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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
FENUGREEK MUCILAGE AS A EXCIPIENT IN THE TABLET DOSAGE FORM” is a
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,
SAC College of Pharmacy,
B.G.Nagara,
Mandya-571448
Karnataka
SRI ADICHUNCHANAGIRI COLLEGE OF PHARMACY
B.G.NAGARA-571448
ENDORSEMENT BY THE HEAD OF THE DEARTMENT
This is to certify that the dissertation entitled “ISOLATION AND EVALUATION OF
FENUGREEK MUCILAGE AS A EXCIPIENT IN THE TABLET DOSAGE FORM” is a
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,
SAC College of Pharmacy,
B.G.Nagara-571448
Karnataka
SRI ADICHUNCHANAGIRI COLLEGE OF PHARMACY
B.G.NAGARA-571448
ENDORSEMENT BY THE PRINCIPAL/HEAD OF THE INSTITUTION
This is to certify that the dissertation entitled “ISOLATION AND EVALUATION OF
FENUGREEK MUCILAGE AS A EXCIPIENT IN THE TABLET DOSAGE FORM” is a
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,
SAC College of Pharmacy,
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,
SAC College of Pharmacy,
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
Department of Pharmaceutics, SACCP
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
LIST OF ABBREVIATIONS
Department of Pharmaceutics, SACCP
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
Department of Pharmaceutics, SACCP
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
Department of Pharmaceutics, SACCP
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
Department of Pharmaceutics, SACCP
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
Department of Pharmaceutics, SACCP
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
Department of Pharmaceutics, SACCP Page 2
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.
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Department of Pharmaceutics, SACCP Page 3
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
Department of Pharmaceutics, SACCP Page 4
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)
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Department of Pharmaceutics, SACCP Page 5
3. Chromosome 20, HNF-4 (MODY1)
4. Chromosome 13, insulin promoter factor-1 (IPF-1; MODY4)
5. Chromosome 17, HNF-1 (MODY5)
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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Department of Pharmaceutics, SACCP Page 6
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
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Department of Pharmaceutics, SACCP Page 9
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
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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).
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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
Department of Pharmaceutics, SACCP Page 12
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
Department of Pharmaceutics, SACCP Page 13
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
Department of Pharmaceutics, SACCP Page 14
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
Department of Pharmaceutics, SACCP Page 15
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
Department of Pharmaceutics, SACCP Page 16
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
INTRODUCTION
Department of Pharmaceutics, SACCP Page 17
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
Department of Pharmaceutics, SACCP Page 18
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
Department of Pharmaceutics, SACCP Page 19
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
Department of Pharmaceutics, SACCP Page 20
(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
Department of Pharmaceutics, SACCP Page 21
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
Department of Pharmaceutics, SACCP Page 22
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
Department of Pharmaceutics, SACCP Page 23
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
Department of Pharmaceutics, SACCP Page 24
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
Department of Pharmaceutics, SACCP Page 25
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
Department of Pharmaceutics, SACCP Page 26
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
Department of Pharmaceutics, SACCP Page 27
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
.
REVIEW OF LITERACTURE
Department of Pharmaceutics, SACCP Page 28
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
REVIEW OF LITERACTURE
Department of Pharmaceutics, SACCP Page 29
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
.
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Department of Pharmaceutics, SACCP Page 30
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
REVIEW OF LITERACTURE
Department of Pharmaceutics, SACCP Page 31
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
REVIEW OF LITERACTURE
Department of Pharmaceutics, SACCP Page 32
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
REVIEW OF LITERACTURE
Department of Pharmaceutics, SACCP Page 33
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
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Department of Pharmaceutics, SACCP Page 34
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.
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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
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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
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Department of Pharmaceutics, SACCP Page 37
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
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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
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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
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Department of Pharmaceutics, SACCP Page 40
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
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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)
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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
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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
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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.
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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.
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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
Stability and storage
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.
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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.
Stability and storage
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.
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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.
Stability and storage
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.
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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.
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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
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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.
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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’
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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
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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.
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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
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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
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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
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Department of Pharmaceutics, SACCP Page 58
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
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Department of Pharmaceutics, SACCP Page 59
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
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Department of Pharmaceutics, SACCP Page 60
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.
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Department of Pharmaceutics, SACCP Page 61
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
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Department of Pharmaceutics, SACCP Page 62
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
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Department of Pharmaceutics, SACCP Page 63
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
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Department of Pharmaceutics, SACCP Page 64
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
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Department of Pharmaceutics, SACCP Page 65
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.
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Department of Pharmaceutics, SACCP Page 66
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
MATERIALS AND METHODS
Department of Pharmaceutics, SACCP Page 67
(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
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Department of Pharmaceutics, SACCP Page 68
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.
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Department of Pharmaceutics, SACCP Page 69
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
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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
Department of Pharmaceutics, SACCP Page 71
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
Department of Pharmaceutics, SACCP Page 72
Spectra 2: FT-IR spectra of Fenugreek Mucilage
Spectra 3: FT-IR spectra of PVP K30
RESULTS
Department of Pharmaceutics, SACCP Page 73
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
Department of Pharmaceutics, SACCP Page 74
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
Department of Pharmaceutics, SACCP Page 75
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
Department of Pharmaceutics, SACCP Page 76
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
Department of Pharmaceutics, SACCP Page 77
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
Department of Pharmaceutics, SACCP Page 78
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
Department of Pharmaceutics, SACCP Page 79
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
F2 White color Flat and circular Odorless
F3 White color Flat and circular Odorless
F4 White color Flat and circular Odorless
F5 White color Flat and circular Odorless
F6 White color Flat and circular Odorless
F7 White color Flat and circular Odorless
F8 White color Flat and circular Odorless
F9 White color Flat and circular Odorless
RESULTS
Department of Pharmaceutics, SACCP Page 80
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
Department of Pharmaceutics, SACCP Page 81
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
Department of Pharmaceutics, SACCP Page 82
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
Department of Pharmaceutics, SACCP Page 83
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
Department of Pharmaceutics, SACCP Page 84
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
Department of Pharmaceutics, SACCP Page 85
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
Department of Pharmaceutics, SACCP Page 86
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
Department of Pharmaceutics, SACCP Page 87
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
Department of Pharmaceutics, SACCP Page 88
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
Department of Pharmaceutics, SACCP Page 89
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
Department of Pharmaceutics, SACCP Page 90
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
Department of Pharmaceutics, SACCP Page 91
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
Department of Pharmaceutics, SACCP Page 92
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
Department of Pharmaceutics, SACCP Page 93
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
Department of Pharmaceutics, SACCP Page 94
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
Department of Pharmaceutics, SACCP Page 95
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
Department of Pharmaceutics, SACCP Page 96
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
Department of Pharmaceutics, SACCP Page 97
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
Department of Pharmaceutics, SACCP Page 98
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
Department of Pharmaceutics, SACCP Page 99
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
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Department of Pharmaceutics, SACCP Page 100
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