31

Melting point 201-2020C

Solubility Practically insoluble in chloroform, ethanol and ether, soluble in water.

Functional Category Binding agent, diluent for dry-powder inhalers, tablet binder, tablet and

capsule diluent.


conditions

Mold growth may occur under humid conditions(80% relative humidity

and above). Lactose may develop a brown coloration on storage, the

reaction being accelerated by warm, damp conditions. It should be

stored in a well-closed container in a cool, dry place.

Incompatibilities Primary amine group, amino acids, aminophylline, amphetamines and

lisinopril.

Safety It is widely used in pharmaceutical formulations as a filler and filler-

binder in oral capsule and tablet formulation.

Application

It is widely used as a filler or diluent in tablets and capsules, and to a

more limited extent in lyophilized products and infant formulas.

Usually, fine grades of lactose are used in the preparation of tablets by

the wet-granulation method. It is also used in combination with sucrose

(approximately 1:3) to prepare sugar-coating solutions.



4.5. METHODOLOGY

4.5.1 PRE-FORMULATION CHARACTERIZATION

A. Solubility studies:

Excess amount of the drug is added to the following,

a) 100 mL distilled water

b) 100 mL Ethanol

c) 100 mL 0.1N HCL pH 1.2

d) 100 mL phosphate buffer pH 7.4

e) 100 mL distilled water : 100 mL Ethanol

After adding maximum amount of the drug shake the each volumetric flask in a shaker for more

than 12 hours, for maximum saturation of the solution. Then remove 5 ml from each of flask and

make dilution as required. Take absorbance at 269nm in UV-Visible spectrophotometer.

B. Melting Point determination:

The melting temperature of drugs was determined using capillary method.

C. Drug – excipients compatibility:

Prior to the development of the dosage forms the compatibility study was carried out. Hence

infrared spectra of the physical mixture of the Pioglitazone and the polymers were taken. Also

the infrared spectrum of the Drug and Polymer was taken individually.



D. Development of analytical method

i. Preparation of 0.1 N HCL solution:

8.5ml of 35% hydrochloric acid was accurately measured and transferred into a 1000ml

volumetric flask and the volume was made upto the mark with distilled water.

ii. Preparation of phosphate buffer pH 7.4

50 mL of 0.2 M potassium di-hydrogen phosphate and 39.1 ml of 0.2 M NaOH were

taken in 200 ml volumetric flask and the volume was made up to the mark with distilled

water.

iii. Preparation of standard Pioglitazone solution

Pioglitazone (10mg) was weighed accurately and dissolve in 60 ml 0.1N HCL .the

solution was diluted up to 100 ml with distilled water, stock solution so prepared was

containing 100 microgram of drug per ml of solvent.

iv. Determination of wavelength of maximum absorbance

Standard Pioglitazone solution (1ml) was pipetted in 10 ml volumetric flask. Then the

volume was adjusted to the mark with 0.1N HCL. The solution (10mg) was scanned and

absorbance was measured in the range of 200-400 nm against blank on Shimadhzu 1800

UV-Visible spectrophotometer. The blank was prepared in similar manner in which

volume of standard drug solution was replaced by equal volume of 0.1 N HCL.

v. Preparation of standard calibration curve of Pioglitazone in 0.1N HCL

Preparation of standard solution:

100 mg of Pioglitazone was accurately weighed in to 100ml volumetric flask and

dissolved in small quantity of 0.1 N HCL. The volume was made up to the mark with the



0.1 N HCL to get a concentration of 1000μg/ml (SS-I). From this 10ml was withdrawn

and diluted to 100ml to get a concentration of 100μg/ml (SS-II).

Preparation of working standard solutions:

From (SS-II) aliquots of 0.4ml, 0.8ml, 1.2ml, 1.6ml, 2.0, 2.4 and 2.8ml were pipetted into

10ml volumetric flasks. The volume was made up with 0.1N HCL to get the final

concentration of 4,8,12,16,20,24 and 28μg/ml respectively.

The λmax was found to be 269 nm from UV spectrum of Pioglitazone in 0.1N HCL,

during scanning from 200-400 nm. Absorbance was measured at 2 nm against 0.1N HCL

as blank on a UV-Visible Spectrophotometer (UV-1800 SHIMADZU). The observations

were recorded in Table No. and the calibration curve was prepared by plotting

absorbance versus concentration of Pioglitazone as shown in figure no.

vi. Preparation of standard calibration curve in pH 7.4 phosphate buffer

Take standard Pioglitazone solution and make up dilution 0-20 μg/ml in 10ml volumetric

flasks and volume was adjusted to mark by pH 7.4 phosphate buffer. Absorbance at

269nm was read at against blank solution.

E. Isolation of seed mucilage

The seeds were washed with water to remove dirt and debris, and dried. The dried seeds were

crushed and powdered in ball mill. To 20g of seed powder, 200ml of cold distilled water was

added and slurry was prepared. The slurry was poured into 800ml of boiling distilled water.

The solution was boiled for 20 minutes under stirring condition in a water bath. The resulting

thin clear solution was kept overnight so that most of the proteins and fibers settled out. The

material was squeezed from an eight-fold muslin cloth bag to remove the marc from the solution.



Acetone was added to the filtrate to precipitate the mucilage in a quantity of three times the

volume of the total filtrate. The mucilage was separated, dried in an oven at a temperature <50

°C, collected, dried-powdered, passed through a sieve (number 80), and stored for further use in

desiccators.

F. Characterization of Mucilage

i. Organoleptic Evaluation

The isolated mucilage was characterized for organoleptic properties such as color, odor,

taste, fracture and texture.

ii. Solubility:

Solubility of isolated mucilage was studied using different types of solvents like water,

alcohol acetone, Polyethylene Glycols, Propylene Glycol, Glycerin, Sorbitol, Ethyl

Alcohol, Methanol, Benzyl Alcohol, Isopropyl Alcohol, etc.

iii. Yield Estimation

Percentage yield in 200 g seeds of Trigonella foenum graecum was calculated.

iv. Phytochemical test:

The phytochemical analysis was carried out to determine the compounds of selected

natural polymer.

v. Determination of Swelling Index:

The natural polymer 1g was taken in a China dish and then 10 ml of distilled water was

added and the mixture was shaken and allowed to stand for 1 hour. After 1 hour the

remaining water in China dish was discarded and the weight increase was rated.

Swelling Index % (SI) = (W2 – W1/W1) x 100

Where, W1= Weight of tablet at time ‘0’ and W2= Weight of tablet at time ‘t’



vi. pH of Mucilage:

The mucilage was weighed and dissolved in water to get a 1% w/v solution. The pH of

solution was determined using digital pH meter.

vii. Micromeritic Evaluation:

1. Bulk density (Bd)

The term bulk density refers to a measure used to describe a packing of particles.

The bulk density was obtained by dividing the mass of a powder by the bulk

volume in cm3 (V). The standard method (USP) was adopted for measurement of

bulk density of both dried powdered mucilage and following equation was used

for calculation.

Bd = M/V

Where, M = weight of samples in grams, V= bulk volume of powder in cm3

2. Tapped density (Td)

The tapped density or poured density attained after mechanically tapping a

container containing the powder sample. The standard method described in USP

was followed and tapped density was calculated using equation given below:

Td = M/ Vp

Where, M = weight of samples in grams and Vp = final tapped volume of powder

in cm3

3. Carr’s index(CI)

An indirect method of measuring powder flow from bulk densities was developed

by Carr. A low Carr’s index implies a good initial packing arrangement, with less

volume of voids. As the value of these indices increases, the flow of the powder



decreases. Carr’s index of each sample was calculated according to equation

given below:

CI =100 (Td- Bd) / Td

4. Hausner’s ratio(HR)

Hausner’s ratio measures the powder ability to settle and permit an assessment of

the relative importance of interparticulate interactions. Hausner’s ratio is

calculated as the ratio of bulk density to tapped density.

HR= V0 / Vf

Where, V0: unsettled apparent volume, Vf: final tapped volume

Table No.6: Effect of Carr’s Index and Hausner’s Ratio on flow property

Carr’s Index (%) Flow Character Hausner’s Ratio

≤10

Excellent 1.00–1.11

11-15 Good 1.12–1.18

16-20 Fair 1.19-1.25

21-25 Passable 1.26-1.34

26-31 Poor 1.35-1.45

32-37 Very poor 1.46-1.59

>38 Very very poor >1.60

5. Angle of Repose

Angle of repose has been defined as the maximum angle possible between the

surface of pile of powder and horizontal plane. The angle of repose for the

granules of each formulation was determined by the fixed height funnel method.



The angle of repose was calculated by substituting the values of the base radius ‘r’

and pile height ‘h’ in the following equation.

Where, h and r are the height and radius of the powder cone respectively.

Table No.7: Effect of Angle of repose (ϴ) on Flow property

4.5.2. FORMULATION DESIGN

Table No.8: Selected Ingredients for formulation with function

Angle of Repose (ϴ) Type of Flow

<20 Excellent

20-30 Good

30-34 Passable

>35 Very poor

Sl.no EXICIPIENT FUNCTION

1 Pioglitazone Model drug

2 Fenugreek Mucilage Binder

3 PVP K30 Binder

4 Magnesium Stearate Lubricant

5 Talc Glidant

6 Lactose Diluent

7 Ethanol Solvent



Table No.9: Formulation developed

Sl.no.

Ingredients

(mg)

F1 F2 F3 F4 F5 F6 F7 F8 F9

1 Pioglitazone 15 15 15 15 15 15 15 15 15

2

Fenugreek

Mucilage

1 1.5 2 2.5 3 3.5 4 4.5 5

3 PVP K30 5 5 5 5 5 5 5 5 5

4 Mg. Stearate 5 5 5 5 5 5 5 5 5

5 Talc 7 7 7 7 7 7 7 7 7

6 Lactose 63 63 63 63 63 63 63 63 63

7 Ethanol q.s q.s q.s q.s q.s q.s q.s q.s q.s

8 Tablet weight 100 100 100 100 100 100 100 100 100



4.5.3. PREPARATION OF PIOGLITAZONE TABLETS BY NON-AQUEOUS WET

GRANULATION METHOD

Non-aqueous wet granulation method was used for all tablet production. Calculation was made

for 100 tablets in each batch.

i. Preparation of Binder Solution: In each case, accurately weighed quantity of FGM with PVP

K30 varying ratios were dissolved thoroughly in sufficient quantity of Ethanol by mixing using

glass rod for 5 min. This mixture was then used as binder solution in the preparation of granules.

ii. Preparation of damp mass: In each case, accurately weighed quantities of Pioglitazone,

Magnesium stearate and Lactose were mixed in a mortar and the binder solution was added to

obtain a damp coherent mass. The damp mass was sieved with 1.7mm sieve and dried at 50oC in

oven for 30mins.

ii. Punching of Tablets: The dried granular mass was passed through a 1.0 mm sieve to obtain

uniform sized granules. The different batches of the granules were then mixed with calculated

equal quantities of Talc, and then were compressed into tablets on a pilot press machine (Lab

Press Multi punch machine, India) using 12 mm diameter, flat faced punches at a pressure of

approximately 5kgs/cm.2

4.5.4. PRE COMPRESSION CHARACTERIZATION

1. Bulk density (Bd)

The term bulk density refers to a measure used to describe a packing of particles. The

bulk density was obtained by dividing the mass of a powder by the bulk volume in cm3

(V). The standard method (USP) was adopted for measurement of bulk density of both

dried powdered mucilage and following equation was used for calculation.

Bd = M/V



Where, M = weight of samples in grams, V= bulk volume of powder in cm3

2. Tapped density (Td)

The tapped density or poured density attained after mechanically tapping a container

containing the powder sample. The standard method described in USP was followed and

tapped density was calculated using equation given below:

Td = M/ Vp

Where, M = weight of samples in grams and Vp = final tapped volume of powder in cm3

3. Carr’s index(CI)

An indirect method of measuring powder flow from bulk densities was developed by

Carr. A low Carr’s index implies a good initial packing arrangement, with less volume of

voids. As the value of these indices increases, the flow of the powder decreases. Carr’s

index of each sample was calculated according to equation given below:

CI =100 (Td- Bd) / Td

4. Hausner’s ratio(HR)

Hausner’s ratio measures the powder ability to settle and permit an assessment of the

relative importance of interparticulate interactions. Hausner’s ratio is calculated as the

ratio of bulk density to tapped density.

HR= V0 / Vf

Where, V0: unsettled apparent volume, Vf: final tapped volume

5. Angle of Repose



Angle of repose has been defined as the maximum angle possible between the surface of

pile of powder and horizontal plane. The angle of repose for the granules of each

formulation was determined by the fixed height funnel method. The angle of repose was

calculated by substituting the values of the base radius ‘r’ and pile height ‘h’ in the

following equation.

Where, h and r are the height and radius of the powder cone respectively.

6. Determination of flow rate

Ten grams (10g) (w) of the granules were passed through dry glass funnel and allowed to

flow through the funnel orifice. The time taken for the powder to flow through the orifice

(t) was noted and the flow rate was computed as

= w / t

7. Friability

The granule strength was determined by friability test using the Roche friabilator. The

apparatus was rotated at 25rpm for 4 min. A sample of 10 g (WA) granules was placed in

friability testing machine. After the drum movement stopped, the granules were sieved

through a 60-mesh and the residue remaining on the sieve were weighed (WB). The

friability was calculated using the following equation

F% = [( WA-WB ) / WB ] 100



8. Percent compressibility (%C)

It is an important measure that can be obtained from bulk density measurements. It is the

simple test to evaluate the V0 and Vf of powder and the rate at which it packed down. The

following formula was used to compute the percent compressibility.

%C = 100 (V0-Vf) / V0

Where, Vf = packed bulk density and V0 = apparent bulk density

9. Particle size distribution

Standard sieve method as per IP was adopted to study particle size distribution. Clean

standard sieves were taken and arranged in such a manner that the coarsest sieve remains

on the top and finest sieve remains on the bottom. Butter paper was placed at the bottom

of arranged sieve set. 10gm of the FSM granules were spread on top sieve. The set of

sieve were rotated semicircular motion with intermediate jerking for 10mins.

Disassemble the sieve set. Separated powder was collected from each sieve and weighed

separately. The oversize and undersize particles for each sieve was noted.

4.5.5. POST COMPRESSION CHARACTERIZATION

i. Appearance:

Organoleptic properties such as color and odor were evaluated. Ten tablets from each

batch were randomly selected and their colors were visually compared and odour was

checked.

ii. Dimensions:

Thickness and diameter of the tablet was measured using Digital Vernier caliper. Five

tablets of the formulation were picked randomly and measured individually.



iii. Hardness:

Hardness was measured using fpizer hardness tester. For each batch five tablets were

used.

iv. Friability:

Twenty tablets were weighed and placed in the Roche friabilator and apparatus was

rotated at 25 rpm for 4 minutes. The tablets were de-dusted and weighed again. The

percentage friability was calculated using the formula:

Friability = {1-(Wt /W)} ×100

Where, F = Friability in percentage

W = Initial weight of tablets

W t= Weight of tablets after friabiation.

v. Weight variation:

Twenty tablets were randomly selected from each batch and weighed, the average weight

was calculated and then they were weighed individually to calculate standard deviation.

vi. Drug content estimation

Delayed release tablets of Pioglitazone equivalent to 100 mg are weighed and dissolved

in little amount of phosphate buffer (7.4pH) in volumetric flask and volume is made upto

100 ml with the buffer pH 7.4 and subsequent dilutions are made and absorbance is

measured at 269 nm and drug content is calculated using standard curve. Each test is

performed in triplicate.

vii. Weight variation test: 20 tablets were selected at random from the lot, weighed

individually and the average weight was determined. The percent deviation of each



tablets weight against the average weight was calculated. The test requirements are met;

if not more than two of the individual weights deviate from the average weight by not

more than existing 5%.

Table 10: Weight variation tolerances for uncoated tablets

Sl.no Average weight of tablets in mg Max % differences allowed

1 130 or less 10%

2 130-324 7.5%

3 More than 324 5%

viii. Disintegration test

The disintegration time was measured using disintegration test apparatus as per the USP.

One tablet was placed in each tube of the basket. The basket with the bottom surface

made of a stainless-steel screen (mesh no.10) were immersed in water bah a 37± 2°C.

The time required for complete disintegration of the tablet in each tube was determined

using a stop watch.

ix. In-vitro dissolution studies:

Dissolution of the tablets was carried out on USP XXIII dissolution type II apparatus

using paddle. The dissolution medium consisted of 900 ml of pH 1.2 buffer (0.1N HCL)

for first two hours and the phosphate buffer pH7.4 from 3- 10 hours maintained and the

temperature of the medium was set at 37±0.5ᵒ C. The rotational speed of the paddle was

set at 50 rpm. 5ml of sample was withdrawn at predetermined time interval of 1 hour up

to 10 hour and same volume of fresh medium was replaced. The withdrawn samples were



diluted to 10ml with pH 7.4, filtered and analyzed on UV spectrophotometer at 269 nm

using pH 7.4 as a blank. Percentage cumulative drug release was calculated.

Details of dissolution test:

Dissolution test apparatus : USP type II

Speed : 50 rpm

Stirrer : Paddle type

Volume of medium : 900 ml

Volume withdrawn : 5 ml

Medium used : pH 0.1N HCL (pH 1.2) and phosphate buffer pH7.4

Temperature : 37±0.5ᵒ C

4.5.6. Data analysis:

To analyze the mechanism of release and release rate kinetics of the dosage form the data

obtained were fitted into Zero order, First order, Higuchi matrix and Korsmeyer’s and peppas

models. Based on the ‘R’-value the best fit model was selected.

Zero order kinetics:

Drug dissolution from pharmaceutical dosage forms that do not disaggregate and release the

drug slowly, assuming that the area does not change and no equilibrium conditions are

obtained can be represented by the following equation

Qt = Q0+K0 t

Qt= amount of drug dissolved in time t



Q0 = initial amount of the drug in the solution

K0= zero order release constant

First order kinetics:

To study the first order release rate kinetics the release rate data were fitted to the following

equation:

Log Q t= log Q0+ + K 1t/2

Where,

Qt= amount of drug released in time t

Q0= initial amount of drug in the solution

K1= first order release constant.

Higuchi model:

Higuchi developed several theoretical models to study the release of water soluble and low

soluble drugs incorporated into semisolids and or solid matrices. Mathematical expressions

were obtained for drug particles dispersed in a uniform matrix behaving as the diffusion

media and the equation is:

Qt= KH.t1/2

Where, Qt= amount of drug released in time t.

KH= Higuchi dissolution constant.

Krosmeyer and Peppas release model:

To study this model the release rate data are fitted to the following equation:

Mt / M∞= K · t n

Where, Mt / M∞= fraction of drug release



K = release constant

t = release time and

n = diffusional coefficient.

Table No.11: Mechanism of Drug Release as per Korsmeyer Equation/ Peppa’s Model

Sl. No ‘n’ value Drug release

1. 0.45 Fickian release

2. 0.45 <n<0.89 Non- Fickian release

3. n>0.89 Case II transport

4.5.7. STABILITY STUDIES

Stability can be defined as the capacity of drug product to remain within specifications

established to ensure its identity, strength, quality, and purity.

A. Importance of stability studies

Stability studies are important for the following reasons.

1. This is an assurance given by the manufacturer that the patient would receive a uniform

dose throughout the shelf life.

2. The drug control administration insists on manufacturers on conducting the stability

studies, identity, strength, purity and quality of the drug for an extended period of time in

the conditions of normal storage.

3. Stability testing prevents the possibility of marketing an unstable product. Both physical

and chemical degradation of drug can result in unstable product.



B. Purpose of stability studies

Stability studies are done to understand how to design a product and its packaging such that

product has appropriate physical, chemical and microbiological properties during a defined

shelf life when stored and used.

Stability conditions according to ICH guidelines

Table No.12: Drug substances intended for normal storage

Study Storage conditions Minimum period of time

Long term 25ºC ±2 ºC/60%RH±5%RH 12 Months

Intermediate 30 ºC±2 ºC/65%RH±5%RH 6 Months

Accelerated 30 ºC±2 ºC/65%RH±5%RH

40 ºC±2 ºC/65%RH±5%RH 6 Months

The optimized formulation was subjected for stability study. The selected formulations

were packed in aluminum foil in tightly closed container. They were then stored at 40ºC / 75%

RH and evaluated for their physical appearance.

4.5.8. In vitro Cytotoxicity Studies

I. Principle: The ability of the cells to survive a toxic insult has been the basis of most

Cytotoxicity assays. This assay is based on the assumption that dead cells or their

products do not reduce tetrazolium. The assay depends both on the number of cells

present and on the mitochondrial activity per cell. The principle involved is the cleavage

of tetrazolium salt 3-(4, 5 dimethyl thiazole-2-yl)-2, 5-diphenyl tetrazolium bromide



(MTT) into a blue coloured product (formazan) by mitochondrial enzyme succinate

dehydrogenase. The number of cells was found to be proportional to the extent of

formazan production by the cells used.

II. Preparation of Test Solutions

For Cytotoxicity studies, each weighed test drugs were separately dissolved in distilled

DMSO and volume was made up with DMEM supplemented with 2% inactivated FBS to

obtain a stock solution of 1 mg/ml concentration and sterilized by filtration. Serial two

fold dilutions were prepared from this for carrying out cytotoxic studies.

III. Determination of cell viability by MTT Assay

Cell lines and Culture medium

L-6 (Rat, Skeletal muscle) cell culture was procured from National Centre for Cell

Sciences (NCCS), Pune, India. Stock cells of L-6 were cultured in DMEM supplemented

with 10% inactivated Fetal Bovine Serum (FBS), penicillin (100 IU/ml), streptomycin

(100 g/ml) and amphotericin B (5 g/ml) in an humidified atmosphere of 5% CO2 at

37 C until confluent. The cells were dissociated with TPVG solution (0.2% trypsin,

0.02% EDTA, 0.05% glucose in PBS). The stock cultures were grown in 25 cm2

culture

flasks and all experiments were carried out in 96 microtitre plates (Tarsons India Pvt.

Ltd., Kolkata, India).

IV. Procedure: The monolayer cell culture was trypsinized and the cell count was adjusted

to 1.0 x 105

cells/ml using DMEM containing 10% FBS. To each well of the 96 well

microtitre plate, 0.1 ml of the diluted cell suspension (approximately 10,000 cells) was



added. After 24 h, when a partial monolayer was formed, the supernatant was flicked off,

washed the monolayer once with medium and 100 l of different test concentrations of

test drugs were added on to the partial monolayer in microtitre plates. The plates were

then incubated at 37o

C for 3 days in 5% CO2 atmosphere, and microscopic examination

was carried out and observations were noted every 24 h interval. After 72 h, the drug

solutions in the wells were discarded and 50 l of MTT in PBS was added to each well.

The plates were gently shaken and incubated for 3 h at 37o

C in 5% CO2 atmosphere. The

supernatant was removed and 100 l of propanol was added and the plates were gently

shaken to solubilize the formed formazan. The absorbance was measured using a

microplate reader at a wavelength of 540 nm. The percentage growth inhibition was

calculated using the following formula and concentration of test drug needed to inhibit

cell growth by 50% (CTC50) values is generated from the dose-response curves for each

cell line.



4.5.9. In vitro glucose uptake assay

Diabetes mellitus is associated with insulin deficiency and decreased glucose uptake in

skeletal muscles. The increased plasma free radicals observed in diabetes mellitus may impair

insulin action, thus contributing to the generation of hyperglycemia. Defects in GLUT- 4 and

GLUT-1 may explain the insulin resistant glucose transport. Skeletal muscle is a major tissue for

blood glucose utilization and a primary target tissue for insulin action. Insulin increases glucose

uptake in skeletal muscle by increasing functional glucose transport molecules (GLUT-4) in the

plasma membrane. Glucose transport in skeletal muscle can also be stimulated by contractile

activity. Free radical impairs insulin stimulated GLUT-4 translocation and exerts an inhibitory

effect on muscle contractility that is major pathological feature of diabetes. L6 cells represent a

good model for glucose uptake because they have been used extensively to elucidate the

mechanisms of glucose uptake in muscle, have an intact insulin signaling pathway, and express

the insulin-sensitive GLUT-4.

Procedure:

Glucose uptake activity of test substance was determined in differentiated L6 cells. In brief, the

24 hr cell cultures with 70-80% confluency in 40mm petri plates were allowed to differentiate by

maintaining in DMEM with 2% FBS for 4-6 days. The extent of differentiation was established

by observing multinucleation of cells. The differentiated cells were serum starved overnight and

at the time of experiment cells were washed with HEPES buffered Krebs Ringer Phosphate

solution (KRP buffer) once and incubated with KRP buffer with 0.1% BSA for 30min at 370C.

Cells were treated with different non-toxic concentrations of test and standard drugs for 30 min



along with negative controls at 370C. 20µl of D-glucose solution was added simultaneously to

each well and incubated at 370C for 30 min. After incubation, the uptake of the glucose was

terminated by aspiration of solutions from wells and washing thrice with ice-cold KRP buffer

solution. Cells were lysed with 0.1M NaOH solution and an aliquot of cell lysates were used to

measure the cell-associated glucose. The glucose levels in cell lysates were measured using

glucose assay kit (ERBA). Three independent experimental values in duplicates were taken to

determine the percentage enhancement of glucose uptake over controls.

RESULTS You may never know what results come of your

action, but if you do nothing there will be no Results.

RESULTS


5. RESULTS

5.1 PRE-FORMULATION CHARACTERIZATION

A. Solubility studies:

Table No.13: Solubility determination

Solvent Used Observation

In Distilled Water Insoluble

In Ethanol Soluble

In 0.1N HCL pH 1.2 Soluble

In Phosphate buffer pH 7.4 Soluble

Water : Ethanol Soluble but precipitates

B. Melting Point determination

The melting point of pioglitazone was found to be 190oC-192

0C.

C. Drug - Excipients Compatibility

Spectra 1: FT-IR spectra of Pioglitazone Drug

RESULTS


Spectra 2: FT-IR spectra of Fenugreek Mucilage

Spectra 3: FT-IR spectra of PVP K30

RESULTS


Spectra 4: FT-IR spectra of PGZ + FGM + PVP K30

D. Development of analytical method

i. Standard Pioglitazone

Graph No.1: Maximum wavelength of Pioglitazone

RESULTS


ii. Standard Calibration curve for Pioglitazone in 0.1N HCL

Table No.14: Spectrophotometric Data for the Estimation of Pioglitazone in 0.1N HCL

Sl No Conc

(µg/ml) Abs

1 0 0

2 4 0.076

3 8 0.145

4 12 0.226

5 16 0.298

6 20 0.369

7 24 0.438

8 28 0.534

Graph No.2: Calibration Curve of Pioglitazone in 0.1N HCL

y = 0.1866x R² = 0.9988

0

0.1

0.2

0.3

0.4

0.5

0.6

0 0.5 1 1.5 2 2.5 3

Ab

sorb

ance

Concentration (µg/ml)

RESULTS


iii. Standard Calibration curve for Pioglitazone in 7.4 Phosphate Buffer

Table No.15: Spectrophotometric Data for the Estimation of PGZ in 7.4pH Phosphate buffer

Sl No Conc (µg/ml) Abs

1 0 0.00

2 4 0.082

3 8 0.144

4 12 0.206

5 16 0.25

6 20 0.322

7 24 0.377

8 28 0.435

Graph No.3: Calibration Curve of Pioglitazone in 7.4 Phosphate buffer

y = 0.159x R² = 0.9935

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0 0.5 1 1.5 2 2.5 3

Ab

sorb

ance

Concentartion

RESULTS


E. Characterization of Mucilage

i. Organoleptic Evaluation

Table No.16: Results of Organoleptic Characters determination

Sl.no Test Inference

1 Color Dark Yellowish Brown

2 Odour Agreeable

3 Taste Bitter

4 Texture Rough & Irregular

5 Fracture Rough

ii. Solubility:

Table No.17: Results of Solubility determination

Sl.no Solvent Observation

1 Hot water Soluble

2 Cold water Insoluble(forms gel)

3 Inorganic solvents Insoluble

iii. Yield Estimation:

Table No.18: Results of Yield estimation

Wt of sample

taken (gm)

Wt of extract

obtained (gm)

Solvent used Qty of Solvent

taken (liter)

Yield value(gm)

200 20 Acetone 5 10

RESULTS


iv. Phytochemical Screening:

Table No.19: Results of Phytochemical tests of isolated mucilage

Sl.no Identification Test For Name of test Observation Inference

1 Carbohydrates Molisch’s Test + A violet colored ring

formation at junction of

2liquids

2 Proteins &Amino Acids Ninnhydrin Test - -

3 Mucilages Ruthenium Red

Test

+ Red colour formation

4 Starches Iodine Test - -

5 Alkaloids Dragendroff’s

Test

- -

6 Glycosides Keller-Killani

Test

- -

7 Tannins Ferric Chloride

Test

- -

8 Steriods & sterols Libermann

Burchard Test

- -

RESULTS


v. Determination of Swelling Index:

It was found W1 = 10 and W2 = 15

SI = (15 – 10 / 10) x 100

SI = 50%

vi. pH of Mucilage:

The mucilage was weighed and dissolved in water to get a 1% w/v solution. The pH of

solution was determined using digital pH meter was found to be 7.9.

vii. Micromeritic Evaluation:

Tablet No.20: Results of Micromeritic Evaluation of isolated mucilage

Parameters Results

Bulk density(gm/ml) 0.66±0.043

Tapped density(gm/ml) 0.94±0.098

Carr’s Index 17.4±3.11

Haunser’s Ratio 1.19±0.057

Angle of Repose 29.20

Flow rate Good

RESULTS


5.2. Evaluation Parameters:

5.2.1. Physicochemical Properties:

Table No.21: Organoleptic properties evaluation results

Formulation

code

Color Shape Odor

F1 White color Flat and circular Odorless









RESULTS


5.2.2. Pre-compression Parameters

Table No.22: Pre-compression parameters results

Code Bulk density

(g/cm3)

Tapped density

(g/cm3)

Carr’s index% Hausner’s

ratio

Angle of

repose(°)

F1 0.521±0.094 0.625±0.120 17.24±0.03 1.19 28.56±0.04

F2 0.529±0.101 0.626±0.034 16.64±0.094 1.18 28.19±0.067

F3 0.528±0.074 0.62±0.069 16.37±0.065 1.17 27.89±0.051

F4 0.523±0.089 0.632±0.091 15.49±0.074 1.20 26.21±0.079

F5 0.521±0.093 0.623±0.113 14.83±0.093 1.19 27.97±0.084

F6 0.476±0.112 0.555±0.108 14.23±0.034 1.16 27.61±0.099

F7 0.5±0.107 0.588±0.07 14.19±0.107 1.17 25.52±0.021

F8 0.523±0.099 0.62±0.074 14.07±0.099 1.18 25.86±0.044

F9 0.52±0.094 0.6±0.043 13.33±0.102 1.14 23.12±0.042

RESULTS


5.2.3. Post-compression Parameters

Table No.23: Post-Compression Parameter results

Code Weight

variation (mg)

Hardness

(kg/cm2)

Thickness

(mm)

Friability

(%)

Drug content

(%)

Disintegration

Time (sec)

F1 119.91±0.22 3.02±0.10 3.12±0.01 0.39±0.15 93.51±0.57 57

F2 120.33±0.36 3.05±0.09 3.15±0.03 0.36±0.11 95.00±0.42 41

F3 120.21±0.49 3.11±0.04 3.18±0.03 0.33±0.09 96.85±0.32 37

F4 120.92±0.41 3.17±0.007 3.12±0.02 0.43±0.62 95.79±0.27 39

F5 120.16±0.32 3.20±0.05 3.32±0.01 0.42±0.44 97.01±0.89 35

F6 119.95±0.91 3.26±0.03 3.19±0.04 0.32±0.53 96.15±0.42 39

F7 120.09±0.99 3.30±0.10 3.19±0.01 0.34±0.20 97.97±0.84 31

F8 120.11±0.60 3.34±0.14 3.15±0.02 0.40±0.32 97.35±0.42 33

F9 120.01±0.59 3.39±0.05 3.15±0.01 0.27±0.06 98.99±0.42 29

RESULTS


5.2.4 In-vitro Drug release profile of all the formulations:

Volume of dissolution media: 0.1 N HCl for first two hrs and phosphate buffer PH 6.8 upto 8 hrs.

Table No.24: In-vitro drug release profile

SL.NO TIME

(mins)

% CUMULATIVE DRUG RELEASE(CDR)

FORMULATION CODE

F1 F2 F3 F4 F5 F6 F7 F8 F9

1 0 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

2 30 10.594 17.215 28.472 5.959 35.093 5.959 16.553 4.635 12.581

3 60 38.410 35.765 36.433 16.557 39.085 16.557 20.535 7.948 14.574

4 120 45.714 42.406 45.723 29.146 40.431 29.146 25.844 13.912 16.568

5 180 50.375 48.389 47.073 31.811 41.778 31.811 31.817 16.568 18.564

6 240 55.038 53.05 56.369 34.477 45.774 35.139 32.497 19.888 23.209

7 300 65.000 64.998 59.049 49.063 47.124 39.139 39.136 23.872 27.195

8 360 74.306 72.318 63.716 55.712 57.744 42.464 42.469 29.182 32.507

9 420 78.320 76.331 71.697 64.35 60.424 48.447 45.803 34.495 33.849

10 480 87.633 82.994 79.02 71.669 63.768 56.419 48.447 40.474 37.841

RESULTS


Graph No.4: In-vitro Cumulative percentage drug released V/S Time for Formulations F1 to F9

5.2.5 Kinetic Release Study

Table No 25: Results of Kinetic data of various models for release study

Formulation

code

Zero order

release

plots

First order

release

Plots

Higuchi’s

plots Korsmeyer’s and Peppas plots

Regression

coefficient

(R2)

Regression

coefficient

(R2)

Regression

coefficient

(R2)

Regression

coefficient

(R2)

Exponential

value

(n)

F1 0.9014 0.9553 0.9682 0.9711 0.7233

F2 0.9158 0.9776 0.9860 0.9738 0.6983

F3 0.8611 0.9743 0.9712 0.9346 0.6657

F4 0.9768 0.9706 0.9520 0.9808 0.7026

F5 0.7248 0.8432 0.8706 0.9792 0.6194

F6 0.7249 0.9582 0.8732 0.9798 0.6615

F7 0.8926 0.9447 0.9883 0.9637 0.6039

F8 0.9888 0.9834 0.9432 0.9788 0.5961

F9 0.9315 0.9548 0.9655 0.9677 0.5551

0.000

10.000

20.000

30.000

40.000

50.000

60.000

70.000

80.000

90.000

100.000

0 100 200 300 400 500 600

% C

DR

Time (min)

F1

F2

F3

F4

F5

F6

F7

F8

F9

RESULTS


Graph No.5: Plot of % Cum. Drug Released Vs. Time

Graph No.6: Plot of Log % Cum. Drug Retained Vs. Time

0.000

10.000

20.000

30.000

40.000

50.000

60.000

70.000

80.000

90.000

100.000

0 100 200 300 400 500 600

% C

DR

Time(min)

Zero order kinetics

F1

F2

F3

F4

F5

F6

F7

F8

F9

0.000

0.500

1.000

1.500

2.000

2.500

0 200 400 600

Log

% d

rug

dru

g re

leas

e

Time

First Order kinetics

F1

F2

F3

F4

F5

F6

F7

F8

F9

RESULTS


Graph No.7: Plot of % Cum. Drug Released Vs. √Time

Graph No.8: Plot of Log % Cum. Drug Released Vs. Log Time

-20.000

0.000

20.000

40.000

60.000

80.000

100.000

0 5 10 15 20 25

% C

DR

Square root of time

Higuchi model

F1

F2

F3

F4

F5

F6

F7

F8

F9

-0.500

0.000

0.500

1.000

1.500

2.000

2.500

0 0.5 1 1.5 2 2.5 3

Log

% C

DR

Log Time

Krosmeyer and Peppas model

F1

F2

F3

F4

F5

F6

F7

F8

F9

RESULTS


5.2.6 Stability Studies

Table No. 26: Results of Stability studies for F5 formulation

Time Evaluation parameters

Color Hardness Drug content

15 days White 3.20 97.01

30 days White 3.18 97.98

45 days White 3.21 96.95

60 days White 3.20 96.69

Table No. 27: Results of Stability studies for F6 formulation

Time Evaluation parameters

Color Hardness Drug content

15 days White 3.26 96.15

30 days White 3.24 96.10

45 days White 3.26 95.85

60 days White 3.25 95.70

Table no 28: Results of %CDR of Formulation of F5 and F6 at 40ºC/75% RH after 10hours

Tested after days

% CDR

F5 F6

15 63.6 56.40

30 63.58 56.3

45 63.78 55.89

60 63.23 56.18

RESULTS


5.3 CytoToxicity Studies:

Table No.29: Results of %Cytotoxicity of F5 and F6 formulations against L6 cell line

Test sample Test Conc

( µg/ml)

% Cytotoxicity CTC50

( µg/ml)

F5

1000 28.02±3.6

>1000

500 17.72±0.4

250 14.50±3.8

125 9.73±4.1

62.5 7.74±4.6

F6

1000 28.52±5.1

>1000

500 15.26±1.1

250 14.10±2.4

125 13.74±4.3

62.5 9.15±2.4

Graph No.9: Cytotoxic effect of F5 and F6 on the L6 Cell line

RESULTS


5.3 In vitro glucose uptake assay:

Table No.30: Result of In vitro glucose uptake studies of F5 and F6 formulations in L-6 cell line

Name of the Test

substances

Test Conc

(mcg/ml)

Protein

content

Glucose uptake percentage

(%)

Control (A) - 697.41 0.01±0.06

Std. Rosiglutozone (B) 100 753.89 127.67±3.54

F5 (C)

500 954.40 74.13±3.37

(D) 250 806.22 61.75±2.20

F6 (E)

500 843.26 21.10±4.11

(F) 250 895.08 13.83±6.67

Graph No.10: Glucose uptake assay of the formulation F5 and F6 on L6 cell line

A B C D E F

DISCUSSION Discussion is an exchange of Knowledge, An argument an exchange of Ignorance.

DISCUSSION


6. DISCUSSION

6.1 Solubility studies:

Solubility analysis is important because the drug has to dissolve in the solvents and also in the

dissolution medium used.

The Pioglitazone is freely soluble in in 0.1N HCL (pH 1.2), 7.4 pH phosphate buffer,

sparingly soluble in ethanol; practically insoluble in Water.

6.2 Melting point:

The melting point of the obtained drug sample was found to be 1940C which is within the

reported range of 192-1950C. It complies with the purity of the drug sample.

6.3 FT- IR Studies:

The FTIR spectroscopy is a useful tool for identifying both organic and inorganic chemicals. It

can be utilized to quantify some components of an unknown mixture and can be used to analyze

liquids, solids and gases.

The FTIR spectrum of the Pioglitazone pure drug was found to be similar to the standard

spectrum of Pioglitazone as in I.P. The individual FT-IR spectra of the pure drug Pioglitazone, as

well as the combination spectra of the drug and polymers are shown in the shown in Spectra

No.1, Spectra No.2 and Spectra No.3, Spectra No.4.

The FT-IR spectrum did not show presence of any additional peaks for new functional

groups indicating no chemical interaction between drug and polymers.

Therefore results showed that there is no significant change in the chemical integrity of the drug

indicating no interaction between the drug molecule and polymers.

DISCUSSION


6.4 Analysis of Pioglitazone

6.1.1 Scanning of Pioglitazone

Pioglitazone was dissolved in both pH 1.2 and pH 7.4, further diluted with the same and

scanned for maximum absorbance in UV double beam spectrophotometer (shimadzu 1800) in the

range from 200 to 400 nm, using pH 1.2 and pH 7.4 as blank. The λmax of drug was found to be

269 nm.

6.1.2 Calibration Curve of Pioglitazone in 0.1 N HCL

The absorbance was measured in UV spectrophotometer at 269nm against 0.1N HCL.

The absorbance so obtained were tabulated as in Table No.14 .Calibration curve was plotted and

shown in Graph No.2 and standard calibration curve with slope 0.1866 and regression value R2

of 0.9988 was obtained.

6.1.3 Calibration Curve of Pioglitazone in 7.4 pH buffer

The absorbance was measured in UV spectrophotometer at 269nm against 7.4 pH buffer.

The absorbance so obtained was tabulated as in Table No.15. Calibration curve was plotted and

shown in Graph No.3.and standard calibration curve with slope 0.159 and regression value R2 of

0.9935 was obtained.

6.5 Characterization of Mucilage:

Isolated mucilage was Evaluated and Characterized for usage as binder in the formulation.

Organoleptic Evaluation : Isolated mucilage was found to be dark yellowish brown in

color, exhibited agreeable odour, bitter taste, rough & irregular texture and rough

fracture. Results were tabulated as in Table No.16.

DISCUSSION


Solubility: Mucilage is freely soluble in hot water, practically insoluble in inorganic

solvents, insoluble in cold water instead forms gel. Results were tabulated as in Table

No.17.

Yield Estimation: Fenugreek mucilage was extracted from 200gms of Fenugreek seeds

using 5liters of Acetone and obtained yield of 20gms. Results were tabulated as in Table

No.18.

Phytochemical Screening: Isolated mucilage was subjected to phytochemical test like

Molisch’s Test, Ninnhydrin Test, Ruthenium Red Test, Iodine Test, Dragendroff’s Test,

Keller-Killani Test, Ferric Chloride Test, and Libermann Burchard Test.

Due to presence of Carbohydrates and Mucilage inference was a violet coloured ring

formation at junction of 2liquids and Red color formation from Molisch’s test and

Ruthenium red test respectively. Results were tabulated as in Table No.19.

Determination of Swelling Index: Swelling Index was determined and was found to be

50%.

pH of Mucilage: The pH of solution in 1% w/v of water was determined using digital pH

meter was found to be 7.9

Micromeritic Evaluation: The isolated mucilage exhibited 0.66±0.043 bulk

Density(gm/ml), 0.94±0.098 tapped density(gm/ml), 17.4±3.11 Carr’s Index, 29.20o

angle of repose and proved to possess good flow property. Results were tabulated as in

Table No.20.

DISCUSSION


6.6 Evaluation of Physicochemical Properties:

All formulations F1 to F9 were evaluated for variable physiochemical properties such as

color, odor and shape. All the formulations were found to white in color, odorless and flat and

circular in shape. Results were tabulated as in Table No.21.

6.7 Evaluation of Pre-compression Parameters:

The results of all formulations F1 to F9 are shown in Table No.22, which were evaluated for

variable parameters such as bulk density, tapped density,% Compressibility index, Hausner’s ratio

and angle of repose.

Angle of repose: Angle of repose of all formulations was between 23 and 28 indicating

reasonable flow property and all formulations were found to fit with respect to flow

property.

Carr’s index: Carr’s index was between 13 to17 indicating all formulations were found

to be within the limits.

Hausner’s Ratio: Hausner’s ratio was between 1.14 and 1.20.

6.8 Evaluation of Post-compression Parameters:

The results of all formulations F1 to F9 are shown in Table No.23, which were evaluated for

variable parameters such as weight variation, hardness, friability, thickness, drug content,

disintegration.

Weight variation: The results of weight variation of tablets for all formulations was

found to be in the range of 119.91±0.22 to 120.11±0.60 mg indicating that the weight

variation is within the pharmacopoeia limits.

Hardness: Hardness was found to be in the range of 3.02±0.10 to 3.39±0.05.

DISCUSSION


Friability: Friability ranges from 0.27±0.06 to 0.43±0.62 indicating that the friability of

all formulations was less than 1%.

Thickness: Thickness of all formulations found to be in the range of 3.12±0.01 to

3.19±0.04.

Drug content: The percentage drug content of all formulations was found in the range of

93.51±0.57 to 98.99±0.42, which was all within the acceptable limits of official

standards.

6.9 In-vitro drug release: The in-vitro release study was carried out in three different dissolution

media namely 0.1N HCL (acidic buffer pH 1.2) for 2 hrs and then medium was replaced by

simulated intestinal fluid for next 6hrs (phosphate buffer 7.4pH).

The amount of drug released from formulations F1, F2, F3, F4, F5, F6, F7, F8 and F9 in

0.1N HCL after 2hrs were 45.714%, 42.406%, 45.723%, 29.146%, 40.431%, 29.146%,

25.844%, 13.912% and 16.568% respectively. The amount of drug released from formulations

F1, F2, F3, F4, F5, F6, F7, F8, F9, F10, F11, F12 in intestinal condition after 8hrs were 87.633%,

82.994%, 79.02%, 71.669%, 63.768%, 56.419%, 48.447%, 40.474% and 37.841% respectively.

Results showed that the drug release from the formulations decreased with increase in the

amount of polymer added in each formulation. Formulation F8 and F9 shows slow release

compared to all formulations.

The in-vitro drug release data of all 9formulations are shown in Table No.24 and Graph No.4.

DISCUSSION


6.10 Release kinetics:

The results obtained from in-vitro drug release were plotted adopting five different

mathematical models of data treatment as follows:

1. % Cum. Drug Release Vs. Time (Zero order rate kinetics).

2. Log % Cum. Drug Retained Vs. Time (First order rate kinetics).

3. % Cum. Drug release was plotted against √T (root time). (Higuchi model)

4. Log % Cum. Drug Release Vs. Log Time (Peppas exponential equation)

The curve fitting results of the release rate profile of the designed formulation are shown in

the Graph No.5,6,7 and 8 which gave an idea on the release rate and the mechanism of

release. The values were compared with each other for model and drug equation as shown in

Table No.25 based on the highest regression values (r2), fitting of the release rate data to

various models revealed that all the formulations (F1 to F9) follows first order release

kinetics with regression values ranging from 0.9432 to 0.9834.

All the formulations were subjected to Korsmeyer-Peppas plots, ‘n’ value ranges from

0.5551 to 0.7233 indicating that the drug release was by non-fickian diffusion mechanism.

6.11 Stability studies:

Stability study was conducted for the formulations F5 and F6 at 40 /75 RH and room

temperature for 2 months. Then the tablets were analysed for physical change, drug content

estimation and in-vitro dissolution studies at an interval of 15, 30, 45 and 60 days. Results show

that after analyzing there was no change in case of physical appearance, no significant

differences in the drug content and dissolution study. It was found that formulations found to be

stable throughout the study period. The results of stability studies are given in the Table No.26,

27 and 28.

DISCUSSION


6.12 CytoToxicity Studies:

Optimized formulations F5 and F6 were selected and evaluated for its cytotoxic activity

by MTT assay. In cytotoxicity study on the L-6 cell line, the optimized formulations F5 and F6

have shown low cytotoxicity activity. The result of cytotoxicity study is seen in the Table No.29

and Graph No.9.

Formulation F5 showed cytotoxicity of 7.74% at the lower concentration (62.5µg/ml) and

cytotoxicity of 28.02% at the higher concentration (1000µg/ml). Formulation F6 showed

cytotoxicity of 9.15% at the lower concentration (62.5µg/ml) and cytotoxicity of 28.52% at the

higher concentration (1000µg/ml). Both the samples at higher concentration (1000µg/ml) show

less cytotoxic effect.

By the cytotoxicity study we can say that the formulations F5 and F6 have not shown the

significant cytotoxic activity against all the L-6 cell lines. Thereby both the formulations F5 and

F6 have been selected for further in-vitro anti-diabetic studies.

DISCUSSION


6.13 In-vitro glucose uptake assay:

Cytotoxicity studied formulations F5 and F6 were selected and subjected for in-vitro

glucose uptake study. The result of cytotoxicity study is seen in the Table No.30 and Graph

No.10.

Formulation F5 shows % glucose uptake in the range of 74.13% - 61.75% at concentrations of

500 µg/ml and 250 µg/ml respectively. Formulation F6 shows % glucose uptake in the range of

21.10% - 13.83% at concentrations of 500 µg/ml and 250 µg/ml respectively. Therefore

formulation F5 has moderate anti-diabetic activity than formulation F6. In the Glucose uptake

activity the both the optimized formulations F5 and F6 have shown dose dependent activity

however formulation F5 shows more glucose uptake when compared to the Std, hence

formulation F5 has moderate activity on L-6 cell line

CONCLUSION It is more fun to arrive to a conclusion than to

Justify it.

CONCLUSION


6. CONCLUSION

The present study reports a novel attempt to Isolate and Evaluate Fenugreek mucilage and to

formulate delayed release tablets of the Pioglitazone by using Fenugreek mucilage as natural

polymer, delayed release tablets of the Pioglitazone were prepared by non-aqueous wet

granulation method. Various evaluation parameters were assessed, with a view to obtain delayed

release of Pioglitazone.

Details regarding Isolation and evaluation of Fenugreek mucilage and formulation and

evaluation of delayed release tablets of Pioglitazone have been discussed in previous chapters.

From the study following conclusions could be drawn,

Biocompatible polymer Fenugreek mucilage was isolated and evaluated.

The FT-IR spectrum did not show presence of any additional peaks for new functional

groups indicating no chemical interaction between drug and polymers.

Natural polymer Fenugreek was used as binder to formulate delayed release tablets.

Pre-compressional parameters bulk density, tapped density, angle of repose, Hauser’s

ratio are in the range of given in official standard, indicated that granules prepared by wet

granulation method were free flowing.

The post-compression parameters were evaluated for hardness, friability, weight

variation, disintegration time, and drug content were within the acceptable official limits.

Cumulative percentage drug release significantly decreased with increase in natural

polymer concentration.

CONCLUSION


The overall curve fitting into various mathematical models were found to be on an

average and were best fitted to first order kinetic model and the drug release from the

formulation was by non-fickian diffusion mechanism.

Formulations F5 and F6 were selected as optimum formulation.

Formulations F5 and F6 were stable and compatible at the selected temperature and

humidity in storage for 60 days.

From the stability studies it was found that there was no significant change in the drug

content, and in-vitro drug release characteristics of the tablet.

Formulation F5 and F6 were evaluated for its cytotoxic activity by MTT assay and

showed moderate anti-diabetic activity over the standard.

Thus, the formulated delayed release tablets seem to be a potential candidate.

SCOPE OF THE STUDY:

Further detailed stability studies and in-vivo bioavailability studies are to be done to establish the

efficacy of these formulations.

In-vitro–in-vivo correlations are to be done to establish the guarantee of efficacy and

bioavailability of the formulation.

SUMMARY

SUMMARY


8. SUMMARY

The present study reports an attempt to Isolate and Evaluate Fenugreek mucilage and to

formulate delayed release tablets of the Pioglitazone by using Fenugreek mucilage as natural

polymer.

Compatibility studies by FTIR indicate that no significant interactions between drug and

excipients. Tablets were prepared by non-aqueous wet granulation method. Nine formulations

(F1-F9) of delayed release were prepared using excipients like FGM, PVPK30, Magnesium

stearate, Lactose, Talc in combination. All the formulations were evaluated for pre-compression

and post-compression parameters. Drug excipient interaction was determined by FTIR. Short

term stability studies of formulated delayed release tablets were carried out at 400C / 75% RH for

2 months. The release kinetics was found to following first order kinetics as the value for ‘r’ is

(0.9432 to 0.9834) found to be high in comparison to zero order (0.7248 to 0.9888) and

Higuchi’s square root of time (0.8706 to 0.9883) ‘n’ values in between 0.5551 to 0.7233 shown

non-fickian release and drug. Stability studies at 40 0C / 75 % RH for 2months indicated that

there are no significant loss in drug content, release profile and physical appearance. By the

cytotoxicity study we can say that the formulations F5 and F6 have not shown the significant

cytotoxic activity against all the L-6 cell lines. In the Glucose uptake activity the both the

optimized formulations F5 and F6 have shown dose dependent activity however formulation F5

shows more glucose uptake when compared to the Std. Rosioglitazone, hence formulation F5 has

moderate anti-diabetic activity on L-6 cell line. Thereby both the formulations F5 and F6 have

been selected for further in-vitro anti-diabetic studies.

In summary, the release profiles of delayed release tablet formulations were quite

promising for further studies.

BIBLIOGRAPHY

BIBLIOGRAPHY


9. BIBLIOGRAPHY

1. Definition and diagnosis of diabetes mellitus and intermediate hyperglycemia, World

Health Organization 2006:p.1-46.

2. Kajal G, Rajan R, Anindya KM, Bikramanditya C, Arunabha N et al. Evaluation of

physicochemical properties and in-vitro release profile of Glipizide matrix patch.

Brazilian Journal of Pharmaceutical Sciences. 2010; 46(2):213-18.

3. Insulin and Oral Hypoglycemic Agents. In Barar FSK, Essentials of

Pharmacotherapeutics, S. Chand and Co. Ltd., New Delhi: 548-56.

4. Insulin, Oral hypoglycemic drug and glucagon. In: Tripathy KD, Essentials of Medical

Pharmacology, New Delhi: Jaypee Brother, Medical Publishers (P) Ltd., 2003; 335-53.

5. WHO report on NCD’S Burden: mortality, morbidity and risk factors, Chapter 1.

6. Insulin and Oral Anti diabetic Drug. In Satoskar RS, Bhandarkar SD, Ainapure SS.,

Pharmacology and Pharmacotherapeutics. Popular Prakashan; 2000; 874, 889,900-901.

7. Insulin and Oral Hypoglycemic Agents. In Brody, Larner, Minneman. Human

Pharmacology, Mosby Publications 1995, 541-51.

8. Oral Hypoglycemic drug. In Hardman JG, Limbira LE, Gilman AG. Goodman and

Gilman The pharmacological Basis of Therapeutics, New York: McGraw Hill Medical

Publishing Division, 2001; 1586-87.

9. Diabetes Care: American Diabetes Association: Jan 2014: Volume 37, Supplement 1.

10. National diabetes statistics report, 2014. Diagnosis of Diabetes and Prediabetes.

11. National diabetes statistics report, 2014. Centers for Disease Control and Prevention

website. www.cdc.gov/ diabeteS/pubs/statsreport14 Htm. Updated June 13, 2014.

Accessed June 16, 2014.

BIBLIOGRAPHY


12. Inzucchi SE, Bergenstal RM, Buse JB, Diamant M, Mnagement of Hyperglycemia in

type2 diabetes:A patient centered approach. Diabetes care journal(2012) vol.35.

13. Benett PH. Diabetes: Definition and Pathogenesis. In: Ronald Kahn C, Weir GC,

Joslin‘s Diabetes Mellitus. 13th ed. New Delhi: Waverly Private Limited; 1996. 193-7.

14. G.S Sainani et al., Diabetology chapter-16 in API text book of medicine. 6th

edition.

2001, Published by Associated of Physicians of India: p.987-1017.

15. Tripathi KD. Essentials of Medical Pharmacology. 6th

edition. New Delhi: Jaypee, 2003:

p.266-67.

16. U.Smith et al, Pioglitazone: Mechanism of action, International Journal of Clinical

Practice. Supplement 2001(121):13-18.

17. Kamboj S., Gupta G.D. and Oberoy J. (2009) Matrix Tablets: An Important Tool for

Oral Controlled-Release Dosage Forms - A Review. Indian Journal of Novel Drug

Delivery 2, 110 – 118.

18. Chandana N, Gopinath H, Bhowmik D, Williamkeri, Thirupathi Reddy A, Modified

release dosage form, Journal of Chemical and Pharmaceutical Sciences (2013):6(1).

19. Venkatraman S., Davar A., Chester A., Kleiner L. and Wise D.L. (2000) an overview of

controlled release systems, Handbook of Pharmaceutical Controlled Release

Technology. New York: Marcel Dekker, Inc.

20. Nayak AK, Pal D, Das S. Calcium pectinate-fenugreek seed mucilage mucoadhesive

beads for controlled delivery of metformin HCL in Carb Polym. 2013; 96: 349-57.

21. Mundhe M.R, Pagore R.R, Biyani K.R. Isolation and Evaluation of Trigonella Foenum

Graecum Mucilage as gelling agent in Diclofenac Potassium Gel. Int. J. Ayur and Herb

medic. 2012 April; 2(2):300-06.

http://europepmc.org/search?page=1&query=JOURNAL:%22Int+J+Clin+Pract+Suppl%22

http://europepmc.org/search?page=1&query=JOURNAL:%22Int+J+Clin+Pract+Suppl%22

BIBLIOGRAPHY


22. Malviya R, Srivastava P, Kulkarni GT. Application of mucilages in drug delivery-A

review. Adv in Biomed Res. 2011; 5(1):01-07.

23. Nayak AK, Pal DK, Pany DR, Mohanty B. Evaluation of Spinacia oleracea L. leaves

mucilage as an innovative suspending agent. J Adv Pharm Tech Res.2010; 1(3):338–41.

24. Prajapati VD, Jani GK, Moradiya NG, Randeria NP. Pharmaceutical applications of

various natural gums, mucilages and their modified forms in Carb Polym. 2013; 92:

1685-99.

25. Sabale V, Patel V, Paranjape A. Natural Gums and Mucilages as Bioadhesive

Components in Oral Controlled Drug Delivery Systems: An Overview. World J. Pharm

Res. 2012; 2(1):166-88.

26. Nathiya S, Durga M, Devasena T, Therapeutic role of Trigonella foenum-graecum

[Fenugreek] – A Review, Int. J. Pharm. Sci. Rev. Res., 27(2), July – August 2014;

Article No. 12.

27. Xuea W, Leib J, Lia X, Zhanga R. Trigonella foenum graecum seed extract protects

kidney function and morphology in diabetic rats via its antioxidant activity. Nut Res J.

2011; 31:555–62.

28. Gondaliya RP, Shah PD, Nerkar PP, Mahajan HS, Ige PP. Buccal gel of verapamil HCL

based on fenugreek mucilage and xanthum gum:in vitro evaluation. American J. Pharm

Tech res. 2013;3(1):410-19

29. Roberts KT, Cui SW, Chang YH, Graham T. The influence of fenugreek gum and

extrusion modified fenugreek gum on bread, Food Hydrocolloids.2012; 26: 350-58.

BIBLIOGRAPHY


30. Patil S, Garima Jain, Holistic approach of Trigonella foenum-graecum in

Phytochemistry and Pharmacology- A Review, Current Trends in Technology and

Science,vol3(1).

31. Charles OO, Soetan KO, A Review of the Health Benefits of Fenugreek (Trigonella

foenum-graecum L.): Nutritional, Biochemical and Pharmaceutical Perspectives,

American journal of social issues and humanities: 2014: 2276 – 6928.

32. Sweetman, C.S., Martindale the complete drug reference, London, Pharmaceutical Press,

333, 2002.

33. Tripathi, K.D., Essential of medical pharmacology, New Delhi, Jaypee brother’s medical

publishers (p) ltd., 247-51, 2003.

34. Tan, M.H., Johns, D., Strand, J., Halse, J., Madsbad, S. and Eriksson, J.W,Sustained

effect of pioglitazone vs. glibenclamide on insulin sensitivity,glycemic control and lipid

profile in patient with type II diabetes, Diabet Med.,21, 859 -866, 2004.

35. Nokhodchi A, Nazemiyeh H, Khodaparast A, Sorkh-Shahan T, Valizadeh H, Ford JL.

An in vitro evaluation of fenugreek mucilage as a potential excipient for oral controlled-

release matrix tablet. Drug Dev. Ind Pharm. 2008 Mar;34(3):323-29

36. Nayak AK, Pal DK, Pany DR, Mohanty B. Evaluation of Spinacia oleracea L. leaves

mucilage as an innovative suspending agent. J Adv Pharm Tech Res. 2010; 1(3):338–41.

37. Yadav IK, Jain DA. Design and development of trigonella foenum graceum seed

polysacharide mucilage based matrix tablets of diclofenac sodium. World J Pharm Res.

2012; 1(4):1170-82.

BIBLIOGRAPHY


38. V. Senthil, D. Sripreethi. Formulation and Evaluation of Paracetamol Suspension from

Trigonella Foenum Graecum Mucilage. J Adv Pharm Edu & Res. 2011; 1(5):225-33.

39. Sav AK, Kapadi P, Shukla S, Amin P. Extended release formulation of theophylline

using modified fenugreek gum as a hydrophilic polymer. Int J Pharm Sci & Res. 2014;

5(1):116-22.

40. Hadriche OB, Bouaziz M, Jamoussi K, Simmonds MSJ. Comparitive study on

hypocholesterolemic and antioxidant activities of various extracts of fenugreek seeds,

Food Chemistry. 2013; 138: 1448-53.

41. Nitalikar MM, Patil RA, Dhole SD, Sarkarkar DM, Evaluation of fenugreek seed husk

as tablet binder, Int J Pharm Res & Dev. 2010;2(8):oct-003.

42. Tavakoli N, Varrshosaz J, Ghannadi A, Bavarsad N, Evaluation of Trigonella foenum-

graecum seeds gum as a novel tablet binder, Int J Phar & Ph Sci. 2012;4(1): 0975-1491.

43. Waugh J, Keating GM, Plosker GL, Easthope S , Robinson DM. Pioglitazone- a review

of its use in type 2 diabetes mellitus. Drugs 2006; 66(1):85-109.

44. Gillies PS, Dunn CJ. Pioglitazone. Drugs Dis Ther 2000;60(2):333-43.

45. Singh C, Jain AK, Agarwal K. Formulation and evaluation of extended release tablet of

Pioglitazone by melt granulation technique. Indian Drugs 2008 June; 45(6):461- 468.

46. N.N.rajendran, Natarajan R, Subashini, Hitesh Patel. Formulation and evaluation of

sustained release bilayer tablets of metformin Hcl and Pioglitazone Hcl international

Journal of current pharmaceutical research issue- 0975-7066 volume 3, issue 3, 2011.

BIBLIOGRAPHY


47. Hatorp V, Hansen KT, Thomsen MS. Influence of drugs interacting with CYP3A4 on

the pharmacokinetics, pharmacodynamics, and safety of the prandial glucose regulator

repaglinide. J Clin Pharmacol, 2003; 43:649-660.

48. K.P.R. Chowdary, P. Dwarakanadha Reddy, Formulation development studies on β-

cyclodextrin complexation of pioglitazone matrix tablets International Journal of

Advances in Pharmaceutical Research 2011 ISSN: 2230 – 7583.

49. Pioglitazone monographs .Facts and comparisons. Facts and comparison. St

.Louis.2002.

50. M. Rangapriya, V. Manigandan, R. Natarajan and K. Mohankumar. Formulation and

Evaluation of floating tablets of Pioglitazone hydrochloride. Vol. 1 (3) Jul-Sep 2012.

ISSN: 2277-5005.

51. G. Chinna devi and K. P. R. Chowdary. Formulation And In Vitro Evaluation Of

Floating Tablets Of Pioglitazone For Oral Sustained Release Employing Olibanum Gum

and HPMC Int. J. Chem. Sci.: 10(1), 2012, 281-290

52. Omaima AS, Hammad MA, Megrab NA, Zidan AS. Formulation and optimisation of

Mouth Dissolve Tablet contain Rofecoxcib solid dispersion. AAPS Pharm Sci Tech

2006;7(2):E1-E6.

53. Damodharan N, Manimaran V, Sravanthi B, Formulation development and evaluation of

delayed release doxycycline tablets, Int Journal Pharm and Pharma Sci: 2010:2(1).

54. Suresh PKumar, Navaneetha SK, Pavani S, Surendarnath Y, Divya S, Sahithi Y,

Formulation and evaluation of rabeprazole sodium delayed release tablets: Der

Pharmacia Lettre, 2012, 4 (1):287-296.

BIBLIOGRAPHY


55. Damodharan N, Manimaran V, Sravanthi B, Formulation development and evaluation of

delayed release doxycycline tablets:2010:2(1).

56. V.Kalvimoorthi, N.Narasimhan, Formulation development and evaluation of aspirin

delayed release tablets, Inte J Ph.Sci Review & Res., 2014:7(1):076.

57. http://en.wikipedia.org/wiki/Pioglitazone.

58. http://www.drugbank.ca/drugs/DB01132.

59. Ainley W, Weller PJ. A Handbook of pharmaceutical excipients. Pharmaceutical press,

American pharmaceutical association, 2nd

Edn, 1994:392-99.

60. http://en.wikipedia.org/wiki/polyvinylpyrrolidone.

61. Ainley W, Paul J W. Hand book of pharmaceutical excipients. 2th

Edn. Pharmaceutical

press, American pharmaceutical association 1994:280-82.

62. Raymond CR, Paul SJ, Sian CO. Handbook of pharmaceutical excipients. 5th

ed. Great

Britain; Pharmaceutical press and American Pharmaceutical Associaiton;2006:430-3.

63. Ainley W, Paul J W. Hand book of pharmaceutical excipients. 2th

Edn. Pharmaceutical

press, American pharmaceutical association 1994:519-21.

64. http://en.wikipedia.org/wiki/talc.

65. Raymond CR, Paul SJ, Sian CO. Handbook of pharmaceutical excipients.5th

ed. Great

Britain; Pharmaceutical press and American Pharmaceutical Associaiton;2006;389-95.

http://en.wikipedia.org/wiki/Pioglitazone


http://en.wikipedia.org/wiki/polyvinylpyrrolidone

http://en.wikipedia.org/wiki/talc

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