Chitosan Loaded Mucoadhesive Microspheres of Gliclazide - Journal

R G U H S
Journal of
Pharmaceutical
Sciences
July - September 2011 / Vol 1 / Issue 2
ISSN: 2249-2208
Abstracted and Indexed in Geneva Foundation for Medical Education and Research (GFMER)
and Pharmaceutical Sciences Open Access Resources (PSOAR)
Rajiv Gandhi University of Health Sciences, Karnataka
w w w . r j p s . i n

R G U H S
Dr. Divakar Goli
Principal & Professor of Biotechnology
Acharya B M Reddy College of Pharmacy
Bangalore
Dr. Gopal Krishna Rao
Prof. & Head, Dept. of Pharmaceutical Chemistry
Al-Ameen College of Pharmacy
Bangalore
Dr. Murugan V.
Principal,Professor of Pharmaceutical Chemistry
Dayanand Sagar College of Pharmacy
Bangalore
Dr. Md. Naseeruddin Inamdar
Prof & Head, Dept. of Pharmacology
Al-Ameen College of Pharmacy
Bangalore
Dr. Nagaraj
Professor
Dept. of Pharma Analysis
PES College of Pharmacy
Bangalore
Journal of Pharmaceutical Sciences (RJPS)
EDITORIAL BOARD
Dr. K.S Sriprakash
Vice-Chancellor
RGUHS
Dr. D. Prem Kumar
Registrar
RGUHS
Dr. Niranjan
Registrar (Evaluation)
RGUHS
Editor-in-Chief
Prof. B. G. Shivananda
Principal
Al-Ameen College of Pharmacy, Bangalore
Executive Editor
Dr. Roopa S. Pai
Professor of Pharmaceutics
Al-Ameen College of Pharmacy, Bangalore
Associate Editor
Dr. Raju B. Koneri
Dean & Professor of Pharmacology
Karnataka College of Pharmacy, Bangalore
MEMBERS
Dr. Nithin Mahurkar
Prof & Head, Dept. of Pharmacology
HKE College of Pharmacy
Dr. Raman Dang
Professor, Dept. of Pharmacognosy
Al-Ameen College of Pharmacy
Bangalore
Dr. Rama Raj Urs
Librarian
RGUHS, Bangalore
Prof. Ramesh C.
Board of Studies Chairman
Under Graduate
RGUHS
Dr. Sanjay Pai P.N.
Professor
Dept. of Pharma Chemistry
Goa College of Pharmacy
Panaji, Goa
Dr. Pranesh Gudur
Director I/C Prasaranga
RGUHS, Karnataka
Dr. Shoba Rani R. Hiremath
Prof & Head, Dept. of Pharmacy Practice
Al-Ameen College of Pharmacy
Bangalore
Prof. Dr. Sirse Kranti Kumar
Board of Studies Chairman
Post Graduate
RGUHS
Dr. Srinath M.S.
Dean
Faculty of Pharmacy
RGUHS
Dr. Swamy P.V.
Professor
Dept. of Pharmaceutics
HKE College of Pharmacy
Dr. Vishwanath B.A
Principal
Bangalore Institute of Pharmacy
Education and Research, Bangalore

RJPS
Issn: 2249-2208
R G U H S
Journal of
Pharmaceutical
Sciences
(An Official Publication of RGUHS)
July - September 2011 / Vol 1 / Issue 2
Rajiv Gandhi University of Health Sciences, Karnataka
4th ‘T’ Block, Jayanagar, Bangalore 560041
Phone: 080-26961934, 26961935, E-mail: rguhsjps@gmail.com
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RJPS
Editorial Board Welcomes
Dr. K.S. Sriprakash
Vice-Chancellor
RGUHS, Bangalore
Dr. K.S. Sriprakash has taken over charge as 6th Vice-Chancellor of
RGUHS w.e.f 14-06-2011. He was Director of Minto Ophthalmic
Hospital, Regional Institute of Ophthalmology, Bangalore and Chief
of Department of Vitre-retina. He has undergone training in Vitre-
retina in Wills Eye Hospital, USA and Schieie Eye Institute, USA.
Dr. K.S. Sriprakash has 29 years of experience and was instrumental
in organising community ophthalmic services.

RGUHS
Journal of Pharmaceutical
Sciences
Scientific Tools
RJPS
Contents
Preamble
Gowraganahalli Jagadeesh..................................................................................................................................................................................... 96
Creative, Critical Thinking and Logic in Research
Fredricka Reisman ..................................................................................................................................................................................... .. 97 - 102
Review Article
Need for Inclusion of Scientific Writing Skill Subjects in Indian Post Graduate Pharmacy Course
Patil J.S, Kotnal R.B, Birajdar R.P, Marapur S.C and Kadam D.V .............................................................................................................. 103 - 106
Research Article
A Novel Spectrophotometric Estimation of Pramipexole in Bulk Drug and Formulations
Shobha Manjunath, Satish Middi and Venkatesh Chouhan ........................................................................................................................ 107 - 110
Validated UV-Spectrophotometric Estimation of Entecavir in Bulk and Formulations
Malipatil S.M, Bharath S Athanikar and Mogal Dipali. ..................................................................................................................................111 - 116
Antihyperlipidemic Effect of Ethanolic Extract of Hibiscus rosa sinensis Flowers in Hyperlipidemic Rats
Sikarwar Mukesh S. and Patil M.B ...............................................................................................................................................................117 - 122
A Study on Drug-Drug Interaction of Diltiazem with Nateglinide in Diabetic Animals
Suresh D.K, Raza Hasan, Hamza Sheth, Md. Saifuddin Khalid and Mohiuddin M ..................................................................................... 123- 126
Influence of Vitamin C with Lansoprazole in Pylorus Ligation Induced Ulcer Model in Rats
Nitin M, Prasad K, Girish M, Ather Javed, Chetan M and Krunal S. ............................................................................................................127 - 130
Assessment of Safety and Efficacy of Doxycycline and Azithromycin Preparations in Patients with Acne Vulgaris
Mahendra Kumar B.J, Ramakrishna S, Kranti Basavant Patil, Sandeep A, Bhimaray S Krishnagoudar and Katti Ravi Venkappa .......... 131 - 135
Antidiarrhoeal Activity of Aqueous Extract of Mimosa pudica Leaves
Md. Saifuddin Khalid, Shah Jinesh Kumar, Suresh D.K., Rajnish Kumar Singh, Reddy Narasimha I.V. and Shaikh Azhar Hussain ........ 136 - 140
Assessment of Various Combination of Drugs Used in Treatment of Lower Respiratory Tract Infection
Imran Ahmad Khan, Shobha Rani R.H, Geeta S, Mahvash Iram ............................................................................................................... 141 - 145
Formulation and Evaluation of Mucoadhesive Buccal Drug Delivery System of Metoprolol Tartrate by Using Central
Composite Design
Prakash Rao B and Gandhi Purvesh ........................................................................................................................................................ 146 - 156
Development and Evaluation of Mucoadhesive Buccal Films of Nebivolol
Bushetti S.S, Mane Prashant P and Kardame S.S ..................................................................................................................................... 157 - 162
Chitosan Loaded Mucoadhesive Microspheres of Gliclazide: In vitro And In vivo Evaluation
Senthil A, Thakkar Hardik R, Ravikumar and Narayanaswamy V.B ...........................................................................................................163 - 171
Effect of Different Acids on the Formation of E and Z Isomers of Dothiepin
Gopal Krishna Rao, Ramesha A.R, Amit Kumar Jain and Sanjay Pai P.N ................................................................................................ 172 - 175
Instructions to Authors

RJPS
RGUHS Journal of Pharmaceutical Sciences Scientific Tools
Preamble
Scientific research is essentially an intellectual investigation undertaken to gain
new information, close gaps in knowledge, and understand concepts to confirm
an idea. Research, whether basic or applied, should have a reasonable possibility
for success. The thrill of scientific discovery and the transfer of technology from
the lab bench and its potential application to a patient's bedside is the hallmark
accomplishment of one's research career. It is a reward of unmatched happiness.
For that to happen, one needs clear vision and imagination.
Research is both a social and cooperative venture. It is important to establish a
positive climate for research by organizing appropriate resources and making
them readily available. To accomplish this, assistance from all corners is needed.
After all, it takes a whole research community to mold a scientist. In addition, it
needs an ever-growing array of scientific tools, and the expertise of an
experienced mentor to steer the knowledge and enthusiasm of a novice
researcher into the proper direction of becoming an established research
investigator. In this endeavor, scientific journals, like mentors, play a vital role in
educating researchers at all levels. The objective of the newly created section
'Scientific Tools' will deliver concise, but regular, descriptions of skills required to
take the journey a step forward in improving one's scientific enterprise.
The first article in the series launches the basic concepts of scientific research by
introducing application of the creative process needed for correctly choosing a
research topic or idea. Dr. Reisman reviews essentials of creativity and critical
thinking in developing many ideas and later how to converge them. We wish to
continue our scientific journey into the areas of research processes, research
methods, study designs, data analysis, scientific communication and much more
in an attempt to help everyone who does professional business in biomedical
research. Check this section promptly for a description of a new tool when each
new issue is received.
Gowraganahalli Jagadeesh
US Food and Drug Administration
Silver Spring, Maryland, USA
gowra.jagadeesh@fda.hhs.gov
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

RGUHS Journal of Pharmaceutical Sciences
Creative, Critical Thinking and Logic in Research
Fredricka Reisman
Goodwin College of Professional Studies, Drexel/Torrance Center for Creativity and Innovation, Drexel University, Philadelphia, PA 19104,
USA
Creative, Critical Thinking and Logic in Research
In order for me, a western mathematics and creativity
researcher and educator, to respond to the exciting invitation
to write an article dealing with creativity for this prestigious
journal published by the Rajiv Gandhi University of Health
Sciences, I needed to investigate the essence and focus of the
audience. I needed to become familiar with the context in
which my article was to contribute. My investigation led me to
the meanings behind the Emblem of the Rajiv Gandhi
University of Health Sciences, which to my delight sets the
stage for creative (Human Energy), critical thinking (the
Human Soul) and logic (Knowledge And Enlightenment) as
cornerstones for pharmaceutical research.
The Emblem
The Emblem of The Rajiv Gandhi University of Health
Sciences is a symbolic expression of the confluence of both
Eastern and Western Health Sciences. A central wand with
entwined snakes symbolizes Greek and Roman Gods of
Health called Hermes. Mercury is adapted as symbol of
modern Medical Science. The pot above the snake depicts
Amrutha Kalasham of Dhanvanthri, the father of all health
sciences. The wings above it depicts Human Soul called
Hamsa (Swan) in Indian Philosophy. The rising sun at the top
symbolises knowledge and enlightenment. All of them set
inside the state map of Karnataka. The two twigs of leaves in
RGUHS Journal of Pharmaceutical Sciences
Received: 14/8/2011, Modified: 27/8/2011, Accepted: 28/8/2011
97
Scientific Tools
Western Philosophy symbolizes Olive branches, which is an
expression of Peace, Love And Harmony. In Hindu
Philosophy, it depicts the Vanaspathi (also called as Aushadi)
held in the hands of Dhanvanthri, which are the source of all
medicines. The lamp at the bottom depicts human energy
(Kundalini). The script “Devahitham Yadayahu” inside the
lamp is taken from Upanishath Shanthi Manthram
(Bhadram Karnebhi Shrunuyanadev…) which says “May we
live the full span of our lives allotted by God in perfect health”
which is the motto of the Rajiv Gandhi University of Health
Sciences.
Link to Creative, Critical Thinking and Logic
This link from emblem to reality is assuring that pharmacy
students, faculty and practitioners, in addition to being
excellent learners and researchers, are also creative problem
solvers, first-rate scientists, and effective clinicians. In looking
at the traits of highly creative people listed below, we see that
many of these traits are salient to creative science researchers
and practitioners in the Pharma industries. In fact, a recent
1
publication considered the question of why an
understanding of creativity and critical thinking is important
for biomedical scientists, especially those new to their science
career paths.
“Understanding how the big breakthroughs occur can lead the newly
minted scientist to efficiently engage in creative research that results in a
novel, appropriate and useful discovery, to obtain funding support, and
to navigate the publication channel…”
1
F. K. Reisman, 2010
In addition, a recent survey of 1,500 chief executives
2
conducted by IBM's Institute for Business Value identified
"creativity" as the most important leadership competency for
corporate success of the future. Note that the desired
competency of leaders is “creativity----not operational
effectiveness, managerial discipline, influence, or even
dedication.” Until recently, creativity was viewed as an
essential element of research or product development, not the
crucial characteristic of leadership.
Traits of Highly Creative People
Following are traits representative of highly creative people
which embody members of the pharmaceutical industries
and that form an assessment checklist as shown in Table 1:
Next, in Table 2 are evaluation criteria for assessing creative
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

Table 1: Creative Traits Assessment
Creative Trait
1. A high level of curiosity
2. Willingness to learn from experience
3. Preparedness to take risks
4. Persistence in situations of failure
5. High levels of energy
6. Tolerate contradictions, ambiguities, and uncertainties in work
7. Resist premature closure
8. May see problems or challenges as more complex because of seeing more alternatives
9. Embrace change
1 2 3 4
Table 2. Evaluation Form Used by Preceptors or Supervisors
Evaluation of Problem-Solving Abilities Instructions: Please provide the following information on
your students or employee. Complete a separate evaluation form for each individual.
Preceptor/Supervisor: ___________________
Student/Employee: __________________
Date:___________________
1. Please rate individual's overall problem (clinical or nonclinical)-solving abilities by circling the letter that
most closely describes the student/employee.
a. High, able to solve difficult problems, equivalent to an experienced researcher or pharmacist-clinician.
b. Very good, able to solve moderate problems, but with some inadequacies with difficult problems.
c. Good, able to solve simple and moderate problems, but unable to solve difficult problems.
d. Fair, able to solve simple problems but unable to solve problems of greater difficulty.
e. Poor, unable to solve simple problems.
Fredricka Reisman - Creative, Critical Thinking and Logic in Research
Please evaluate yourself or other individuals on each of the following traits of highly creative people, using the scale below. Check ( ) the
number corresponding to your evaluation. 4 = Outstandingly creative, 3 = Competently creative majority of time, 2 = Likehood of improvement
with training, 1 = Lacks evidence of creative thinking
Nonclinical/Clinical Problem-solving Competence 1 2 3 4
a. Recognizing the existence of a problem.
b. Defining the nature/requirements of a problem.
c. Generating more than one set of steps that may solve a problem.
d. Knowledge acquisition to solve a problem.
e. Organizing information about a problem.
f. Critical and logical thinking process related to a problem.
g. Allocating mental and physical resources to solving a problem.
h. Monitoring the outcome related to the solution of a problem.
i. Personal attributes required for problem-solving (e.g., values, attitudes, emotions, confidence).
Please evaluate student/employee on each of the following aspects of nonclinical or clinical problem solving, using the scale below. Check
( ) the number corresponding to your evaluation. 4 = Outstanding, 3 = Competent, 2 = Improvement needed, 1 = Incompetent
98

problem solving in pharmacy students or employees by their
3
preceptors or supervisors :
A mobile-based self-assessment is the Reisman Diagnostic
4
Creativity Assessment (RDCA) . The RDCA is a free Apple
application that may be downloaded to an iPhone, iPad or
iTouch via iTunes. The RDCA is built upon 11 creativity
factors, shown in Table 3, that are most prevalent in the
research literature as representative of creative thinking. The
RDCA is built upon the Torrance Tests of Creative Thinking
5
(TTCT) , which in turn stems from Guilford's creativity
6
research . The TTCT remains the most widely used test of
creativity and the most referenced of all creativity tests.
However, it must be scored by trained evaluators, takes two
hours for administration, focuses on prediction of creative
performance, and is costly.
The RDCA may be self - scored, takes about 10 minutes to
complete, and at this point in time is free. This is a self-report
Likert-type assessment designed to be used diagnostically to
identify one's creative strengths, rather than to predict
creativity. The RDCA assesses an individual's self-perception
on 11 major creativity factors. The results may be used to
determine which factors already are strong, which factors one
personally wishes to strengthen, which they are satisfied with,
and which are most important for strengthening their
creativity through selected exercises.
You can decide if you wish to strengthen an area by practice.
For example, to increase fluency, practice generating many
scenarios, such as brainstorming possible drug trial outcomes,
identifying possible contraindications, etc., within a short
timeframe such as three minutes. To increase flexibility,
generate many categories of possible trial techniques, such as
realigning the trial expectations, reallocating resources, or
redefining success. To increase originality, practice coming up
with many possible trial scenarios, based on the trial research
to date. To increase elaboration, add detail to possible
resolutions.
Fredricka Reisman - Creative, Critical Thinking and Logic in Research
The RDCA is electronically scored and you may email
yourself your results. The 11 creativity factors assessed and
their definitions are shown in Table 3.
Examples of Integrating Creativity into Pharma
industry
The latest trends within the pharmaceutical industries engulf
creativity from many perspectives:
Ÿ Marketing, drug-delivery systems and package design that
please the customer;
Ÿ Making new and original connections among pharma
products and diseases;
Ÿ Interacting with patients;
Ÿ Dealing with challenges resulting from patents expiring
and generic drugs flooding the market;
Ÿ Pharmacoeconomics of a new drug; and
Ÿ Investigation of stem cell biology whereby small molecules
that target cancer stem cells may lead to a potential cancer
therapy innovation including pan-active compounds such
as some kinase inhibitors.
In India, AstraZeneca packages its cholesterol lowering drug
®
Crestor in transparent packs so that patients can visually
®
examine the pills. New Arimidex packaging resembles a
cosmetics case because the old version reminded women that
they were cancer patients, not cancer survivors. Proteus
Biomedical's intelligent pills contain soluble microchips linked
to detectors that remind patients if they have not swallowed
their medication.
Six Elements for Establishing Corporate Creativity
7
Robinson & Stern presented six conditions for a creative
corporate environment that may be applied to the Pharma
Table 3: RDCA Factors and Definitions
Creativity Factor Definition
Originality Unique and novel
Fluency Generates many ideas
Flexibility Generates many categories of ideas
Elaboration Adds detail
Tolerance of Ambiguity Comfortable with the unknown
Resistance to premature closure Keeps an open mind
Convergent Thinking Comes to closure, evaluative, critical, logical thinking
Divergent thinking Generates many solutions (related to fluency)
Risk Taking Adventuresome
Intrinsic Motivation Inner drive
Extrinsic Motivation Needs reward or reinforcement
99
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

industries. The first element is alignment that involves ensuring
that the interests and actions of all employees are directed
toward a company's key goals. Companies can function with
relatively poor alignment, but they cannot be consistently
creative unless they are strongly aligned. The effects of
alignment on corporate creativity are apparent when a
company is either well aligned or misaligned.
The second element of corporate creativity is self-initiated
activity that allows employees to pick a problem they are
interested in and feel able to solve it. The previous examples of
integrating creativity into Pharma worksites are indicative of
someone realizing that knowledge of the psychological and
personality needs of customers are as important as the
medicinal chemistry expertise, and resulting in focus on
design in packaging and marketing.
The third element of corporate creativity is unofficial activity
that occurs in the absence of direct official support, and with
the intent of doing something new and useful. When an idea is
new to a company, it is often resisted and opposed. This is
analogous to Sternberg and Lubart's Investment Theory of
8
Creativity :
Investment Theory asserts that creative thinkers are like good investors:
They buy low and sell high. Whereas investors do so in the world of
finance, creative people do so in the world of ideas. Creative people generate
ideas that are like undervalued stocks (stocks with a low price-to-earnings
ratio), and the public generally rejects both the stocks and the ideas. When
creative ideas are proposed, they often are viewed as bizarre, useless, and
even foolish, and summarily are rejected. The person proposing them often
is regarded with suspicion and perhaps even with disdain and derision.
Unofficial activity gives ideas a safe haven where they have the
chance to develop until they are strong enough to overcome
that resistance. The introduction of packaging design in
®
placing Crestor in transparent packs was a creative approach
to offset the problem of drug counterfeiting.
The fourth element is serendipity. A serendipitous discovery is
one made by fortunate accident in the presence of wisdom or
insight. Creativity often involves recombining or making
connections between things that may seem unconnected. The
®
Arimidex packaging came about from connecting a woman's
emotional state as a cancer survivor and her aesthetic nature
and sense of fashion. Serendipity has played a significant role
throughout the history of drug discovery, especially in
9
cardiovascular medicine . The anticoagulant properties of
dicoumarol were discovered after farmers observed that their
cattle were dying of internal hemorrhaging after feeding on
10
rotting sweet clover and digitoxin was identified when the
condition of a patient suffering from congestive heart failure
dramatically improved after being given an herbal remedy
Fredricka Reisman - Creative, Critical Thinking and Logic in Research
100
11
containing extracts of the foxglove plant . In fact, many of
the most effective pharmacological agents in use today arose
through serendipity.
The fifth element of corporate creativity is diverse stimuli. A
stimulus may provide fresh insight into something a person
has already set out to do, or it may change their course of
action. It is important for an organization to provide
opportunities for its employees to tell others about the stimuli
they have received and the possibilities these stimuli suggest to
them. It is here that the real leverage lies as in the cases where
design and creativity became important industry changers in
how they packaged and marketed their Pharma products.
The sixth and final element of corporate creativity is withincompany
communication; especially, unanticipated
communication. Every organization carries out planned
activities and establishes lines of communication to support
them. But these official channels are of limited usefulness for
corporate creativity. Corporations need to promote
unanticipated exchanges of information. A company's
creative potential needs systems in place to promote
unanticipated exchanges of information for these illuminate
creative and serendipitous connections.
Critical Thinking and Logic as Essentials of
Creative Thinking
Usually, creative thinking is associated with brainstorming
(generating many ideas), novelty, and uniqueness of ideas.
Critical thinking is analytical, judgmental and involves
evaluating choices before making a decision. When you are
thinking critically, you are using logic, reason and convergent-
1
type thinking. As I point out in another publication :
“…creativity involves creative thinking as a process of
sequential interaction of two types of thinking – divergence
and convergence as depicted in Figure 1. Divergent thinking is
the ability to elaborate and think of diverse and original ideas
with fluency and speed (e.g., brainstorming). Convergent
thinking involves narrowing ideas by evaluating the previously
generated ideas that emerged in the divergent portion of the
sequence (e.g., settling upon an idea from a selection of
1
ideas).” F. K. Reissman, 2010
Creative Ideas Versus Innovative Ideas
First, let's distinguish between creativity and innovation.
Creativity involves generating unique, original and novel
ideas. Innovation is the implementation of these ideas. Merely
generating ideas without bringing them to fruition in the form
of a product or service is uneconomical and a waste of talent.
These two parameters play important roles in choosing a
research project, identifying a thesis topic, or improving
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

Figure 1. Creative thinking process
Divergent
customer service in a clinical or retail Pharma setting.
One of the most difficult things in research as in any other
endeavour is identifying a research question, a real problem
(as opposed to a superficial or incorrect problem), or the cause
of an organizational dilemma. Key is having in-depth
knowledge related to the problem - often the salient issue
arises from a knowledge-base developed in writing a review of
the relevant literature.
Thus, how many have said they "invented" the latest new
gadget or fad, only they never found the time to actually
develop the product. They had the idea, but never took the
idea to fruition. That's the difference between creativity and
innovation.
Elements of Creativity
12
Strong and Davis list four creativity elements; namely,
valuable (perceived as having worth and genuinely contributing
to society), intentional (result of a deliberate effort), novel (new or
has at least some element of originality), excellent (significant
effort expended to make it the best it can be). Thus, the
creative product or service must be new and judged to be
valuable according to designated criteria. Further, creative
products are the result of purposive behaviour and to become
13
excellent, creative effort takes time . It is noted that creativity
usually is not purely original, but rather that it stems from
some prior knowledge and something is considered creative
through implementation (innovation) and enhancement,
although not necessarily a completely new idea. For example,
the Romans improved upon Greek culture, and the Greeks in
12
turn built upon Mesopotamian and Egyptian cultures .
Elements of Innovation
A broadly accepted definition for innovation is: To turn a creative
idea into products and services of value and profit. The basic goal of
all innovation is positive change, to make someone or
something better. There are two basic types of innovation.
Incremental Innovation: Also called continuous innovation, this
Fredricka Reisman - Creative, Critical Thinking and Logic in Research
Convergent
Divergent Convergent Divergent Convergent
type improves upon existing products/services. From a result
standpoint, incremental innovations can range from very
small to huge increases in productivity, revenues and profits.
Breakthrough (Radical, Disruptive) Innovation: This type of
innovation develops new products/services that do not exist.
Many times this type of innovation emerges from scientific
discoveries or R&D organizations. But, while a breakthrough
innovation might mean getting a patent, it does not guarantee
huge profits.
Out of many hundreds of creative ideas, only a few may ever
be implemented. For those precious few, we know them as
innovation - or simply, applied creativity.
So creativity is the idea, and innovation is the idea applied or
implemented.
Future for Creative, Critical Thinking and Logic in
India's Pharmaceutical Industries
In September 2004, a global innovation survey by the
14
Economist Intelligence Unit identified India “as an R&D
hotspot, defined as a place where (1) companies are able to tap
into existing scientific and technical expertise networks, (2)
there are good links to academic research facilities, (3) the
environment supports innovation and (4) it is easy to
commercialize.” The Economist further states:
Costs of pharmaceutical innovation in India are estimated as low as oneseventh
of their levels in Europe, and the country's clinical research
industry is currently worth $100 million growing around 40 to 50
percent annually, although some forecasts say it could be worth as much as
$1 billion to Indian firms in 2008.
The research enterprise in India is exemplified by numerous
15
R & D investments including the following :
Ÿ AstraZeneca is conducting research into tuberculosis (TB) at the
AstraZeneca Research Foundation India in Bengaluru. India's estimated
8.5 million TB patients mean clinical trials can be conducted easily and
economically.
101 RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

Ÿ GSK and Ranbaxy are partnering where GSK will provide drug
research leads and Ranbaxy will conduct preclinical studies; GSK will
take the drug through human trials.
Ÿ Pfizer is exploring setting up an Academy for Clinical Research in
Mumbai since costs of clinical trials in India are around one-tenth the
U.S.
In addition to drug studies, investigated is the correlation
16
between brain chemistry and creative cognition , the
17
relationship between creativity and academic achievement ,
18-21
and the relationship between creativity and intelligence .
New technologies are opening pathways for
biochemical and neurological research as these
impact creativity research, and the role of creativity
22
in design and marketing research is at the
forefront of the Pharma industries. As Einstein
cautioned: “you cannot solve problems by using the kind of
thinking that produced the problem in the first place.”
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2. IBM 2010 Global CEO Study. Creativity selected as most crucial factor
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12. Strong B, Davis M. The history of creativity in the arts, science and
nd
technology: 1500-present. 2 edition. Dubuque, Iowa: Kendall Hunt
Publishing Company; 2011.
13. Wallace DB, Gruber HE. Creative people at work. UK:Oxford
University Press, 1989.28-9.
14. Reddy P. Global Innovation in Emerging Economies. New York:
Routledge; 2011.108.
15. Gopi PG, Subramani R, Santha, T. et al. Estimation of burden of
tuberculosis in India for the year 2000. Ind J Med Res 2005;122:243-
8.
16. Jung RE, Gasparovic C, Chavez, RS. et al. Biochemical support for
the “threshold” theory of creativity: a magnetic resonance
spectroscopy study. J Neurosci 2009;29:5319 –25.
17. Naderi H, Abdullah R, Aizan HT, Sharir J, Kumar V. Relationship
between creativity and academic achievement: a study of gender
differences. J Am Sci 2010;6:181-90
18. Weisberg RW. Creativity: beyond the myth of genius. New York:WH
Freeman & co; 1993.
19. Sternberg RJ. Wisdom, intelligence, and creativity synthesized.
Cambridge: Cambridge university press; 2003.
20. Kassem A. The creativity chemical [online]. 2011 [Cited 2011 August
11]. Available from: URL: http://theilluminatedbrain.com/drugschemicals/the-creativity-chemical.
21. Kharkhurin AV. The Impact of Culture on the Relationship between
Bilingualism and Creative Potential [online]. [Cited 2011 August 11].
Available from: URL: http://academic.brooklyn.cuny.edu/psych/
tovyharhur/research/publications/jccp1.pdf
22. Jack A. A pharmaceutical experiment in design [online]. 2010 [Cited
2011 August 11]. Available from: UR: http://www.ft.com/cms/s/0/
4ed58ce0-b091-11df-8c04-00144 feabdc0. html#ixzz1U5wNrIvd.
Address for Correspondence
Fredricka K. Reisman, Ph.D., Professor, Goodwin College of Professional
Studies, Director, Drexel/Torrance Center for Creativity and Innovation, Drexel
University, 3001 Market Street Suite 110, Philadelphia, PA 19104, USA
President, American Creativity Association
E-mail: reismafk@drexel.edu
102 RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

A B S T R A C T
RGUHS Journal of Pharmaceutical Sciences
Need for Inclusion of Scientific Writing Skill Subjects in Indian Post Graduate
Pharmacy Course
1 2 1 1 1
Patil J.S* , Kotnal R.B , Birajdar R.P , Marapur S.C and Kadam D.V
1 2
Dept. of Pharmaceutics, Dept. of Pharmaceutical Chemistry, B.L.D.E.A's College of Pharmacy, BLDE University Campus,
Bijapur- 586103, Karnataka, India.
This article emphasize more on why we need stuffing of scientific writing skills in curriculum rather than how to write scientific articles.
Writing scientific articles is a great obstacle for many people, or perhaps for most people. Scientific writing provides vital information with
the creation and dissemination of research knowledge. In recent years, most of the Indian pharmacy research students are unable to
publish their scientific data in peer reviewed journals. Reasons for this failure are numerous, few among these are not knowing how to
begin, poor language and drafting skills. Lacking in basics of scientific writing skills and no motivation by the research
guides/supervisors is the matter of concern today. In India, all the universities offering post graduate (PG) course in pharmacy are
concentrating more on teaching the subject contents of respective specializations. The PG course is a blend of study of specialized
subjects in part- I and research programme in part-II. The students need to write thesis of their dissertation work at the end. For writing the
thesis students require scientific writing knowledge. Hence, it necessitates studying the subjects on scientific writing skills at part-I level.
In this present discussion an attempt was made to explore the reasons which emphasize the inclusion of scientific writing subject/s in PG
course and also tried to suggest the possible course structure which may become more appropriate for our present context.
Keywords: Scientific writing, publication, course structure, post graduate, pharmacy.
INTRODUCTION
Good scientific writing skills open up many opportunities to
the researcher: publications, conference or seminar
attendance. They also lead to better patents, better research
partnerships and better funded research. Clarity and
efficiency in scientific writing bears witness to the quality of a
researcher; it influences career promotion. As we are seeing
many avenues for scientific writing, medical writing in
healthcare, medical and R & D sector viz; pharmaceutical
industry and Clinical Research centers. It is essential to have
subject in the post graduate level of pharmacy course. For a
researcher, scientific writing is a rewarding knowledge both
professionally and socially. The idea and skill of writing help
to publish scientific data in the peer reviewed journals.
Publication of research articles is the measure of his/her
productivity which may lead to upgrade the professional
1,2
status especially for those from academic field . Publishing
the research results in scientific journals reaches the audience
in larger extent and it contributes a significant influence on
career development. The scientific writing is a well written
report describing the results of overall original research work.
Publication is the crucial end point of a research work to share
RGUHS Journal of Pharmaceutical Sciences
Received: 3/1/2011, Modified: 3/8/2011, Accepted: 1/9/2011
103
Review Article
important information with the scientific community which
results in personal contentment and professional
3
advancement . Writing is a skill born from practice. Before
becoming a good writer it is essential to become an avid and
careful reader. Most reputed institutes consider quality
publications as a measure of research productivity and basic
indicators of accountability. In recent days, number of good
publications by Indian pharmaceutical scientists in reputed
journals is drastically declining. The reasons for such failure
are plenty. But this is really a serious concern and our
educators need to think urgently to rectify this problem.
Although, few research/academic institutions routinely
conducting short term workshops and seminars on scientific
writing skills in their institutions but the number of virtual
beneficiaries are less. Most of the pharmacy institutions
offering PG programme in India are teaching basic subjects
of the respective specialization. Although a great deal of
importance is given to teach such specialization subjects, no
attention is paid in teaching scientific writing skills. It may be
said without any exaggeration that scientific writing skill is one
among the most crucial problems that plague the research
oriented educational scene in India and there is almost no
place for learning, coaching and motivation for scientific
writing skills which is essential for PG students. In most of the
institutions almost 90% students leave the college after
completion of the course without even communicating their
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

Patel J.S et al./ Need For Inclusion of Scientific Writing Skill Subjects in Indian Post Graduate Pharmacy Course
scientific results for possible publication. Further, it is a matter
of great regret that, scientific writing is not considered as one
of the essential study tool for PG pharmacy students. That is
why even though a large number of PG students are coming
out every year, the number of publications in peer reviewed
journals are very less. This aspect not only reducing the
professional reputation and recognition but also reduces the
scope of pharmacy post graduates heading high academic
positions, editorial and scientific review opportunities.
Most modern universities in the world have begun to modify
the course structure or entire study programme in various
disciplines according to the present need and importance.
Instructors tailor such course to many individuals, including
4 5
medical students , osteopathic residents and scientific
6
researchers . Even in India, students studying in
universities/institutions exposing to learning scientific writing
skills through seminars and workshops in their campus have
the benefit of a better eminence. However, most pharmacy
institutions still continue with primitive and callous fashion of
teaching and not thinking on importance and significances of
scientific writing skills. This seems that there is need for
inclusion of subject/s on scientific writing skills as a part of
PG curriculum. Thus, the present study was done with the
aim of discussing the need for inclusion of such subject/s with
a possible proposal of course design.
“But in science the credit goes to the man (or woman) who convinces the
world, not to the man (or woman) to whom the idea first occurs”. Sir
Francis Darwin.
Reasons for inclusion of scientific writing subject/s
Motives for the publication vary widely. Some students having
a special driving force and well guided by their research
supervisors are finally publish their scientific results in reputed
journals. But this is all depends on special talent and skills.
Scientific writing is easier when it is an integral part of the
study and it is harder when it require a student to think and
prepare a scientific paper. To motivate all the students towards
publication habits it is essential to make scientific writing as a
curriculum part. In the present Indian context, motives to
scientific writing and publishing habits are poor. In the present
discussion we focused mainly on the reasons which made to
discourage the publishing habits in our PG students. These
include,
In recent days, professional and technical teaching
community suffering badly with grammatical English
language. This is because most of us not considering that the
language is necessarily grammatical in professional teaching
field. This situation lacking behind in flourished writing
activities.
104
A poor motives and encouragement to the student
community pertaining to scientific writing and publishing
habits. This may be because of the fact that the teachers/
research guides themselves hesitate to write the papers due to
their poor language and lack of writing skills.
Though some people interest to publish the papers of their
research results, a primary obstacle is how to begin, even
though the approaches to and procedure for writing a
7
scientific paper is well defined .
A poor or no clear vision for the institutions which are
currently working on commercial base, such institutions has
absolutely failed to attract a talented research supervisor who
has basics of teaching and interest in research. This ultimately
failed to achieve minimum scientific and educational
standards in our research based educational system. The
Indian universities seem to have not made publication as
mandatory requirement for the PG students in the
curriculum, although it is already exist in some universities for
Ph D programme.
Benefits of scientific writing knowledge and
publications
Scientific writing skill and research publications give publicity
to the scientist which may help him in many of the following
ways. The recognition and publicity that gained by
publications may result in getting consulting work and
assignments such as resource person. Publishing the research
results in scientific journals contributes a significant influence
on career development. Publication may lead to professional
recognition and job promotion. Publication is the crucial end
point of a research work which results in personal satisfaction
and professional advancement.
In the recruitment of faculty most reputed institutes consider
quality publications as a measure of research productivity and
basic indicators of accountability. Good scientific writing
skills can bring many personal rewards such as getting
research grants.
Suggested course design
Acquiring good writing skill is a difficult task for the student
community especially those who have not studied the 'English'
as a first language. An issue always faced in teaching any
specializations of professional communication is the bridge
between technical knowledge and rhetorical skills. Teaching
subjects like scientific writing skills is always suggested to
concentrate on coordinating the theoretical knowledge and
rhetorical skills, there by students are made more skillful
candidates both in theory as well as in practical. All research
process always begins with a standard protocol and concludes
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

with writing the scientific data in a systematic manner. A
scientific writing describing an instance of the scientific
process reflects the way that experiments are devised and
8, 9
carried out . In this context, Indian universities are required
to design the practical oriented course structure on scientific
writing and research methodology subjects for post graduate
pharmacy students. For this, we have to begin with
constituting an expert committee comprising renowned
educationists and subject experts who can design and tailor
such subject suitable for our PG students. The Educationists
constructing syllabi for such subjects have to describe the
benefits and contributions of the subjects for their career
advancement in a clear way. The course design proposed by us
comprise two distinct parts covering the study on theoretical
aspects as well as practical assignments and workshops
pertaining to impart the knowledge of scientific writing skills.
Section- I: Theory Part
Patel J.S et al./ Need For Inclusion of Scientific Writing Skill Subjects in Indian Post Graduate Pharmacy Course
Theory part comprises the comprehensive contents on
fundamentals and applied aspects of scientific writing skills.
The course structure has different sections;
Start with study of literature review procedures; because
research programmes always begin with thorough literature
review which provides a strong base for the proposed study.
The next important section is study on writing skills of
research protocol, because research protocol is a brief plan of
proposed work with established methods needs to submit for
approval of the study. Hence, it is essential to study the design
and writing skills of research protocol along with study on
planning and execution of the research programme.
The theory part also contains a detailed study on abstract
writing, knowledge on anatomy of a good abstract, use of
rhetorical language in framing the abstract as well as whole
scientific paper, criteria to identify the target journal, type of
manuscript and authorship.
Further, the course also cover chapters on fundamental rules
and techniques involved in framing the result and discussion
parts of a scientific writing, statistical analysis of the data
obtained, and use of various statistical softwares.
This part also includes the systematic study of bibliographic
writing because it is vital part of a scientific writing. Different
journal follow unique reference style. The study covers
introduction of various reference styles. Hence, a thorough
study on bibliographic writing helps the students in preparing
the thesis or scientific paper.
Finally the course also cover study on preparation of posters
and power point slides for oral presentation. Study of basic
procedures involved in the preparation of poster and oral
105
presentations at various scientific conferences, seminars and
conventions.
To make all these study components more familiar to students
the commonest ways of teaching and to explore their views
are in-depth counseling and group discussions. In depth
counseling as one-to-one basis and group discussions are
suggested to cover in the syllabus with respect to the study
content.
In present day academic scenario it is worth important to
follow the ethical principles in scientific publication rather
than publishing good number of papers. Hence, it is very
much essential to have a chapter on ethical principles to be
stringently followed by every research students while involving
in the scientific writing work.
To ensure successful teaching of this course, a periodic
internal assessment and university examinations are to be
conducted through a systematic evaluation procedures.
Section-II: Practical Part
In the practical part of the subject design the vigorous
practical assignments and workshops are suggested to
conduct. Students receive a thorough introduction to the
various theoretical aspects on scientific writing that provides
the necessary context for practical writing assignments that
complements the lectures and discussions.
A definite number of students in a group are to be allotted the
specific scientific writing assignments. In these assignments
students are supplied the results of different published papers
asking them to interpret the data and write the results and
discussion.
The journal articles provide source material from which the
10
students can craft the research report using a template
provided in the theory class. Each assignments challenges the
students to selectively organize the information found in
published results.
The practical training provides student to practice the
abstract, methodology, result-discussion, and reference
writing. A series of stringent practical workshops are to be
recommended on various aspects of scientific writing
including the preparation of a scientific paper for publication
as well as thesis writing exercises.
The students are made much aware about the existing
reputed national and international journals and their
reputation, importance of impact factor and its calculation
and study on better understanding of instructions to authors
followed by peer reviewed journals.
The institutes are also suggested to invite the reputed experts
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

working at editorial levels of various journals as resource
persons to provide valuable knowledge pertaining to scientific
writing skills.
During the study course, the students are to be motivated to
publish at least a review article or short communication in a
reputed peer reviewed journal.
At the end of the programme when the students start their
research work in part II of the course the knowledge of
scientific writing skills studied in part I make them more
confident to proceed. Finally the students are expected to be
able to write a scientific paper in a lucid and elegant manner
after completion of their research programme.
CONCLUSION
Patel J.S et al./ Need For Inclusion of Scientific Writing Skill Subjects in Indian Post Graduate Pharmacy Course
Writing is not an easy task for most students. The ability to
write in 'second' language is mainly depends on ability of a
student to understand and use grammar. Students who
studied English as a second language often commit many
errors while writing. This may be due to incomplete
knowledge of the English language and its complexities. This
is exactly true in case of Indian students who pursue post
graduate study in any of the professional courses. Hence,
there is an urgent need to introduce the subjects on scientific
writing skills at the post graduate level in pharmacy education
which in turn not only help them to write scientific papers in a
elegant manner but also helps to establish the good and
acceptable writing and communication skills along with
enhancing credibility, competence and professionalism
among the budding research scientists.
106
REFERENCES
1. Hamilton C W. How to write and publish scientific papers:
Scribing information for pharmacists. Am J Hosp Pharm 1992;
49:2477-84.
2. Fye W B. Medical authorship: traditions, trends, and tribulations. Ann
Intern Med 1990; 113:317-25.
3. Richard D B. Anatomy of research paper. Respiratory Care 2004;
49(10):1222-8.
4. Tollan A, Magnus J H. Writing a scientific paper as a part of medical
curriculum. Med Educ 1993; 27(5):461-4.
5. Coleridge S T. Teaching residents to write a research paper. J Am
Osteopath Assoc 1993; 93(9):936-40.
6. Stephens P A, Campbell J M. Scientific writing and editing: a new role
for the library. Bull Med Libr Assoc 1995; 83(4):478-82.
7. Hulth E J. How to write and publish papers in the medical sciences.
2nd ed. Baltimore: Williams and Wilkins; 1990.
8. Charles w. Van W III. Writing a scientific paper. Nutr Clinic Pract 2007;
22:636-40.
9. Van way C W III. On scientific writing. J Parenter Enteral Nutr 2007;
31:449-50.
10. Bone R J. Appendix A. Template for a clinical trial report. In: medical
writing in drug development: A practical guide for pharmaceutical
research. New York: Haworth Press; 1998.
Address for Correspondence
J S Patil, Dept. of Pharmaceutics, BLDEA's College of Pharmacy, BLDE
University Campus, Bijapur-586 103, Karnataka, India.
E-mail: pharmajspatil@gmail
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

A B S T R A C T
RGUHS Journal of Pharmaceutical Sciences
A Novel Spectrophotometric Estimation of Pramipexole in Bulk Drug and
Formulations
Shobha Manjunath*, Satish Middi and Venkatesh Chouhan
Department of Pharmaceutical Analysis, H.K.E. Society's College of Pharmacy, Gulbarga- 585105, Karnataka State (India)
Three simple, sensitive and selective spectrophotometric methods have been developed and validated for the estimation of
Pramipexole in bulk drug and pharmaceutical formulations. In Method A Pramipexole exhibits absorption maximum at 261.8 nm in
ethanol, in Method B it shows a sharp peak at 249.4 nm in first order derivative spectrum with n=1 and Method C is based on calculation
of area under curve(AUC) for analysis of Pramipexole in the wavelength range of 255-265 nm. The drug follows the Beer-Lambert's law
-1
in the concentration range of 9-45 µg mL for the three methods. Results of the analysis were validated statistically and by recovery
studies it was found to be satisfactory.
Keywords: Pramipexole, UV Spectrophotometry, Derivative Spectroscopy, Area Under Curve.
INTRODUCTION
Pramipexole is a new drug in therapy of Parkinson's disease.
Chemically it is (s)-2-amino-4,5,6,7-tetrahydro-6-
(propylamino) benzothiazole, a non-ergotine dopamine
agonist, initially introduced for the treatment of early and
advanced parkinson's disease and recently approved in US
and Europe for the treatment of idiopathic restless legs
1
syndrome in adults . Parkinson's disease is chronic
neurodegenerative disease characterized by bradykinesia,
predominantly affecting the elderly. It occurs when certain
nerve cells (neurons) in a part of brain called substantia nigra
die or become impaired. Normally, these neurons produce a
vital chemical known as dopamine which allows smooth,
2
coordinated function of the body's muscles and movement .
It is not official in any of the Pharmacopeias. It is listed in the
3 4
Merck Index and Martindale: The complete drug reference .
5
Literature survey reveals that a few methods based on HPLC ,
6
LC-MS are reported for the determination of Pramipexole in
biological fluids. Analytical procedures were reported for the
determination of dissociation constant values of Pramipexole
7 8
by HPLC method, an RP-HPLC and simple VISIBLE
9
spectrophotometric method have also been reported for
estimation of Pramipexole in bulk drug and formulations.
Since no UV method has been reported, the objective of the
work is to develop new UV spectrophotometric method for its
estimation in bulk drug and pharmaceutical formulations
with good accuracy, simplicity, precision and economy.
RGUHS Journal of Pharmaceutical Sciences
Received: 29/1/2011, Modified: 27/2/2011, Accepted: 1/3/2011
107
Original Research Article
MATERIALS AND METHODS
Pure sample of Pramipexole was supplied as gift sample by
Sun Pharmaceutical Ltd, Jammu and Kashmir, India and was
used as received. Ethanol was used as solvent (precaution was
taken by keeping the lid on volumetric flasks and caps on
cuvetts to prevent evaporation of ethanol). Shimadzu-1700
UV-visible spectrophotometer was used with 1cm matched
quartz cells. Tablets of 1mg strength were procured from local
pharmacy of two brands that is Parpex and Pramipex.
Accurately about 10 mg of the pure drug was weighed and
dissolved in sufficient quantity of distilled ethanol and the
volume was made upto 100 ml with distilled ethanol to give
-1
standard stock solution (100 µg mL ).
Method A: UV Spectrophotometry
Aliquots of standard stock solution were pipetted out and
suitably diluted with distilled ethanol to get the final
-1
concentration of 9, 18, 27, 36 and 45 µg mL of standard
solutions. The absorbance of the solutions was measured at
261.8 nm against solvent blank (Fig. 1). A calibration curve
was plotted taking the absorbance against the concentration
of the standard solutions (Graph 1). The amount of drug was
computed from calibration curve.
Method B: First order derivative spectra
Aliquots of standard stock solution were pipetted out and
suitably diluted with distilled ethanol to get the final
-1
concentration of 9, 18, 27, 36 and 45 µg mL of standard
solutions. The solutions were scanned in the spectrum mode
from 400-200 nm wavelength range and the first order
derivative spectra were obtained at 249.4 nm (Fig. 2). The
absorbance difference at n=1 was calculated by the inbuilt
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

software of the instrument which is directly proportional to
the concentration of the standard solutions (Graph.2). The
method was applied for the known concentration and was
found to be satisfactory for the analysis of tablet formulations.
Method C: Area Under Curve (AUC)
Shobha Manjunath et al./ A Novel Spectrophotometric Estimation of Pramipexole in Bulk Drug and Formulations
This method is applicable when there is no sharp peak or
broad spectra are obtained. It involves the calculation of
integrated value of absorbance with respect to the wave
length between two selected wave lengths λ1 and λ 2.
Area
calculation processing item calculates the area bound by the
curve and the horizontal axis. The horizontal axis is selected
by entering the wavelength range over which the area has to
be calculated. This wave length range is selected on the basis
of repeated observations so as to get the linearity between
AUC and concentrations. Suitable dilutions of standard stock
-1
solution (100 µg mL ) of the drug were prepared and scanned
in the spectrum mode from the wavelength range 400-200 nm
(Fig. 3). From the spectrum, AUC in the range of 255-265 nm
was selected for analysis. The calibration curve was plotted as
-1
concentration (9-45 µg mL ) against AUC (Graph 3). The
method was checked by analyzing the samples with known
concentration for the AUC. As per the results obtained were
satisfactory, the method was applied for pharmaceutical
formulations.
For the estimation of Pramipexole in tablet formulations by
the three methods, fifty tablets of each brand were weighed
and triturated to fine powder. Tablet powder equivalent to
5mg of Pramipexole was weighed and dissolved and further
diluted with sufficient quantity of distilled ethanol to get stock
-1
solution of concentration 100 µg mL . For method A, analysis
of Pramipexole in both tablet formulations Parpex (T 1)
and
Pramipex (T 2 ) was done using various sample solutions at
261.8 nm against reagent blank in quantitation mode for six
times. For method B, analysis of T 1 and T2 was done for first
order derivative spectra using various sample solutions at
249.4 nm against reagent blank for six times. For method C
the sample solutions were scanned in the spectrum mode and
AUC calculations were done in the wavelength range of 255-
265 nm for both the tablet formulation T 1 and T 2 for six times
and concentrations calculated using the calibration curve.
Methods A, B and C were validated for linearity, accuracy.
RESULTS AND DISCUSSION:
The Optical Characteristics such as Absorption maxima,
Beer's law limits, Molar absorptivity are presented in (Table1).
The regression analysis using the method of least squares was
made for the slope (b), intercept (a) and correlation coefficient
(r) obtained from different concentrations and the results are
summarized in (Table 1). The percent relative standard
deviation and percent range of error (0.05 and 0.01 level of
confidence limits) calculated from the eight measurements, ¾
of the upper Beer's law limits of Pramipexole are given in
(Table 1).
Relative standard deviation for analysis of six replicate
samples of two brands T 1 and T 2 in method A, B and C are
given in (Table 2). The percentage recovery was found to be
within range. The results obtained by proposed method are in
good agreement with the label claims (Table 2).
Table 1: Optical Characteristics and Other Parameters
Parameters Method A Method B Method C
λl max (nm) 261.8 249.4 255-265
-1
Beer ’ s Law Limit (µg mL ) (C) 9 – 45 9 - 45 9 – 45
-1 -1
Molar absorptivity (L mole cm )
3
5.050 x 10
3
4.349 x 10
4
4.7603 x 10
Regression Equation (Y*) Slope (b) 0.0165 0.014 0.001
Intercept (a) 0.0006 0.013 -3.398
Correlation Coefficient ® 0.9998 1.003 1.822 %
RSD
Range of errors**
0.1983 0.0527 0.0278
Confidence limits with 0.05 level ±0.0007 ±0.0001 ±0.298
Confidence limits with 0.01 level ±0.0011 ±0.0002 ±0.441
-1
Limit of Detection (LOD)( µg mL ) 0.1783 0.0482 0.013
-1
Limit of Quantification (LOQ) ( µg mL ) 0.5404 0.1463 0.041
-1
Y*= bC+a where C is the concentration of Pramipexole in µg mL and Y is the absorbance at the respective λ max.
** For eight measurements.
108
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

Shobha Manjunath et al./ A Novel Spectrophotometric Estimation of Pramipexole in Bulk Drug and Formulations
Table 2: Evaluation of Pramipexole in Pharmaceutical Preparations
Method Brand Labelled amount (mg) Amount obtained (mg)* %Recovery
A T1 1 0.986 99.73
T2 1 0.983 99.62
B T1 1 0.989 99.83
T2 1 0.987 99.82
C T1 1 0.991 99.87
T2 1 0.986 99.73
*Average of Six determinations
Fig.1: Selection of Wavelength for Pramipexole. Method A
Graph.1: Calibration curve of Pramipexole Method A
Fig.2: First order derivative spectrum of Pramipexole with
n=1. Method B
109
Graph.2: Calibration Curve of Pramipexole Method B
Fig.3: Wavelength range selected for AUC. Method C
Graph.3: Calibration Curve of Pramipexole Method C
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

CONCLUSION
Methods A, B and C for the estimation of Pramipexole in
tablet dosage form were found to be simple, accurate. Beer-
Lambert's law was obeyed in the concentration range of 9-
45µg/ml for the three methods. The values of standard
deviation were satisfactory. As the drug Pramipexole showed a
broad spectrum, the derivative spectroscopy method applied
has advantage that it locates the hidden peaks in the normal
spectrum when the spectrum is not sharp and it also
eliminates the interference caused by the excipients and the
degradation products present, if any, in the formulation. The
AUC method is also advantageous as it is applicable to the
drugs which show the broad spectra without a sharp peak.
Hence these methods can be useful in the routine analysis of
Pramipexole in bulk drug and formulations.
ACKNOWLEDGEMENT
We are thankful to Sun Pharmaceuticals Ltd, Jammu and
Kashmir for providing us the gift sample of the pure drug and
to the Principal, H.K.E.Society's college of pharmacy,
Gulbarga for providing research facilities.
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Shobha Manjunath et al./ A Novel Spectrophotometric Estimation of Pramipexole in Bulk Drug and Formulations
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2007;65(9-10): 633-5.
8. VLN.SeshagiriRao J, Anantha Kumar D, Shaik mastanamma,
Srilakshmi K. Int.J.Chem.sci. 2009;7(4):2789-94.
9. Gurupadayya BM, Vishwajith V, Srujana N. World j chem. 2009;
4(2):157-60.
Address for Correspondence
Shobha Manjunath, Department of Pharmaceutical Analysis, H.K.E.
Society's College of Pharmacy, Gulbarga- 585105, Karnataka, India
E-mail: Shobamanjunath8@gmail.com
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

A B S T R A C T
RGUHS Journal of Pharmaceutical Sciences
Validated UV-Spectrophotometric Estimation of Entecavir in Bulk and
Formulations
Malipatil S.M*, Bharath S Athanikar and Mogal Dipali
Original Research Article
Department of Pharmaceutical Analysis, H.K.E.S's Matoshree Taradevi Rampure Institute of Pharmaceutical Sciences, Gulbarga-585105,
Karnataka, India
Three new, simple and cost effective UV-spectrophotometric methods were developed for the estimation of Entecavir in bulk and
pharmaceutical formulations. Entecavir was estimated at 253.6 nm in UV-spectroscopic method (Method A), 242.6 nm in first order
derivative spectroscopy (Method B) and scanned at 258.0 - 248.0 nm in Area Under Curve method (Method C). Linearity range was
2 2 2
found to be 2-18 μg/ml (Correlation coefficient r = 0.9999 in method A, r = 0.9999 in method B and r = 0.9998 in method C) in all the three
4 -1 -1 4 -1 -1 4 -1
methods. The molar absorptivity was found to be 1.5x10 L mol cm in method A, 1.108X10 L mol cm in method B and 3.17x10 L mol
-1
cm in in method C. These methods were tested and validated for various parameters according to ICH guidelines. The proposed
methods were successfully applied for the determination of Entecavir in pharmaceutical formulation (tablets). The results demonstrated
that the procedure is accurate, precise and reproducible (% relative standard deviation

tablet dosage form. For the selection of solvent the criteria
employed were sensitivity of the method, ease of sample
preparation, solubility of the drug, cost of the solvent and
applicability of the method to various purposes. Absorbance
of the Entecavir in selected solvent at respective wavelength
was determined and molar absorptivity and Sandell's
sensitivity was calculated according to the standard formulae
(Table 1).
Calibration standards
Accurately about 100 mg of the pure drug was weighed and
dissolved in double distilled water and the volume is made up
to the volume 100 ml to give standard stock solution
(1000μg/mL) and from this solution 10ml of sample was
transferred in to separate 100ml volumetric flask and the
volume was made up to the mark 100ml with double distilled
water to get concentration 100 µg/ml as a second stock.
Aliquots of standard stock solution were pipetted out and
suitably diluted with double distilled water to get the final
concentration of 2, 4, 6, 8, 10, 12, 14, 16 and 18 μg/ml of
standard stock solution.
UV-Spectrophotometric method (Method A)
Malipatil S.M et al./ Validated UV-Spectrophotometric Estimation of Entecavir in Bulk and Formulations
For the selection of analytical wavelength, 10 μg/ml working
standard solution was prepared by appropriate dilution of
standard stock solution in double distilled water and double
distilled water was used as blank solution. This solution was
scanned in the spectrum mode from 400 nm to 200 nm. From
the spectra of drug (Fig. 2), λ max of Entecavir, 253.6 nm was
selected for the analysis. A calibration curve was plotted by
taking the absorbance against the concentration of standard
stock solutions (Graph 1). By using the calibration curve, the
concentration of the sample solution can be determined.
First order derivative spectroscopy (Method B)
In this method, 10 μg/ml working standard solution was
prepared by appropriate dilution of standard stock solution in
double distilled water and double distilled water was used as
blank solution. This solution was scanned in the spectrum
mode from 400 nm to 200 nm wavelength ranges and the first
order derivative spectra were obtained at n=1, a sharp peak at
242.6 nm (Fig. 3). The absorbance difference at n=1 (dA/dλ)
was calculated, which is directly proportional to the
concentration of the standard stock solution. A calibration
curve was plotted by taking the absorbance difference (dA/dλ)
against the concentration of standard stock solutions (Graph
2). By using the calibration curve, the concentration of the
sample solution can be determined.
Table 1: Optical characteristics, statistical data of the correlation coefficient and validation parameters for
Entecavir by UV spectroscopy, first order derivative & AUC method
Parameters Method A Method B Method C
λ max 253.6 nm 242.6 nm 258.0-248.0
-1 -1
Molar absorptivity (L mol cm )
Regression equation (Y = a+bc)
4
1.5x10
4
1.108x10
4
3.17x10
Slope(b) 0.0548 0.0040 0.2450
Intercept(a) -0.0007 -0.0002 0.0615
Standard deviation 0.001 0.0004 0.0001
RSD% 0.1824 1.1503 0.00402
2
Correlation coefficient (r ) 0.9999 0.9999 0.9998
Sandell ’ s Sensitivity
-2
(µg cm / 0.001 abs unit)
% Range of errors
0.003 0.0011 0.0012
Confidence limit with 0.05 level ±0.000788 ±0.000364 ±0.0000788
Confidence limit with 0.01 level
Validation parameters
±0.00166 ±0.000539 ±0.000116
Selectivity
and Specificity (t-test)
0.305 to 0.290 0.0157 to 0.0255 1.731 to 1.146
Linearity (µg / ml) 2-18 µg/ml 2-18 µg/ml 2-18 µg/ml
Limit of detection [LOD] (µg / ml) 0.0601 0.1823 0.001346
Limit of quantitation [LOQ] (µg / ml) 0.1234 0.3740 0.004081
Robustness (mean % recovery ± S.D.) 100.98 ± 0.001 103.41 ± 0.004 101.86 ± 0.0001
Y=bC+a where C is the concentration of Entecavir in µg/ml and Y is absorbance unit.
(Each value is a result of nine separate determinations)
112
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

Area Under Curve method (Method C)
Malipatil S.M et al./ Validated UV-Spectrophotometric Estimation of Entecavir in Bulk and Formulations
This method is applicable when there is no sharp peak or
when broad spectra are obtained. It involves the calculation of
integrated value of absorbance with respect to the wavelength
between two selected wavelengths λ 1 and λ 2.
Area calculation
processing item calculates the area bound by the curve and the
horizontal axis. The horizontal axis is selected by entering
wavelength range over which the area has to be calculated.
This wavelength range is selected on the basis of repeated
observations so as to get the linearity between area under
10
curve and concentration. For the selection of analytical
wavelength, 10 μg/ml working standard solution was
prepared by appropriate dilution of standard stock solution in
double distilled water and double distilled water was used as
blank solution. This solution was scanned in the spectrum
mode from 400 nm to 200 nm (Fig. 4). From the spectra of
drug, area under the curve in the range of 258.0-248.0 nm
was selected for the analysis. The calibration curve was
plotted as AUC against concentration of standard stock
solution (Graph 3). By using the calibration curve, the
concentration of the sample solution can be determined.
ESTIMATION OF ENTECAVIR FROM TABLET
DOSAGE FORMS
For the estimation of Entecavir in tablet formulation by three
methods, 25 tablets of each brand were weighed and
triturated to fine powder. Tablet powder equivalent to 25mg
of Entecavir was weighed and dissolved and further dilution
was carried out with quantity sufficient of double distilled
water. This was then filtered through the Whatmann filter
paper no. 41 to get the stock solution of concentration 100
Table 2: Accuracy and precision data for the developed methods
Level
Method A
Range (µg/ml)
a
S.D
b
% RSD
LQC 2.0-4.0 0.0004 0.3071
MQC 8.0-10.0 0.0011 0.2446
HQC
Method B
16.0-18.0 0.0008 0.0969
LQC 2.0-4.0 0.00007 0.2551
MQC 8.0-10.0 0.00004 0.3718
HQC
Method C
16.0-18.0 0.00003 0.7774
LQC 2.0-4.0 0.000053 0.00659
MQC 8.0-10 0.000149 0.006609
HQC 16.0-18.0 0.000035 0.000839
a b
standard deviation, % relative standard deviation LQC Lower
Quantifiable Concentration, MQC Middle Quantifiable
Concentration, HQC Higher Quantifiable Concentration (Each
value is a result of six separate determinations)
μg/ml. Further this stock solution was suitably diluted to get
10 μg / ml and these samples were analysed using proposed
methods (Table 4).
ANALYTICAL VALIDATIONS
Specificity and Selectivity
Entecavir solutions (10 μg/ml) were prepared in double
distilled water along with and without common excipients
[lactose monohydrate, microcrystalline cellulose,
crospovidone, povidone, and magnesium stearate, titanium
Table 3: Results of intermediate precision study
Concentration Inter-day repeatability Intra-day repeatability Inter-instrument*
(in µg/ml) % RSD (N=8) % RSD (N=8) repeatability % RSD (N=8)
Day 1 Day 2 Day 3
Method A
2 0.7932 0.6903 0.4764 0.50 0.73
10 0.1363 0.2140 0.098 2 0.11 0.29
18
Method B
0.0752 0.1181 0.0767 0.10 0.15
2 0.1234 1.3137 1.7788 0.1238 0.62
10 0.2885 3.4077 2.2308 0.2880 0.55
18
Method C
0.9855 1.0137 1.2948 0.9855 0.49
2 0.0279 0.0203 0.0130 0.013 0.021
10 0.0602 0.0466 0.0029 0.002 0.008
18 0.0059 0.0025 0.0026 0.001 0.002
* Instrument 1: Shimadzu UV-Visible spectrophotometer 1700, Instrument 2: Systronic-118 UV-Visible spectrophotometer
113
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

dioxide, hypromellose, polyethylene glycol 400, polysorbate
80, and iron oxide red (used only in 1mg tablets)]. All the
solutions were scanned in the range of 400-200 nm and
checked for change in absorbance at respective wavelengths.
In a separate study, drug concentration of 10 μg / ml was
prepared independently from pure drug stock and
commercial sample stock in selected media and analysed (N =
5). Paired t-test at 95% level was performed to compare the
means of absorbance (Table 1).
Accuracy
As a part of determining accuracy of the proposed methods,
different levels of drug concentrations (LQC, MQC and
HQC) were prepared from independent stock solution and
analysed (N=6). Accuracy was assessed as the standard
deviation and percentage relative standard deviation studies
were found to be satisfactory (Table 2). To give additional
support to accuracy of the developed assay method, standard
addition method was done. The percent recovery of the
added pure drug was calculated as,
% Recovery = [(C - C ) / C ] X 100
v u a
Malipatil S.M et al./ Validated UV-Spectrophotometric Estimation of Entecavir in Bulk and Formulations
Where C is the total drug concentration measured after
v
standard addition
C is the drug concentration in the formulation
u
C is the drug concentration added to the formulation (Table
a
4).
Precision
Repeatability was determined by using different levels of drug
concentrations (same concentration levels taken in accuracy
study), prepared from independent stock solution and
analysed (N=6) (Table 2). Inter-day and intra-day variation
and instrument variation were taken to determine
intermediate precision of the proposed methods (N=6). The
% relative standard deviation of the predicted concentrations
from the regression equation was taken as precision (Table 3).
Linearity
To establish linearity of the proposed method, nine separate
series of solutions of the drug (2-18μg / ml) were prepared
from the stock solution and analysed.
Limit Of Detection (LOD) and Limit Of
Quantitation (LOQ)
The LOD and LOQ of the Entecavir by proposed methods
were determined by using calibration standards. LOD and
LOQ were calculated as 3.3σ/S and 10σ/S, respectively,
where S is the slope of the calibration curve and σ is standard
deviation of the y-intercept of regression equation (Table 1).
Table 4: Results of standard addition method
Method Concentration Concentration % level of Total % Analytical
of drug in of pure drug pure drug concentration of recovery ± SD
formulation added (µg/ml) added drug found
(µg/ml) (µg/ml)
Method A 10 8 80 17.98 99.75 ± 0.01
10 10 100 20.12 101.20 ± 0.15
10 12 120 22.24 102.00 ± 0.01
Method B 10 8 80 18.02 100.25 ± 0.01
10 10 100 19.90 99.90 ± 0.52
10 12 120 21.88 99.00 ± 0.08
Method C 10 8 80 18.10 101.25 ± 0.12
10 10 100 20.21 102.10 ± 0.10
10 12 120 22.27 102.25 ± 0.03
(Each value is a result of three separate determinations)
Table 5: Application of the proposed spectrophotometric methods for the determination of Entecavir in tablet
dosage forms
Method A Method B Method C
Commercial products Amount Assay Amount Assay Amount Assay
found (mg) (%) found (mg) (%) found (mg) (%)
Entehep (0.5 mg) 0.498 99.6 0.501 100.2 0.502 100.4
Baraclude (0.5 mg) 0.499 99.8 0.502 100.4 0.503 100.6
Entehep (1.0 mg) 0.996 99.6 0.997 99.7 0.998 99.8
Baraclude (1.0 mg) 0.998 99.8 0.999 99.9 0.997 99.7
114
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

Robustness:
Robustness of the proposed method was determined by
stability of the Entecavir at room temperature for 8 hours.
Three different concentrations (LQC, MQC and HQC) were
prepared in double distilled water. Mean percentage recovery
was determined (Table 1).
RESULTS AND DISCUSSION
Malipatil S.M et al./ Validated UV-Spectrophotometric Estimation of Entecavir in Bulk and Formulations
Fig 2 Graph I
Fig 3 Graph 2
Fig 4 Graph 3
The methods discussed in the present work provide a
convenient and accurate way for the analysis of Entecavir in
115
its pharmaceutical dosage form. Absorbance maxima of
Entecavir at 253.6 nm (Method A) and in the first order
derivative spectra, sharp peak at 242.6 nm (Method B) were
selected for the analysis. Method C was area under curve
(AUC) and the wavelength range for quantitation was 248.0-
258.0 nm. Linearity for detector response was observed in the
concentration range of 2-18 μg/ml for all three methods.
Percentage label claim for Entecavir in tablet analysis, by all
the three methods, was found to be in the range of 99.6% to
100.60% Standard deviation and correlation coefficient for

eight determinations of tablet sample, by all the methods, was
found to be less than ± 1.0 indicating the precision of the
methods. Accuracy of proposed methods was ascertained by
recovery studies and the results are expressed as % recovery.
Percentage analytical recovery for Entecavir, by all the
methods, was found in the range of 99.75% to 102.25%
values of standard.
CONCLUSION
In summary, the proposed methods were less time consuming,
rapid, accurate, precise and inexpensive and can be used for
routine analysis of Entecavir in bulk and pharmaceutical
formulations. The sample recoveries in all the tablets were in
good agreement with their respective label claims and thus
suggested non-interference of excipients in the estimation of
tablets and at the same time no need to use any organic
solvents for extraction of Entecavir from the tablet dosage
forms.
ACKNOWLEDGEMENT
The authors are grateful to the Principal of H.K.E. Society's
Matoshree Taradevi Rampure Institute of Pharmaceutical
Sciences for providing excellent research facilities in analysis
department. We are also thankful to Cadila Healthcare Ltd.,
Goa for providing pure sample of Entecavir as gift sample.
REFERENCES
Malipatil S.M et al./ Validated UV-Spectrophotometric Estimation of Entecavir in Bulk and Formulations
1. O' Neil M J, editor. The Merck Index: An Encyclopaedia of Chemicals,
th
Drugs and Biologicals. Merck & Co. Inc: 2006; 14 edition: 613.
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2. Sweetman S C, editor. Martindale The Complete Drug Reference,
th
Pharmaceutical press, London (U.K), 2007; 35 edition: 786.
3. Bertam G Katzaung, editor. Basic and Clinical pharmacology, 2007;
th
9 edition: 814.
4. Zahler R, Slusarchyk W A, EP 481754; eidem,US 5206244 (1992,
1993 both to squibb); Bisacchi G S et al., Bioorg.Med.Chem lett.7, 127
(1997).
5. Honkoop P, de Man R A, Review of Pharmacology and Clinical
Experience. Expert opin. Invest. Drugs 2003; 12: 683-8.
6. Shah T, Locarnini S, Expert Rev. Anti Infect. Ther. 2004; 2: 853-87.
7. Duxi Zhang, Yulin Fu, Jane P Gale, Anne F Aubry and Mark E Arnold.
Journal of Pharmaceutical and Biomedical Analysis. 2009; 49(4):
1027-33.
8. Fang, Yang Y -c, X -h. Chinese Journal of Pharmaceutical Analysis.
2007; 27(8): 1267-8.
9. V Kiran kumar, N Appala Raju. Spectrophotometric estimation of
Entecavir in pharmaceutical dosage formulations. Biomedical and
Pharmacology Journal: December 2008; 1 (2).
10. Akmar Sandip, Kothapalli Latha, et al. Spectrophotometric estimation
of Bisoprolol Fumarate in bulk drug and tablets. Indian J. Pharm Educ.
Res. 2007; 41 (4): 353-7.
Address for Correspondence
Malipatil S.M, Department of Pharmaceutical Analysis, H.K.E.S's Matoshree
Taradevi Rampure Institute of Pharmaceutical Sciences, Gulbarga-585105,
Karnataka, India
E-mail: smmalipatil@gmail.com

RGUHS Journal of Pharmaceutical Sciences
Antihyperlipidemic Effect of Ethanolic Extract of Hibiscus rosa sinensis
Flowers in Hyperlipidemic Rats
A B S T R A C T
Sikarwar Mukesh S* and Patil M.B.
Original Research Article
Department of Pharmacognosy and Phytochemistry, K.L.E'S College of Pharmacy, Ankola-581314, Uttar Kannada, Karnataka, India,
The antihyperlipidemic activity of Hibiscus rosa sinensis flowers ethanolic extract was investigated in triton (400 mg/kg b.w.) induced and
atherogenic diet-induced hyperlipidemic rats in comparison of a known antihyperlipidemic drug simvastatin (10 mg/kg body wt.). Dose
selection was made on the basis of acute oral toxicity study (50 mg to 5000 mg/kg body weight) as per OECD guidelines. Oral
administration of 500 mg/kg body wt. of the ethanolic extract of Hibiscus rosa sinensis flowers exhibited a significant reduction (p

Sikarwar Mukesh S et al./ Antihyperlipidemic Effect of Ethanolic Extract of Hibiscus rosa sinensis Flowers in Hyperlipidemic Rats
the plant flower possesses anti-spermatogenic and
7
androgenic, anti-tumour and anticonvulsant activities . It
helps in inducing abortion, provide treatment for headache.
Young leaves are sometimes used as a spinach substitute. It
also showed anti implantation, anti-inflammatory, antipyretic,
anti-spasmodic, anti-spermatogenic and anti-viral
activities. The decoction of the roots is used for coughs and
colds. The infusion of the petals of the flowers soothes and
protects the alimentary tract, relieves inflammation and
lowers body heat. In fevers, an infusion of the flowers helps to
reduce body temperature. The application of crushed flowers
soothes external wounds and sores. Flowers can also be made
into a kind of pickle or used as a purple dye for coloring foods
such as preserved fruits and cooked vegetables. The leaves
make a gentle laxative and soften inflamed parts. Root is
8
edible but very fibrous. It's also good for hair treatment .
To the best of our knowledge no scientific data regarding the
antihyperlipidemic effect of Hibiscus rosa sinensis flowers are
available except in the treatise of Ayurvedic medicine. Thus,
the present study was undertaken to evaluate the
antihyperlipidemic effect of Hibiscus rosa sinensis flowers.
Several studies showed that systemic administration of triton
WR1339 (ionic surfactant) in fasted rats causes elevation in
plasma lipid level. Initially, there is a sharp increase in lipid
level reaching a peak two to three times the control value by 24
hours after the administration of triton injection phase I
(synthetic phase),this hyperlipidemia falls within next 24 hr
i.e. 48 hrs after triton administration, phase II (Excretion
phase). This increase in plasma lipid by triton is thought to be
due to increased hepatic synthesis of cholesterol or removal of
very low density protein (VLDL) from the blood due to their
physical alteration by triton. Antihyperlipidemic drugs
interfering with cholesterol synthesis were shown to be active
in phase I while drug interfering with cholesterol excretion
and metabolism were active in phase II. Triton-induced
hyperlipidemia is rather simple and rapid method for
evaluation of test substance and can be considered as the
useful method for preliminary screening of
2
antihyperlipidemic drugs .
The search for new drug with the ability to reduce or regulate
serum cholesterol and triglyceride concentrations has gained
momentum over the years, resulting in a plethora of
publications reporting significant activity of a variety of
natural and synthetic agents. Molecular modification of
naturally occurring compounds has also given rise to potent
agents like pravastatin and simvastatin; the former prepared
by replacement of the methyl group of naturally occurring
lovastatin by a hydroxyl group and the latter a methylated
derivative of compaction. In continuation of our search for
118
plant derived antihypercholesterolemic and hypolipidemic
agents, we direct our attention to some Indian medicinal
plants of which antihyperlipidemic activity has not been
scientifically validated.
MATERIALS
Plant material
Flowers of Hibiscus rosa sinensis were collected in and around
local forest area of Ankola in Western Ghats, Karnataka and
authenticated by the Botanist Prof. G. S. Naik, Department of
Botany, G. C. Science and Art College, Ankola. A voucher
herbarium specimen number GCSAC/HRS/01 was also
preserved in the same college. The collected flowers were
dried and powdered to coarse consistency in cutter mill. The
powder was passed through 40 # mesh particle size and stored
in an airtight container at room temperature.
Atherogenic diet and chemicals
Experimental diet consists of well pulverized mixture of
Cholesterol (2%), Cholic acid (1%), peanut oil (10%), sucrose
(40%) and normal laboratory diet (47%).
A suspension of Triton –WR 1339 (S D Fine chemicals) in
0.15 M NaCl was used for inducing hyperlipidemia in
experimental rats. Simvastatin (Dr. Reddy's Laboratories,
Hyderabad), Diagnostic kits for estimation of were purchased
from Merck Diagnostics India Ltd. Anesthetic Ether (Ozone
International, Mumbai), Distilled Water and All other
chemicals were of Analytical grade.
Animals
Adult Albino rats of wistar strain (150-200 g) of either sex were
procured and housed in the animal house of K L E S College
of Pharmacy, Ankola with 12 h light and 12 h dark cycles.
Standard pellets obtained from Goldmohar rat feed, Mumbai
India, were used as a basal diet during the experimental
period. The control and experimental animals were provided
food and drinking water ad libitum. Ethical clearance was
granted by institutional ethical committee in resolution no.
1/18/2007 held on 23rd November 2007 at J N Medical
college, Belgaum (Ethical committee IAEC reg. no.:
627/02/a/CPCSEA). All the animal experiments were
conducted according to the ethical norms approved by
CPCSEA, Ministry of social justice and empowerment,
Government of India.
METHODS
Extraction of plant material
Powdered crude drug (2.5 kg of the fresh air-dried) of Hibiscus
rosa sinensis flowers were extracted with 95% ethanol by
adopting simple maceration procedure at room temperature
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

for seven days in conical flask with occasional shaking and
stirring. The extract was filtered and concentrated to dryness
at room temperature to avoid the decomposition of the
9
natural metabolites . Extract was preserved in a refrigerator
till further use. Preliminary phytochemical analysis was
carried out by different methods of phytochemical
10
analysis .A known volume of extract was suspended in
distilled water and was orally administered to the animals by
gastric intubation using a force feeding needle during the
experimental period.
Preparation of dose for dried extract and standard
drugs
Ethanolic extract (500 mg/kg b.w) of the selected plant were
formulated as suspension in distilled water using Tween-80 as
suspending agent. The strength of the suspension was
according to the dose administered and was expressed as
11
weight of dried extract .
Simvastatin 10 mg/kg was used as the reference standard
drug for evaluating the antihyperlipidemic activity which was
made into suspension in distilled water using Tween-80 as a
suspending agent.
Acute oral toxicity studies
Sikarwar Mukesh S et al./ Antihyperlipidemic Effect of Ethanolic Extract of Hibiscus rosa sinensis Flowers in Hyperlipidemic Rats
The acute oral toxicity studies of extract were carried out as
per the OECD guidelines from CPCSEA. Administration of
the stepwise doses of ethanolic extract of Hibiscus rosa sinensis
from 50 mg/kg b.w. up to the dose 5000 mg/kg b.w. caused no
considerable signs of toxicity in the tested animals. One tenth
of upper limit dose were selected as the levels for examination
12
of antihyperlipidemic activity .
Diet-induced hyperlipidemic model:
The animals were selected, weighed then marked for
individual identification. Rats were made hyperlipidemic by
the oral administration of atherogenic diet for 20 days. The
rats were then given plant extract suspended in 0.2% tween 80
at the dose of 500 mg/kg b.w. once daily in the morning
through gastric intubation for 14 consecutive days. During
these days, all the groups also received atherogenic diet in the
same dose as given earlier. The control animals received the
hyperlipidemic diet and the vehicle. At the end of treatment
period, the animals were used for various biochemical
13
parameters .
Triton-induced hyperlipidemic model
Animals kept for fasting for 24 h, were injected a saline
solution of Triton at the dose of 400 mg/kg b.w. intraperitoneally.
The plant extract, at the dose of 500 mg/kg b.w.,
were administered orally through gastric intubation. The first
119
dose being given immediately after triton injection and second
dose 20 h later. After 4 h of second dose the animals were used
13
for various biochemical parameters .
Experimental design
Animals were divided into four different groups with six
animals in each group. Group I served as normal control,
Group II was positive control which was given standard
antihyperlipidemic drug simvastatin (10 mg/kg/day p.o.).
Group III was hyperlipidemic control and this group did not
receive any treatment except standard pellet diet. Group IV
received ethanolic extract of Hibiscus rosa sinensis flowers (500
mg/kg/day, p.o.). Treatment periods for all these groups were
14 days in atherogenic diet-induced hyperlipidemia and 48
hours in case of triton-induced hyperlipidemia.
Collection of blood
Blood was collected by retro-orbital sinus puncture, under
mild ether anesthesia. The collected samples were centrifuged
at 4000 rpm for 10 minutes.
Biochemical and HPTLC analysis
The serum was assayed for total cholesterol, triglycerides,
phospholipids and high-density lipoprotein (HDL) using
standard protocol method. By using Friedwald formula the
concentration of very low density lipoprotein (VLDL) and low
density lipoprotein (LDL) in serum were calculated.
An HPTLC chromatogram of active extract was also done by
using CAMAG TLC SCANNER IV, densitometric
evaluation system with CAT software instrument was used for
scanning of thin layer chromatogram objects in reflectance or
transmission mode by absorbance or by fluorescence at 254 or
366 nm respectively.
Statistical Analysis
The results of the study were expressed as mean± S.E.M.
Data was analyzed by using one way analysis of variance test
(ANOVA) followed by Dunnett's t-test for multiple
comparisons. Values with (P

Sikarwar Mukesh S et al./ Antihyperlipidemic Effect of Ethanolic Extract of Hibiscus rosa sinensis Flowers in Hyperlipidemic Rats
extract of Hibiscus rosa sinensis flowers. The results were
comparable with reference standard simvastatin. There was a
significant elevation in serum lipids and lipoproteins in tritoninduced
hyperlipidemic control (p

Sikarwar Mukesh S et al./ Antihyperlipidemic Effect of Ethanolic Extract of Hibiscus rosa sinensis Flowers in Hyperlipidemic Rats
Table 1: Effect of Hibiscus rosa sinensis ethanolic extract on serum total cholesterol, triglycerides and phospholipids level in triton-induced hyperlipidemic rats
a
Gro Treatment Values are expressed as mg/dl, Mean±SEM
up Cholesterol Triglycerides Phospholipids
6 hr 24 hr 48 hr 6 hr 24 hr 48 hr 6 hr 24 hr 48 hr
I Normal control 60.83±1.327 68.17 ±1.701 64.66±1.54 2 65.33±2.140 67.16±2.023 64.83±1.701 75.5±3.304 78.33±0.918 74±1.366
(vehicle only)
II Hyperlipidemic 106.5±4.089 260.33±5.925 178.5±3.649 101 ±3.000 208.66±5.469 107.83±3.208 106.5±2.3 20 185.83±2. 600 100.83±2. 482
control
III Simvastatin 83.67±1.838 ** 176.17±7.56 9** 73.83±2.82 2** 81.5±1.746 ** 172.5±3.631** 79.66±3.981** 91.66±1.7 26** 139.66±1. 333** 80.33±1.4 06**
10mg/kg
IV Ethanolic extract 87.33±3.333 ** 186.16±3.20 8** 81±1.932** 85.16±1.956** 175±3.088 83.33±2.186 ** 92.33±1.9 26* 135.83±3. 816** 86±2.769* *
500mg (EEHRS)
a * **
mg/kg/day for 48 hrs. Values are means±SEM; N=6. Values are statistically significant at P

hyperlipidemic agent in above mentioned hyperlipidemic
models. In comparison to standard drug simvastatin effect of
ethanolic extract of Hibiscus rosa sinensis flower extract was less
but comparable notably. Present studies reveal that ethanolic
extract of Hibiscus rosa sinensis flowers can be used as effective
antihyperlipidemic agent and can be exploited as
antihyperlipidemic therapeutic agent or adjuvant in existing
therapy for the treatment of hyperlipidemia. Further
experiments are required to prove the mechanism and
advantage of this drug over other drugs.
REFERENCES
Sikarwar Mukesh S et al./ Antihyperlipidemic Effect of Ethanolic Extract of Hibiscus rosa sinensis Flowers in Hyperlipidemic Rats
1. Vogel HG, Drug Discovery and Evaluation: Pharmacological Assays,
rd
3 edition, Springer Berlin Heidelberg publisher, 1997; 1095-107.
2. Yu Pengzhan, Li Ning, Liu Xiguang, Zhou Gefei, Zhang Quanbin, Li
Pengcheng, Antihyperlipidemic effects of different molecular weight
sulphated polysaccharides from Ulva pertusa (Chlorophyta).
Pharmacological Research,2003;48:543–9.
3. Nomura H, Kimura Y, Okamoto O, Shiraishi G. Effects of
antihyperlipidemic drugs and diet plus exercise therapy in the
treatment of patients with moderate Hypercholesterolemia. Clinical
Therapeutics 1996;18(3):196.
4. www.tnsmpb.tn.gov.in/images/Hibiscus%20rosa%20sinensis.pdf
Accessed on Nov. 2007.
5. http://www.stuartxchange.com/Gumamela.html Accessed on Nov.
2007.
6. Nair, R., Kalariya, T., Chanda, S., Antibacterial Activity of Some
Selected Indian Medicinal Flora, Turk J Biol, 2005; 29: 41-7.
7. http://www.motherherbs.com/hibiscus-rosa-sinensis.html Accessed
on Nov. 2007.
122
8. Khemani, L.D. , Sachdewa, A., Effect of Hibiscus rosa sinensis Linn.
ethanol flower extract on blood glucose and lipid profile in
streptozotocin induced diabetes in rats, J. Ethnopharmacol.,
2003:89:61-6.
9. Ministry of Health (India). Pharmacopoeia of India. Government of
India; 1982. p. 650, 948.
th
10. Khandewal KR. Practical Pharmacognosy. 14 ed. Pune (India): Nirali
prakashan; 2005. p.146-57.
11. Alam AMD, Ahuja Alka, Baboota Sanjula, Gidwani SK, Ali J.
Formulation and evaluation of Pharmaceutically equivalent parental
depot suspension of methyl prednisolone acetate. Ind. J. Pharm. Sci
2009;30-33.
12. Committee for the Purpose of Control and Supervision of
Experimental Animals (CPCSEA), OECD Guidelines for the testing of
chemicals, revised draft guidelines 423: Acute Oral toxicity- Acute
toxic class method, revised document. India: Ministry of Social
Justice and Empowerment; 2000.
13. Pande VV, Dubey Sonal. Antihyperlipidemic activity of Sphaeranthus
indicus on atherogenic diet-induced hyperlipidemia in rats. Int. J
Green Pharm 2009:159-61.
14. S a r a v a n a K u m a r, M a z u m d e r Av i j i t , S a r a v a n a n V S .
Antihyperlipidemic activity of Camellia sinensis leaves in Triton WR-
1339 induced albino rats. Pharmacognosy Magazine 2008; 4(13):60-
4.
Address for Correspondence
Sikarwar Mukesh. S, Ph.D. , K L E University, K.L.E.S College of Pharmacy,
Ankola, Karnataka, India
E-mail: mukeshsikarwar@gmail.com

RGUHS Journal of Pharmaceutical Sciences
A Study on Drug-Drug Interaction of Diltiazem with Nateglinide in Diabetic
Animals
A B S T R A C T
Raza Hasan*, Suresh D.K, Hamza Sheth, Md. Saifuddin Khalid and Mohiuddin M
Luqman College of Pharmacy, Old jewargi road, Gulbarga-585102, Karnataka, India
INTRODUCTION
Drug interaction is a chemical or physiological reaction that
can occur when two different drugs are taken together. It can
occur when the effects of one drug are modified by the prior
or concurrent administration of another drug. A drug
interaction may result in beneficial or harmful effects.
However, harmful effects are usually predominated. In
considering the clinical relevance of pharmacokinetic drugdrug
interactions mediated by drug-metabolizing enzymes,
efficacy linked to dosage requirements and/or toxicity can be
considered as appropriate endpoints. It may modify the
1
diagnostic, preventive or therapeutic activity of either drug .
In multiple drug therapy, it is important to determine the
incidence and frequency of occurrence of drug interactions,
in hospitalized patients. Further, it is also useful to find out
2
agents that are most likely to produce hazardous interactions .
A study which was conducted on drug-drug interactions in
selected community pharmacies in which out of 1368
prescriptions evaluated over a span of 3 months, 613
interactions were found in 516 prescriptions, out of which
16.15% interactions were severe, 3.75% interactions were
found where patient was receiving more than 8 drugs and
3
11.58% interactions had a significance level . Almost 783,936
people in the United States die every year from conventional
4
medicine mistakes .
RGUHS Journal of Pharmaceutical Sciences
Received: 2/2/2011, Modified: 21/2/2011, Accepted: 27/2/2011
123
Original Research Article
Aim of this investigation was to study the drug-drug interaction between antidiabetic drugs and antianginal drugs. Interaction of
Nateglinide, the known Meglitinide antidiabetic drugs with Diltiazem (antianginal drug) was evaluated in normal healthy and STZ induced
diabetic rats. The blood samples were collected from normal healthy and diabetic rats at different time interval upto 24 hrs and blood
glucose was estimated. Diltiazem pre-treatment (15 mg/kg for seven days), has not significantly altered the onset of antidiabetic effect of
Nateglinide in healthy and STZ induced diabetic rats but significantly increased the peak antidiabetic effect from 40.80±2.54 %
th
reductions before treatment to 51.9±61.14% reduction after treatment at 6 hr and 46.16±1.25% reduction before treatment to
th
55.80±0.30% reduction after treatment at 6 hr in both healthy and in diabetic rats respectively. Duration of antidiabetic effect was
enhanced from 08hrs to more than 18hrs in both groups. This study indicates that therapeutic drug monitoring is required, and the
therapeutic dose of Diltiazem and Nateglinide, needs to be altered when used concomitantly.
Keywords: Diltiazem, Nateglinide, STZ (Streptozotocin), Antidiabetic activity.
Diabetes mellitus is a chronic metabolic disorder
characterized by hyperglycemia, glycosuria, Hyperlipidemia,
5
negative nitrogen balance and sometimes ketonaemia .
Diabetes is always coinciding with serious complications and
adverse effects. Microvascular and macrovascular disease
account for most of the morbidity and mortality associated
with diabetes. Nearly 80% of deaths in those with type 2
diabetes involve cardiovascular disease, Angina Pectoris or
6
stroke . Diabetic patients are more prone serious
complications, like cardiovascular diseases, hypertension,
arrhythmia, angina pectoris, and fungal infections etc which
7
require long term treatment . The total cost of diabetes to the
US health care system in 2002 was estimated at $132 billion,
the majority of this associated with the treatment of chronic
8
diabetic complications . In such cases multiple drug therapy is
needed to prescribe. So, there is always a need for co -
administration of calcium channel blockers like verapamil,
nifedipine, amlodipine and diltiazem etc along with oral
Antidiabetic agents like Nateglinide or Pioglitazone.
There are reports that Nateglinide is predominantly
eliminated by metabolism via the cytochrome P-450 enzyme
9
3A4 and CYP2C9 . Diltiazem is also metabolized by
Cytochrome P-450 enzyme 3A4 and 2C9 and 2D6 and is
10
known to inhibit Cytochrome P-450 enzyme system , hence
there is a possibility of occurrence of pharmacokinetic type of
drug interactions with concomitantly used drugs. Therefore
the present study was carried out on healthy and diabetic rats
to assess the influence of Diltiazem pretreatment on the
antidiabetic effects of Nateglinide.
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

MATERIALS AND METHODS
Animals
Albino Wistar rats of either sex, weighing 150-200 g, were
used as the test animal. The experimental animals were
procured from Sri Mahavir Enterprises, Hyderabad. The
rats were fed on a standard pellet diet (Hindustan Lever Ltd.,
Bangalore, India) and water ad libitum and maintained at 25°C
with 12 hr light / dark cycle. After laboratory acclimation for 7
days, the rats were starved for 48 h. Prior approval by
institutional ethics committee (reg. no: 346/CPCSEA) was
obtained for carrying out the experiments. The study was
conducted in the Department of Pharmacology of Luqman
College of Pharmacy, Gulbarga.
Drugs
Nateglinide was obtained from Dr Reddy's Lab.; Hyderabad.
Diltiazem was obtained from Sun Pharma, Silvassa, Gujarat.
Nateglinide (50 mg/kg, p.o.) and Diltiazem (15 mg/kg, p.o)
suspensions were prepared using 2% w/v gum acacia as
suspending agent to represent 125 mg/ml and 7.5 mg/ml
respectively.
EXPERIMENTAL PROCEDURES
1. In healthy rats
Raza Hasan et al./ A Study on Drug-drug Interaction of Diltiazem with Nateglinide in Diabetic Animals
The healthy rats were marked conveniently and distributed
randomly into two groups of 6 animals each. All the animals
were over night fasted with water ad libitum. The animals in
group-1 received Diltiazem (15 mg/kg, p.o.) and the animals
in the group-2 received Nateglinide (50 mg/kg, p.o) in acacia
suspension. Blood samples were withdrawn at 0, 0.5, 1, 2, 4, 6,
8, 12, 18 and 24 hours intervals and were analyzed for blood
glucose concentration determination by GOD/POD method
using semi auto Analyzer (BCA201) and expressed as mg/dl
12
of blood .
In the next phase of the experiment, after the washing period
of 10 days, the same animals of group-2 received Diltiazem
th
15 mg/kg, p.o. for seven days. On the 7 day, 6 hours after
administration of Diltiazem, the animals were fasted for 14
th
hours. On the 8 day, Diltiazem was given as usual. One hour
after the treatment, animals of group-2 received Nateglinide
50 mg/kg, p.o. Blood samples were collected thereafter at
above mentioned intervals and glucose levels were estimated.
The percentage blood glucose reductions at various time
intervals were calculated and compiled in ( Table 1).
2. In diabetic rats
Experimental induction of diabetes mellitus
The animals were induced into a diabetic state by
intraperitonially injection of a freshly prepared solution of
Streptozotocin (STZ) (Sigma Chemical Co., St. Louis, MO,
USA) in 0.05 mM citrate buffer (pH 4.5) at a dose of 50
11
mg/kg body weight for a single day . Blood samples were
collected after 24 hrs and blood glucose levels were estimated.
Albino rats which have shown more than 250 mg/dl blood
glucose levels were considered as diabetic. The blood glucose
levels were inspected for further four days. From this it was
Table 1: Effect of Diltiazem and Nateglinide on percentage decrease in blood glucose levels at
different time intervals in healthy albino rats.
Percentage reduction in blood glucose concentration ( mean ± SEM )
Time in Diltiazem Nateglinide Diltiazem (15 mg/kg. p.o.) +
Hr (15 mg/kg. p.o.) (50 mg/kg. p.o.) Nateglinide (50 mg/kg. p.o.)
Fasting – – --
0.5 1.78±0.44 6.84±1.02 8.17±0.52
1.0 2.73±1.64 19.70±1.93 24.23±0.95**
2.0 1.34±0.99 27.76±1.74 32.03±1.16*
4.0 ‐0.30±076 36.45±2.45 42.02±1.13***
6.0 0.41±0.90 40.80±2.54 51.96±1.14***
8.0 0.31±0.44 21.49±3.32 39.21±0.87***
12.0 2.23±0.40 10.64±3.72 32.98±3.21
18.0 ‐0.19±0.91 3.51±2.70 18.59±2.45**
24.0 ‐0.27±0.35 0.95±2.35 5.88±1.18
n=6 * Significant at p< 0.05; ** highly significant at p

confirmed that diabetes was induced in 48 hrs and stabilized
within 7 days. These animals were used for further studies.
The same procedure as mentioned in the healthy rats should
be carried out for further study. Blood samples were collected
thereafter at above mentioned intervals and glucose levels
were estimated. The percentage blood glucose reductions at
various time intervals were calculated and compiled in (Table
2).
Statistical analysis
The data were analyzed by Student't' test. P values lower than
0.05 were considered as statistically significant.
RESULTS
Raza Hasan et al./ A Study on Drug-drug Interaction of Diltiazem with Nateglinide in Diabetic Animals
As shown in (Table 1), treatment with Diltiazem alone did not
alter the blood glucose levels in healthy rats. However,
diltiazem pre-treatment (15 mg/kg for seven days), has not
significantly altered the onset of hypoglycemia (i.e.
st
19.70±1.93 to 24.23±0.95, p< 0.01) at 1 hr but significantly
enhanced the peak hypoglycemia (40.80±2.54 % before
th
treatment to 51.9±61.14% after treatment, p

In our study, diltiazem pre-treatment (15 mg/kg for seven
days), has no significant effect on the onset of action of
nateglinide, whereas peak effect and duration of antidiabetic
effect were significantly enhanced as compared to Nateglinide
(50 mg/kg. p.o.) plain treatment. This suggests that Diltiazem
inhibits the metabolism of these antidiabetic drugs by
inhibiting the enzymes responsible for their metabolism.
There are reports that Nateglinide is mainly metabolized by
09
CYP3A4 and CYP2C9 . Reports also indicate that Diltiazem
10
is an inhibitor of CYP3A4 and CYP2C9 . It is revealed from
the results that Diltiazem, in therapeutic dose enhanced the
antidiabetic effect of Nateglinide. This may be due to
inhibitory effect of Diltiazem on CYP3A4 and CYP2C9.
Our studies in healthy and diabetic rats suggested that drug
interaction occurs between Diltiazem and Nateglinide when
they used concomitantly in normal and pathophysiological
conditions like diabetes mellitus.
The present study indicates clearly that during the
concomitant administration of Nateglinide and Diltiazem at
therapeutic doses, the dose and frequency of administration
of Nateglinide need to be readjusted. Also blood glucose levels
need to be monitored during treatment period as a
precautionary measure, so as to avoid severe hypoglycaemia.
CONCLUSION
In the simultaneous treatment of diabetes mellitus and angina
pectoris in a patient with nateglinide and diltiazem,
therapeutic drug monitoring is required and the dose and
frequency of administration of nateglinide needs to be
adjusted. Diltiazem, by inhibiting cytochrome P450 enzyme
system, potentiates the anti diabetes action of nateglinide
.Hence the dose of nateglinide should be reduced.
ACKNOWLEDGEMENT
Raza Hasan et al./ A Study on Drug-drug Interaction of Diltiazem with Nateglinide in Diabetic Animals
Authors wish to thank management committee, Vocational
Education Society (V.E.S.) for providing all the facilities to
carry out this research work. We also thank Sun Pharma,
Silvassa, Gujarat and Dr Reddy's Lab, Hyderabad for
supplying the drugs.
126
REFERENCES
st
1. Kohler GI. Elements of Clinical Pharmacy. 1 edition. Ahmadabad; BS
Shah Prakashan 2004; 135-48.
2. Sunilkumar B, Lucia P, Miglani BD. Possible drug interactions in
hospitalised patients. The Ind J Hos Pharm 1998; 91-3.
3. Rohit Singhal, Nagavi B G. Adepu Ramesh. Drug interactions in
community pharmacy. Pharma times 2004; 36:20-6.
4. URL: http://wiki.answers.com/Q/How_many_people_die_of_
prescription _ drug_related_deaths_ each_year#ixzz1Aw6B6IaC.
th
5. Tripathi K D. Essentials of medical pharmacology. 6 edition. New
Delhi; Jaypee Brothers medical publishers 2008; 254.
6. Hanefeld M, Fischer S, Julius U, et al. Risk factors for myocardial
infarction and death in newly detected NIDDM: The Diabetes
Intervention Study, 11-year follow-up. Diabetologia 1996;39:
1577–83.
7. National Diabetes Information Clearinghouse (NDIC); national
diabetes statistics; http://diabetes.niddk.nih.gov/dm/pubs/statistics.
8. Hogan P, Dall T, Nikolov P, for the American Diabetes Association.
Economic costs of diabetes in the US in 2002. Diabetes Care 2003;
26:917–32.
9. URL: http://www.drugbank.ca/drugs/DB01132/16/08/2011
10. URL: http://www.drugbank.ca/drugs/DB00343/16/08/2011
11. Parthasarathy R, Iavarasan R, Karrunakaran C M. Antidiabetic
activity of Thespesia populnea bark and leaf extract against
Streptozotocin induced diabetic rats. International Journal of Pharm
Tech Research 2009; 1(4):1069-72.
12. Sunil Kumar K, Patel A, shirode D, Ramachandra Setty S. Influence of
Metronidazole on hypoglycemic activity of Thiazolidinediones in
normal and alloxan induced diabetic rats. Indian Journal of Pharm.
Education 2009; 43(1):91-5.
Address for Correspondence
Raza Hasan, Luqman College of Pharmacy, Old jewargi road, Gulbarga-
585102, Karnataka, India
E-mail: raza_hasan21@yahoomail.co.in
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

A B S T R A C T
RGUHS Journal of Pharmaceutical Sciences
Influence of Vitamin C with Lansoprazole in Pylorus Ligation Induced Ulcer
Model in Rats
Nitin M*, Prasad K, Girish M, Ather Javed, Chetan M and Krunal S
Department of Pharmacology, HKES College of Pharmacy, Sedam Road, Gulbarga-585105, Karnataka, India.
Gastric hyperacidity and ulceration of stomach mucosa due to various factors are serious health problems of global concern. Though
currently available drugs provide adequate relief against ulcer, the problem of relapse has eluded success. Hence an attempt is being
made to find a therapy with more efficacy and better results. The present study was designed to evaluate the combination effect of
vitamin C with lansoprazole against pylorus ligation induced ulcer model in rats. The antiulcer effect of the combination of vitamin C 4.5
mg/200 g and lansoprazole 0.54 mg/200 g b.w orally was compared with the reference standard lansoprazole 0.54 mg/200 g b.w orally.
The ulcer index was calculated and other biochemical parameters of gastric juice were estimated. The ulcer index of combination
showed significant (P < 0.05) reduction while other biochemical parameters like volume, pH, free acidity and total acidity of gastric juice
showed highly significant (P < 0.001) reduction when compared to control and standard lansoprazole. The percentage protection of
combination was 94.3% as compared to standard lansoprazole 82.8%. Thus the combination group was found to be synergistic in nature
when compared to lansoprazole alone.
Keywords: Antiulcer activity, vitamin C, lansoprazole, pylorus ligation, ulcer index.
INTRODUCTION
Gastric ulcer, the most common disorder of GIT has
1
multifunctional causes in its pathophysiology . The
pathophysiology of peptic ulcer has been centralized on an
imbalance between aggressive and protective factors in the
stomach such as acid-pepsin secretion, mucosal barrier,
mucus secretion, blood flow, cellular regeneration,
prostaglandins and epidermal growth factors. Various causes
of gastric ulceration include stress, alcohol, Helicobacter pylori
and use of NSAIDs have been shown to be mediated largely
through generation of reactive oxygen species (ROS),
2
especially the hydroxyl radical . A number of excellent drugs,
developed over the decades have proven useful in controlling
hyperacidity and ulceration but their long term use is reported
to have various side effects. Hence the investigation with an
objective to find a therapy with more efficacy and lesser side
effects.
Vitamin-C is a well known antioxidant that neutralizes
various reactive oxygen species such as superoxide radical,
3
singlet oxygen, hydrogen peroxide , alkoxyl, hydroxyl radical
3
directly by hydrogen donation and also neutralizes the radical
4
form of other antioxidants like glutathione & vitamin-E .
Apart from scavenging free radicals it translates haemeoxgenase-1
mRNA into active protein, which then may exert
gastroprotection by its antioxidant and vasodilative
RGUHS Journal of Pharmaceutical Sciences
Received: 12/3/2011, Modified: 28/7/2011, Accepted: 20/8/2011
127
Original Research Article
5
properties . Hence an attempt was made to study the
combination effect of vitamin C with antisecretory drugs like
lansoprazole. Lansoprazole is a proton pump inhibitor that
+ +
suppresses the gastric acid secretion by inhibiting H K
ATPase pump. The FDA has approved it for treatment and
prevention of recurrence of NSAIDs associated gastric ulcers
6
in patients who continue NSAID use . It is more potent, has
longer duration of action, better bioavailability and lesser
drug interaction than other drugs. Therefore, the present
study is designed to evaluate the combination of vitamin C
and lansoprazole against pylorus ligation induced ulcer
model.
MATERIALS AND METHODS
Albino wistar rats of either sex weighing between 180 to 220 g
were selected for the present study. The animals were
acclimatized for seven days and housed under standard
conditions of temperature (25±2°C) and relative humidity
(30-70%) with a 12:12 light-dark cycle. The animals were fed
with standard pellet diet (Hindustan liver co. Mumbai) and
water ad libitum. Pure drug samples of lansoprazole and
vitamin C were procured from Lee Pharmaceuticals
(Hyderabad.) and Wockhardt Ltd (Aurangabad,
Maharashtra.). The dose calculations were extension of
7
human dose based on body surface area . Animal studies were
performed with prior permission of Institutional Animal
Ethics Committee (IAEC) of H.K.E.S College of pharmacy,
Gulbarga (Protocol No. HKECOP/ IAEC/ 16/ 2009-10/
CPCSEA).
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

Nitin M et al./ Influence of Vitamin C with Lansoprazole in Pylorus Ligation Induced Ulcer Model in Rats
8
In pylorus ligation induced ulcer model , the rats were divided
into 3 groups of 6 animals each. The animals of Group I were
treated with vehicle and the animals of Group II were treated
with standard, i.e., lansoprazole 0.54 mg/200 g b.w orally.
The animals of Group III were treated with vitamin C and
lansoprazole i.e., 4.5 mg and 0.54 mg per 200 g b.w orally
respectively. The drugs were administered daily for 5 days. On
th
5 day, the rats were fasted for 24 h before pyloric ligation.
Care was being taken to avoid coprophagy at the end of 24 h,
the rats were anaesthetized with anesthetic ether. Abdomen
was opened by a midline incision. The stomach was lifted out
and a ligature was placed at the pyloric sphincter without
causing any damage to its blood supply. The stomach was
replaced carefully and abdomen wall was sutured in two
layers. After 6 h, the rats were sacrificed with excess of
anesthetic ether, and the stomachs were dissected out. Gastric
juice was collected and drained into test tubes and then
centrifuged at 1000 rpm for 10 min and the volume of
supernatant was noted. The pH of the gastric juice was
recorded by pH meter. Then the contents were subjected for
the analysis of free and total acidity. The stomachs were
opened along the greater curvature then washed under
running water to see the ulcers in the glandular portion of the
stomach. The number of ulcers per stomach was noted and
scoring was done microscopically with the help of hand lens
9
(10x) . The recording was 0 for normal colored stomach, 0.5
for red coloration, 1 for spot ulcer, 1.5 for hemorrhagic
streaks, 2 for ulcer ≥ 3 ≤ 5 and 3 for ulcer > 5. The mean
ulcer score for each animal is expressed as ulcer index and the
percentage protection was calculated by using the following
10
formula :
% Protection = [(UI control – UI treated) /UI control] x 100
9
Determination of free acidity and total acidity
1 ml of gastric juice was pipetted into 100 ml conical flask. It
was diluted to 10ml with distilled water and 2 –3 drops of
Topfer's reagent was added and titrated with 0.01 N sodium
hydroxide until all traces of red color disappear and the color
of the solution turns to yellowish orange. The volume of the
alkali added was noted. This volume corresponds to free
acidity. Then 2 - 3 drops of phenolphthalein solution was
added and titration was continued until a definite red tinge
reappears. Again the total volume of alkali added was noted.
The volume corresponds to total acidity. Acidity was
calculated by using the following formula:
Volume of NaOH x Normality of NaOH x 100
Acidity = ------------------------------------------------------------- mEq/L/100 gm
0.1
11
Histopathological evaluation
The stomachs were immersed in 10 % formalin solution
embedded in paraffin wax, sections of thickness of about 5
128
µm were cut and stained with haemotoxylin and eosin.
Sections were sections were examined for histopathological
changes such as congestion, haemorrhage, necrosis,
inflammation, infiltration, erosion and ulcers.
Statistical analysis
The results were expressed as mean ± SEM, (n=6). Statistical
analysis was performed using student 't' test. P value less than
0.05 was considered to be statistically significant.
RESULTS
It is evident from Table 1 that the effect of combination group
i.e., vitamin C and lansoprazole showed significant (P < 0.001)
reduction in all biochemical parameters like volume, free
acidity, total acidity and increase in pH of gastric juice, while
ulcer index also showed significant (P < 0.05) reduction when
compared to control and standard lansoprazole. The
percentage protection of combination group was found to be
94.3% when compared to control and standard lansoprazole
alone (82.8%). The histopathological examination using
haematoxylene and eosin staining also revealed the protective
activity of combination group when compared to control and
standard lansoprazole (Fig. 5 to Fig. 8).
DISCUSSION
Ulcer is a recurrent disease affecting large populations in all
geographical regions, and reactive oxygen species have been
implicated in the pathogenesis of a wide variety of clinical
disorders and gastric damage. Peptic ulcers result from an
imbalance between defensive (cytoprotective) and offensive
factors (gastric acid), association with Helicobacter pylori
infection and increased use of NSAIDs like aspirin and
12
indomethacin , causing damage by inhibiting the
13
biosynthesis of cytoprotectve prostaglandins .
7
The Shay model of pylorus ligation is a simple, reproducible
and highly predictable model for the screening and evaluation
of antiulcer drugs. It utilizes neither the exogenous ulcerogens
nor the induced exogenous interfering factors. In this model
gastric ulceration may be due to increased secretion of acid
pepsin which leads to auto digestion of gastric mucosa,
decreased mucosal blood flow and breakdown of mucosal
14
barrier . In addition pyloric ligation may reduce glutathione
15
levels of gastric mucosa and increase the lipid peroxidation .
There is one more report that pepsin is active in lower pH.
Therefore the reduced gastric ulcer in this model may occur
due to the reduction in acid secretion and increased gastric
pH. In this condition the activity of pepsin is minimized and
consequently the digestion of mucosal barrier is prevented.
In the present study, from (Table 1), and (Fig. 1 to Fig. 8) the
significant reduction in ulcer index and other biochemical
parameters of gastric juice like volume, free acidity, total
acidity and increase in pH by combination group suggests that
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

Nitin M et al./ Influence of Vitamin C with Lansoprazole in Pylorus Ligation Induced Ulcer Model in Rats
Table 1: Effect of Vitamin C and Lansoprazole in pylorus ligation induced gastric ulcer model in rats:
Gr. Treatment Dose/200 g Volume of Free Total pH of Ulcer %
No. rat gastric acidity acidity gastric Index Protec
juice (ml) (mEq/l)/ (mEq/l)/ Juice tion
100 g 100 g
1 Control Distilled
water 0.5 ml
8.133 ± 0.11 112.0 ± 0.73 124.3 ± 0.55 1.800 ± 0.07 5.833 ± 0.10 -
2 Lansoprazole 0.54 mg 4.567 ± 0.14 56.67 ± 0.55 67.67 ± 0.55 6.767 ± 0.09 1.000± 0.18 82.8%
3 Vitamin C + (0.54+4.5) 2.467 ± 0.15*** 34.00 ± 1.31*** 44.00 ± 0.96*** 7.467 ± 0.11*** 0.333 ± 0.10* 94.3%
Lansoprazole mg
Values are the mean ± S.E.M.n=6, *P < 0.05 and ***P < 0.001 compared with lansoprazole.
Fig. 1: Stomach epithelium of normal rat in pylorus ligation model
Fig. 1: Stomach epithelium of control rat in pylorus ligation model
Redness and ulcer
Fig.3: Stomach epithelium of standard lansoprazole treated
rat in pylorus ligation model
129
Fig. 4: Stomach epithelium of standard lansoprazole
treated rat in pylorus ligation model
Fig. 5: Histopathological slide of normal rat showing normal
histology.
Fig. 6: Histopathological slide of control rat showing redness,
congestion, hemorrhagic streaks, edema, ulceration, necrosis
and dilation of blood vessels.
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

its cytoprotective mechanism may be due to inhibition of
gastric secretion and neutralization of reactive oxygen species
by one or more mechanisms. Lansoprazole, being a potent
proton pump inhibitor, decreases the excess acid secretion, by
+ +
irreversibly blocking the H , K -ATPase of the parietal cell.
Vitamin C, being an antioxidant neutralizes all the free
radicals, nitrates, nitrites that cause cellular damage. It
maintains the gastric blood flow, intragastric vitamin C levels,
antioxidant enzyme activities, which is impaired due to peptic
ulcer disease. Further it translates haeme-oxgenase-1 mRNA
into active protein, which then may exert gastroprotection by
its antioxidant and vasodilative properties. The study also
showed that the lower level of vitamin-C in the blood the
more likely a person will become infected by Helicobacter pylori,
16
the bacteria that can cause peptic ulcers and stomach cancer .
CONCLUSION
From the present study and available results, it can be
concluded that the combination group of vitamin C and
lansoprazole was found to be synergistic in nature. Vitamin C
per se also has a role in prevention of ulcer. The combination
has more cytoprotective and antisecretory effect when
compared to the standard lansoprazole alone.
REFERENCES
Nitin M et al./ Influence of Vitamin C with Lansoprazole in Pylorus Ligation Induced Ulcer Model in Rats
Fig. 7: Histopathological slide of standard lansoprazole treated
rat showing redness, congestion, hemorrhage, mild
edema and dilation of blood vessels.
1. Surana SJ, Tatiya AU, Jain AS, Ushir YV. Antiulcer activity of
Eranthemum Roseum (VAHL) R.Br on ethanol induced ulcer in albino
rats. Int J Pharmacol Biol Sci 2007; 1(1): 65-9.
2. Bandyopadhyay, Debashis, Chattopadhyay, Aindrila. Reactive
Oxygen Species-Induced Gastric Ulceration: Protection by Melatonin.
Curr. Med. Chem 2006;13 (10):1187-202.
3. Padayatty SJ, Arie K, Yaohui W et al. Vitamin C as an antioxidant:
evaluation of its role in disease prevention. J Am Coll Nutr 2003;22
(1):18-35.
4. Dhrubo JS. Herbal Antioxidants: A Great Hope for future. Pharma
Times 2008; 40(12): 22-36.
5. Becker JC, Nina G, Christian W et al Gastro Protection by Vitamin C- a
heme oxygenase-1-dependent mechanism. Biochem. Biophys. Res.
Commun. 2003; 312(2):507-12.
130
Fig. 8: Histopathological slide of Vitamin C and Lansoprazole
treated rat showing no redness, no congestion, no edema but
mild dilation of blood.
6. Goodman and Gilman. The Pharmacological Basis of Therapeutics.
th
11 Ed. New York McGraw Hill: Medical Publishing Division; 2006;
1005-6.
7. Laurence DR, Bacharach AL. Evaluation of drug activities and
pharmacometrics. London and New York. Academic press; 1:160-61.
8. Shay M, Komarov SA, Fels D, Meranze D, Gruenstein H and Siplet. A
simple method for uniform production of gastric ulceration in rat.
Gastroenterology 1945;5:43-61.
rd
9. Kulkarni SK. Handbook of Experimental Pharmacology, 3 Ed. New
Delhi: Vallabh Prakashan; 2005.
10. Fathihah B, Mahmood AA, Sidik K and Salmah I. The antiulcer and
cytoprotective effect of ageratum conyzoides–honey combination in
rats, Department of Molecular Medicine, Faculty of Medicine,
University of Malaya, Kuala Lumpur, JUMMEC 2003-2005; 8: 28-32.
11. Deshpande SS, Shah GB, Parmar NS. Antiulcer activity of Tephrosia
purpurea in rat. Indian J Pharmacol 2003; 35:168-72.
12. Grover JK, Vats V. Proton pump inhibitors. Trop Gastroenterol
1999;20:16-28.
13. Rainsford KD. The effect of 5-lipoxygenase inhibitors and leukotrien
antagonists on the development of gastric lesion by non steroidal anti-
inflammatory drugs in mice. Agents Actions 1987; 21:316-9.
14. Goel RK and Bhattacharya SJL. Gastroduodenal mucosal defense
and mucosal protective agents. Indian J Exp Bio 1991;29:701-14.
15. Mahendran P, Sabitha KE, Shyamaldevi CV. Prevention of HCl-
ethanol induced gastric mucosal injury in rats by Garcinia cambogia
extract and its possible mechanism of action. J Expl Biol 2002:58-62.
16. Camillle MRNot Vancouver. Vitamin C may protect against ulcer-
causing bacteria. University of California, San Francis (UCSF) news
office. 2003.
Address for Correspondence
Dr. NITIN MAHURKAR, Professor and HOD, Department of Pharmacology,
HKES College of Pharmacy, Sedam Road, Gulbarga-585105, Karnataka, India.
E-mail: allnitin@yahoo.co.in
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

RGUHS Journal of Pharmaceutical Sciences
Assessment of Safety and Efficacy of Doxycycline and Azithromycin
Preparations in Patients with Acne Vulgaris
1
Mahendra Kumar B.J*, Ramakrishna S , Kranti Basavant Patil, Sandeep A, Bhimaray S Krishnagoudar and
Katti Ravi Venkappa.
A B S T R A C T
Clinical Pharmacy Department, S A C College of Pharmacy, B.G.Nagara-571448, Karnataka
1 Principal, CR College of Pharmacy, Koratagere, Tumkur, Karnataka
INTRODUCTION
Acne vulgaris is a chronic inflammatory dermatosis which is
notable for open and/ or closed comedones (blackheads and
whiteheads) and inflammatory lesions including papules,
1
pustules, or nodules. Acne vulgaris is the most common skin
disorder in the United States, affecting 40 to 50 million
people. Acne vulgaris affects approximately about 80% of the
total population between the age of 12 to 25 years, with no
gender, race, or ethnicity prevalence. Acne depending on the
ages varies and usually begins at puberty. A form of acne
called adult acne may first occur after the mid 20s, affecting
females more than males, and with lesions generally
distributed in the lower facial area around the mouth, chin
2
and jaw line.
Localization of acne vulgaris on the facial area, especially in
adolescent population, significantly impacts self-esteem.
Although acne is self limiting, it can persist for years and can
result in disfigurement and scarring. Acne may also be
associated with anxiety, depression, and higher than average
unemployment rates. As the emotional impact of acne is not
always easy to assess clinically, it is important for the health
care professionals to educate patients on various causes of
acne, discussing treatment regimens, and counseling on
2
proper medication use.
RGUHS Journal of Pharmaceutical Sciences
Received: 27/1/2011, Modified: 27/2/2011, Accepted: 4/3/2011
131
Original Research Article
Acne vulgaris is the most common skin disease, affects 80% of population between the age of 12 and 25 years with no gender, race or
ethnicity prevalence. Oral and topical agents advocated for the treatment. Antibiotics are the mainstay of acne treatment. The objective
of the study is to assess efficacy and safety of azithromycin and doxycycline with topical clindamycin for patients with acne vulgaris. This
study is a prospective, observational, investigational and randomisation study carried out in Dermatology department of a tertiary care
teaching hospital in a north Karnataka for a period of 8 months. Patients enrolled in the study were divided in to two groups- Group-I and
Group-II. The Group-I received Tablet Azithromycin 500 mg with Topical Clindamycin where Group-II received Tablet Doxycycline 100
mg with Topical Clindamycin. Clinical assessment was done at 30 days interval for 3 months. The mean severity index of Group-I
rd
declined from 113.36 to 09.92 at 3 follow-up and Group-II from 107.32 to 28. In Group-I out of 25 patients 2 patients reported ADR where
from Group-II out of 25 patients 6 patients reported ADR.
Keywords: Acne vulgaris, Azithromycin, Doxycycline, efficacy and safety assessment.
Antibiotics are a main stay of acne treatment, and
clindamycin phosphate is the most widely used topical
antibacterial agent. Although there are several effective
therapies for acne vulgaris, lack of adherence to the treatment
regimen is believed to contribute to treatment failure or less
3
than optimal result.
There are number of oral antibiotics that are used to treat
acne including minocycline, tetracycline, and doxycycline.
The efficacy and possible side effects of these various oral
antibiotics has been the subject of numerous studies for at
least the last 20 years in an effort to understand, which
products are likely to produce better results with the least
4
amount of possible side effects.
While effective therapeutic options exist for the treatment of
acne, treatment compliance with acne medications has been
shown to be as low as 12.5%. In fact poor patient compliance
has been identified as the main reason for acne treatment
failure. Accurate diagnosis, appropriate therapy and good
compliance with directions for therapy are all important
5
components in the treatment of disease.
Antibiotics used for the treatment of acne vulgaris are
associated with some of the mild to severe side effects and
require interventional studies to compare the efficacy and
safety profiles of these medications to ensure the safe usage of
medications. Therefore, this study is being conducted to assess
the efficacy and safety of azithromycin and doxycycline with
topical clindamycin for patients with acne vulgaris.
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

Methodology
Study setting: The study was carried out at dermatology
outpatient department of tertiary care teaching hospital
situated in the north Karnataka.
Study design: This was a prospective, observational,
investigational, randomized study.
Study criteria: The acne vulgaris patients were enrolled
into the study by considering following inclusion and
exclusion criteria.
Inclusion criteria:
• Patients of either sex newly diagnosed with acne vulgaris.
• Patients aged from 10 to 38 years.
• Patients with acne vulgaris coming on outpatient
department basis in the department of dermatology.
• Willingness to participate into study.
Exclusion criteria:
• Patients who are known cases of acne vulgaris.
• Special patient population (Pregnant women, Geriatric).
• Patients with other co morbidities (include skin disease).
Source of data
• Case records from dermatology department.
• Patient data collection form.
• Safety and efficacy (severity index) assessment
questionnaire.
Study procedure
Patients were enrolled by considering inclusion and exclusion
criteria and grouped into two groups (group I & II) using
simple envelop randomisation technique. Group I patients
were treated with Azithromycin 500 mg daily before meals for
three consecutive days in 10 days duration of 30 days, the
remaining 21 days being drug free. Group II were treated with
Doxycycline 100 mg once daily after meals. Topical
clindamycin (cream) twice daily was used in all the enrolled
patients (Group I & II). Patients were clinically assessed at an
interval of 30 days for up to 3 months. At each interval,
clinical assessment was done on the severity index of the
disease and safety assessment was also done in consultation
with dermatologist. The final safety assessment was done and
severity index was calculated at the end of the third follow up.
Statistical Analysis
Mahendra Kumar B.J et al./ Assessment of Safety and Efficacy of Doxycycline and Azithromycin Preparations in Patients with Acne Vulgaris
't' test and 'ANOVA' test were used for calculating significant
difference in consulting with biostatistician of the institution.
132
RESULTS
During the study period of eight months 8325 patients visited
outpatient department of Dermatology, among which 121
patients were diagnosed with acne vulgaris. Out of 121
patients only 53 patients were enrolled into the study by
considering inclusion and exclusion criteria. Out of 53, 29
(54.71%) were males and 24 (45.28%) were females. 50 have
completed the study, remaining 3 patients did not turn up for
the follow up. The incidence in males was slightly higher than
in females, with a male female ration of 1: 0.83. Maximum
prevalence was seen in the 16 – 20 years age group (54.71%) in
both the sexes. Among these, the male prevalence was 58.62%
and female prevalence was 50%. Maximum prevalence in
males was seen at 17 years and in female at 18years. All other
age groups had almost equal sex incidence. The incidence in
the 21–25 years age group was 22.64%. The 26–30 years age
group had an incidence of 11.32%. The 11–15 and 31–35
years age groups had an incidence of 9.34% and 1.88%
respectively. The oldest patient was of 35 years and the
youngest was 13 years old. (Table 1).
The onsets of acne vulgaris were more in the 16 – 20 years
(66.03%) age group. Among these, maximum male patients
had their age of onset at 17 years and maximum females had
their onset at age 18 years. 12 patients (22.64%) had their age
of onset between 11 – 15 years of age. Only 6 patients, with 5
males and 1 female had their age of onset between 21 – 25
years of age. (Table 2).
Most of the patients were students 75.47% (40). Other major
group was that of factory workers (11.32%) and among these,
only one person came in constant contact with oil during his
work. (Table 3).
Out of 53 patients, 51 (96.22%) were unmarried and only 2
(3.77%) were married. Among 53, 25 patients (47.16%) had
acne lesions exclusively on the face. Face was involved in all
the 53 patients. 10 patients (18.86%) had lesions on face, chest
and back. 8 of them (15.06%) had lesions on the face and
Table 1: Age and sex distribution with incidence
Age group Male Female No of
cases
Percentage
11-15 3 2 5 9.43%
16-20 17 12 29 54.71%
21-25 6 6 12 22.64%
26-30 3 3 06 11.32%
31-35 0 1 01 1.88%
Number of 29 24
cases
P ercentage 55% 45%
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

Mahendra Kumar B.J et al./ Assessment of Safety and Efficacy of Doxycycline and Azithromycin Preparations in Patients with Acne Vulgaris
Table 2 : Age of onset
No of cases
Age group Males Females Total Percentage
(years)
11-15 06 06 12 22.64%
16-20 18 17 35 66.03%
21-25 05 01 06 11.32%
Table 3: Occupation
Occupation No. of patients Percentage
Students 40 75.47%
Lecturer 02 03.71%
Librarian 01 01.88%
Labourer 01 01.88%
Housewife 01 01.88%
Worker 06 11.32%
Watchman 01 01.88%
back. 3 patients (5.66%) had lesions on face and chest only.
Other 7 patients (13.20%) had lesions on all the four sites
(Table 4).
All patients were arbitrarily divided into four grades of acne
on the basis of severity. Zero patients (0.00%) belonged to
grade I acne. Majority of patients 24 (45.28%) had grade II
acne. Grade III and IV acne were severe types of acne
consisting of cyst on nodules. 24 (45.28%) and 10 (18.86%)
belonged to grade III and IV respectively. Among these 24
cases of severe acne, 15 were males and 9 were females (Table
5).
About 41 patients gave a history of manual picking among
these, 22 were males and 19 were females. Rest of the patients
did not give any history of manual picking of their lesions
(Table 6).
Majority of the patients (35.84%) gave a history of using
greasy moisturizing creams. 13.20% of patients gave a history
of using talcum powder daily, 11.32% used sandalwood paste,
7.54% used turmeric powder and 1 patient had used pudnia
leave (Table 7).
Skin type: 42 (79.24%) of the patients described their skin
type as oily. 7 (13.20%) of patients called it dry. Rest of them
could describe their skin type neither as oily nor as dry.
Efficacy assessment of group I and group II: Twenty five
patients completed study in group I, who were on treatment
with azithromycin and topical clindamycin. The baseline
score was 113.36 and the mean severity index declined to
91.08, 62.16 and 9.92 along with the first, second and third
follow-ups after the treatment of 30, 60 and 90 days. Decrease
percentage was 19.65% in first follow-up, 45.17% and
91.25% in the second and third follow-ups respectively.
133
Table 4: Distribution of Lesions
Site No. of patients Percentage
Face only 25 47.16%
Face and back 08 15.09%
Face and chest 03 05.66%
Face, chest and back 10 18.86%
Face, chest and back arms 07 13.20%
Table 5: Severity of Acne
Grade No. of patients Percentage
I 00 00%
II 19 35.84%
III 24 45.28%
IV 10 18.86%
Table 6: History of Manual Picking
History No. of patients Percentage
Present 41 77.35%
Absent 12 22.64%
Table 7: Type of Cosmetics and Topical Applications used
Types of application No. of patients Percentage
Greasy creams 19 35.845
Sandalwood paste 6 11.325
Turmeric 4 7.545
Pudina leaves 1 1.88%
Talcum powder 7 13.20%
Twenty five patients completed study in group II, who were on
treatment with doxycycline and topical clindamycin. The
baseline score was 107.32 and the mean severity index
declined to 91.44, 66.32 and 28.00 along with the first, second
and third follow-ups after the treatment of 30, 60 and 90 days.
Decrease percentage was 14.80% in first follow-up, 38.20%
and 73.91% in the second and third follow-ups respectively
(Table 8).
The severity reduction was compared with both drugs and it
was tested with t-test for difference between the means; this
also showed significant change in the reduction of lesions.
The mean severity index of the two drugs was 113.36+19.727
for azithromycin and 107.32+16.790 for doxycycline. After
treatment the mean score reduced to 9.92+5.45 and
28+8.377 respectively. There was a significant difference
between the severity reduction when comparing the effects of
azithromycin and doxycycline (p

Safety assessment: In group I (azithromycin with clindamycin)
out of twenty five patients 2 (08%) patients reported adverse
effects. 1 (04%) diarrhea/loose stools and 1 (04%) abdominal
pain.
In group II (doxycycline with clindamycin) out of twenty five
patients, 6 (24%) patients reported adverse effects 1 (04%).
DISCUSSION
Out of patients who attended the outpatient department
during the study, 121 cases of acne vulgaris were seen. The
incidences of acne, thus amounted to 2.3% out of these, 53
cases were randomly selected for the present study. Maximum
prevalence of acne was in the age group of 16 – 20 years. In
girls, maximum prevalence was at 18 years (50%) and at 17
years (80%) in boys. This finding was in accordance with the
studies done by Bloch. The correct incidence and prevalence
of acne could not be assessed with our study, because the cases
were randomly selected and also due to small size of the study
group. Most of the studies have been done in schools
comprising of adolescents, where as our study was not
restricted to the adolescents. Only patients who attended the
clinic with acne as their presenting complaint were included in
6
the study.
Family History
Mahendra Kumar B.J et al./ Assessment of Safety and Efficacy of Doxycycline and Azithromycin Preparations in Patients with Acne Vulgaris
Table 8: Safety assessment of group-I and group-II
Group -I: Azithromycin with Topical Clindamycin
Sl.No Adverse effects Numbers Percentage
1 Nausea 0 0%
2 Diarrhoea/loose stools 1 4%
3 Abdominal pain 1 4%
4 Others 0 0%
Group -II: Doxycycline with Topical Clindamycin
1 Photo sensitivity 1 4%
2 Oral candidacies 0 0%
3 Vomiting 3 12%
4 Others(rash) 2 8%
Table 9: Comparison between Group-I and Group-II
Group -I: Group -II:
Azithromycin Doxycycline
with Topical with Topical
Clindamycin Clindamycin
Average base line score 113.36 107.32
Average first follow up 91.08 91.44
Average second follow up 62.16 66.32
Average third follow up
P

patients had lesions on the face, 47.16%of the patients had
acne on the back also and 35.84%had lesions on the chest.
Lesion distribution varies with individuals and there is no
definite pattern of lesion distribution described.
Clinical assessment of Acne
Different grading methods have been used for measuring the
severity of acne, but to date, a widely accepted standardised
classification system does not exist. In the present study, in all
the cases the acne lesions were graded according to severity
index described by Michaelsson et al by counting the number
of open or closed comedones (0.5), papules (1), pustules (2),
and infiltrated lesions (3) and cystic lesions (4). The total
severity score of disease was calculated by multiplying each
type of lesion with its severity index and adding them together.
Efficacy Assessment
In a study it was found that a combination of azithromycin
and topical clindamycin was significantly better than
doxycycline and topical clindamycin in the treatment of
inflammatory acne vulgaris. In this study there was 91.25%
improvement in group I (Azithromycin group) in comparison
to 73.91% improvement in group II (Doxycycline group) and
this difference was statistically significant by 'ANOVA' test
(p

RGUHS Journal of Pharmaceutical Sciences
Antidiarrhoeal Activity of Aqueous Extract of Mimosa pudica Leaves
Md. Saifuddin Khalid*, Shah Jinesh Kumar, Suresh D.K, Rajnish Kumar Singh, Reddy Narasimha I.V and
Shaikh Azhar Hussain
A B S T R A C T
Dept. of Pharmacology, Luqman College of Pharmacy, Gulbarga-585 102, Karnataka.
INTRODUCTION
1
Diarrhoea is a killer disease worldwide and unfortunately, it
happens to be amongst the symptoms of many other diseases.
In most rural communities of developing countries including
Asia, diarrhoea poses serious problems particularly for
children due to amongst other reasons, lack of adequate
sanitation and pipe bornewater. Diarrhoea is characterized by
passage of abnormally soft or liquid feces leading to excess loss
2
of fluid, salts and nutrients . Several herbs and shrubs are
3
useful as medicines as reported by many scientists . Many such
herbs, shrubs and plants are known to protect the organs from
the environmental, chemical and occupational challenges.
Traditional medicine practitioners in asia have been known
to treat diarrhoea with a variety of medicinal plants one of
which being Mimosa pudica Linn. Mimosa pudica Linn is one
4
such green leaf shrubs . The leaves of these Mimosa pudica
have been shown to contain tannins and flavonoids which
have been implicated in their antidiarrhoeal activity. The
plant has been used to treat a variety of ailments. The plant
Mimosa pudica used in indigenous medicine for the treatment
of hydrocele, scrofula, conjunctivitis, cuts, wounds,
hemorrhages, bleeding disorders like menorrhagia, dysentery
5
with blood and mucus, piles, in herbal formulations .
However, scientific evidence does not exist in literatures to
Original Research Article
The study intended to investigate the antidiarrhoeal activity of the leaf aqueous extract of Mimosa pudica in wistar albino rats. Castor oil
induced diarrhoeal test, prostaglandin-E induced enteropooling test and gastrointestinal tract transit of charcoal meal test were used to
2
assess the antidiarrhoeal activity of Mimosa pudica. While the acute toxicity study and phytochemical analysis was carried out using well
established protocols and methods. The leaf aqueous extract of Mimosa pudica significantly inhibited castor oil induced diarrhoea, PGE
2
induced enteropooling and has also reduced gastrointestinal motility after charcoal meal administration in rats. Loperamide, a standard
antidiarrhoeal drug, produced similar effects to the leaf aqueous extract of Mimosa pudica on three diarrhoeal model. The phytochemical
analysis of the leaves revealed the presence of tannins, saponins particularly steroidal saponin, and flavonoids. The LD50 of the plant
species obtained was greater than 2000 mg/kg (p.o.). The data obtained indicate that the leaf aqueous extract of Mimosa pudica has
antidiarrhoeal activity. The data also showed that the plant material given orally may be safe and/or non toxic in mice. However, further
investigation on the acute toxicity and on the mechanism of the antidiarrhoeal effect of the plant species needs to be carried out.
Keywords: Mimosa pudica Linn; Anti-diarrhoeal activity; Castrol oil-PGE induced diarrhea; Leaves aqueous extract; Intestinal
2
secretion; Gastrointestinal motility.
RGUHS Journal of Pharmaceutical Sciences
Received: 31/1/2011, Modified: 10/2/2011, Accepted: 19/2/2011
136
corroborate the claims by traditional medicine practitioners
of the therapeutic successes of the plant species.
The main aim of the present study was, therefore, to
investigate the antidiarrhoeal activity of the leaf aqueous
extract of Mimosa pudica to justify its folklore use in diarrhoea.
The acute toxicity, the phytochemical analysis of various
components and the effect of the plant species on the
intestinal length of charcoal meal were also investigated in
rats.
MATERIALS AND METHODS
The fresh leaves of Mimosa pudica Linn were collected from
Luqman college of Pharmacy, Gulbarga. (Karnataka). The
plant herbarium specimen was identified and authenticated
by Mr. P. G. Diwakar, Joint Director, Botanical Survey of
India, Western circle,7, Koregaon Road, Pune -1. (Voucher
No. JINSHMI1)
6
Extraction
The authenticated leaves of Mimosa pudica Linn were dried in
shade and ground to fine powdered coarsely. 80 gm of fine
powder was refluxed in 1 L of boiling water, allowed to cool
and filtered. The filtrate was then frozen at −80°C and freezedried
for 120 hour. A yield of 10.4 gm of dried leaf aqueous
extract was obtained. Fresh extract solutions were prepared
on each day of the experiment by dissolving weighed
quantities of the extract in appropriate volumes of
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

physiological saline. The solution was administered orally
(p.o.) in a volume of 1 ml/100 gm of animals using a bulbed
steel needle.
7
Phytochemical Screening
Preliminary phytochemical screening of the ethanolic extract
of leaves was performed for the presence of alkaloids,
phenolics, flavonoids, saponins, carotenoids, carbohydrates
and glycosides.
AnimalsUsed
Albino wistar rats of either sex weighing between 150 to 200
gm and albino mice of either sex weighing between 20 to 25
gms were procured from registered breeders (346/CPCSEA,
Mahavir Enterprises, Hyderabad.) used for studying antidiarrhoeal
activity and acute toxicity respectively.. The
animals were housed under standard conditions of
temperature (25±2°C ) and relative humidity (30-70%) with a
12:12 light-dark cycle. The animals were fed with standard
pellet diet (VRK Nutrition, Pune) and water ad libitum.
Approval at the Institutional Animal Ethics Committee
(IAEC) of Luqman College of Pharmacy, Gulbarga was
taken for conducting anti-diarrhoeal activity.
8
Acute Toxicity Study :
The acute toxicity of aqueous extracts of Mimosa pudica leaves
was determined in female albino mice. Animal were fasted
overnight prior to the experiment. Fixed dose (Annexure-2d)
method of CPCSEA, OECD guideline No. 420; was adopted
th th
for the study. 1/5 and 1/10 of LD50 cut off (2000 mg/kg)
values taken as screening dose.
Anti-diarrhoeal Activity:
9
Castor oil induced Diarrhoea
In the present study animals were divided into four groups of
six rats each. Group-I was administered vehicle and served as
control. Group-II served as standard and received loperamide
(1mg/kg). Group-III and IV were given orally aqueous
extract (200 and 400 mg/kg) of Mimosa pudica leaves
respectively. They were fasted overnight before the test with
free access to water.
After 30 minute of administration of above dose all the rats
were given with 1ml of castor oil orally. The numbers of wet
fecal dropping were measured for six hours.
10
Prostaglandin-E2 induced Enteropooling
Md. Saifuddin Khalid et al./ Antidiarrhoeal Activity of Aqueous Extract of Mimosa Pudica Leaves
In this test the animal were divided into 5 groups of six rats
each. The animals were deprived of food and water for 18
hours prior to the experiment. Group-I received only 1ml of
5% v/v ethanol in normal saline and then treated with 2% of
137
w/v aqueous gum acacia suspension and served as vehicle
control, Group II treated with PGE (100 mg/kg, p.o.) and
2
served as PGE control, Group- III served as standard and
2
received loperamide (5 mg/kg), group IV and V were
administered orally aqueous extract (200 and 400 mg/kg)
respectively. Immediately after the extract treatment each rat
was administered PGE (100 mg/kg in 5% v/v. ethanol in
2
normal saline, orally) in the group III, IV and V. All the rats
were killed after 30 minute and the whole length of the
intestine from the pylorus to caecum was dissected out and its
contents were collected in a test tube and volume was
measured.
11
Gastrointestinal Motility Test
In this study also the animals were divided into four groups of
six rats each. They were fasted for 24 hours before the test with
free access to water. The Group-I served as control (vehicle)
while the Group II was administered standard drug atropine
sulphate (5mg/kg) intraperitoneally, Group III and IV was
treated with aqueous (200 & 400 mg/kg) extracts. After 30
minute they were administered 1 ml of charcoal meal (3% of
charcoal in 2 % aqueous tragacanth) after half an hour the
rats were sacrificed and intestinal distance moved by the
charcoal meal from pylorus to caecum was measured.
Statistical Analysis
Results were expressed as mean ± SEM, (n=6). Statistical
analysis were performed with one way analysis of variance
(ANOVA) followed by Dunnet's t test. P value less than

1. Castor-oil induced diarrhoea:
Md. Saifuddin Khalid et al./ Antidiarrhoeal Activity of Aqueous Extract of Mimosa Pudica Leaves
produced a considerable amount of stool. Mimosa pudica
(200–400 mg/kg, p.o.) significantly (p

2. Prostaglandin-E induced enteropooling model:
2
Table:2 Effect of aqueous extracts of Mimosa pudica leaves on mean volume of intestinal fluid (ml) and their %
protection in prostaglandin-E 2 induced diarrhoea in rats
Groups Treatment Dose Mean volume of intestinal % of
fluid (ml) � SEM inhibition
I. Vehicle 1 ml of 5% v/v ethanol and 1.567 ± 0.08819 —
control normal saline p.o.
II. PGE control PGE 100 � g/kg 3.367 ± 0.09189 —
2
2
III. Standard 5 mg/kg 1.690 ± 0.03890 *** 79.21%
IV. AEMP 200mg/kg 2.467 ± 0.1256* 40.01%
V. AEMP 400mg/kg 2.155 ± 0.07482** 52.65%
AEMP: Aqueous Extract of Mimosa pudica, The values are Mean SEM, n = 6, *p < 0.05, **p < 0.01 and *** p < 0.001 vs control
Fig. 2: Effect of aqueous extracts of Mimosa pudica leaves on mean volume of intestinal fluid in prostaglandin-E 2
induced enteropooling model
3. Gastro-intestinal motility Test:
Md. Saifuddin Khalid et al./ Antidiarrhoeal Activity of Aqueous Extract of Mimosa Pudica Leaves
A: Vehical Control
B: PGE Control
2
C: Standard (Loperamide 5 mg/kg p. o.)
D: Aqueous extract of Mimosa pudica leaves (200 mg/kg p. o.)
E: Aqueous extract of Mimosa pudica leaves (400 mg/kg p. o.)
Table: 3 Effect of aqueous extracts of Mimosa pudica leaves on mean movement of charcoal and their %
inhibition in gastro-intestinal motility test in rats
Group Treatment Dose Mean length Mean distance Mean% movement of % of
of intestine travelled by charcoal charcoal±SEM inhibition
±SEM (cm) meal±SEM (cm) (cm)
I. control --- 106±0.7303 82.33±1.764 75.69 ± 0.4357 ----
II. Standard 5 102.7±1.02*** 53.33±0.8819*** 50.79 ± 0.8274*** 50%
(Atropine Sulphate)
III. AEMP 200 105±0.8944* 74.17±1.400* 67.46 ± 0.1014* 32.07%
IV. AEMP 400 103.8±1.014* 63.50±0.7638** 65.13 ± 0.1841* 34.65%
AEMP: Aqueous Extract of Mimosa pudica, The values are Mean SEM, n = 6, *p < 0.05, **p < 0.01 and *** p < 0.001 vs control
Fig 3: Effect of aqueous extracts of Mimosa pudica leaves on mean % movement of charcoal meal in
gastro-intestinal motility test
A: Control
B: Standard (Atropine sulphate 5 mg/kg p. o.)
C: Aqueous extract of Mimosa pudica leaves (200 mg/kg p. o.)
D: Aqueous extract of Mimosa pudica leaves (400 mg/kg p. o.)
139
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

DISCUSSION
The ability of Mimosa pudica to reduce the number of animals
exhibiting diarrhoea and the number of diarrhoeal episodes is
12
taken as an antidiarrhoeal activity . Castor oil, an irritant or
stimulant laxative, is hydrolysed in the upper small intestine to
ricinoleic acid, a local irritant, that irritates the mucosa of the
gastrointestinal tract resulting in increase in intestinal
13
motility . Nitric acid mechanism has also been shown to be
involved in castor oilinduced diarrhoea. The data obtained in
the present study show that loperamide, a standard
antidiarrhoeal agent, profoundly inhibited castor oil-induced
diarrhoea and also inhibited in rats. Loperamide, an opioid
derivative, has been shown to slow intestinal motility by its
action on µ receptors on neurons in the submucosal neural
plexus of the intestinal wall and its anti-muscarinic activity in
13
the gastrointestinal tract . It is not surprising, therefore, that
loperamide protected rats against castor oil-induced
diarrhoea. Mimosa pudica significantly reduced the number of
animals exhibiting diarrhoea and the number of diarrhoeal
episodes and also inhibited the intestinal propulsion of
charcoal meal in the present study. It is probable that the plant
extract may be exerting its antidiarrhoeal activity by slowing
intestinal motility. The extract significantly inhibited the
PGE 2 induced intestinal fluid accumulation (enteropooling).
PGE 2 also inhibit the absorption of glucose, a major stimulus
14
to intestinal absorption of water and electrolytes . These
observations tend to suggest that extract reduced diarrhoea by
inhibiting PGE 2 induced intestinal accumulation of fluid. The
extracts appear to act on all parts of intestine. Thus, it
decreased the intestinal propulsive movement in charcoal
meal test. The mechanism of this inhibition of motility may
be due to the non-specific spasmolytic activity of the extract.
Furthermore, the standard chemical tests carried out in this
study showed that the leaves of the plant species contain
tannins, saponins particularly steroidal saponin, and
flavonoids. It is pertinent to note that tannins have been
15
reported in several studies to have antidiarrhoeal effect . The
previous study reported that tannin containing drugs are
widely used for the treatment of diarrhoea and related
16
disorders . In view of the above publications, it is, therefore,
not surprising that the standard chemical tests in this study,
showed the presence of tannins in Mimosa pudica which also
may probably contribute to its antidiarrhoeal activity.
CONCLUSION
Md. Saifuddin Khalid et al./ Antidiarrhoeal Activity of Aqueous Extract of Mimosa Pudica Leaves
On the basis of the present results and available reports, it can
be concluded that the anti-diarrhoeal activity elucidated by
aqueous extract of Mimosa pudica leaves could be mainly due to
its inhibitory effect both on gastrointestinal propulsion and
fluid secretion. The inhibitory effect of the extracts justifies
the use of the plant as a non-specific anti-diarrhoeal agent in
folk medicine.
140
ACKNOWLEDGEMENT
The authors are thankful to Dr. P. G. Diwakar, joint director,
botanical survey of India, Pune, for identification and
authentication of plant, Dr. S.K. Srinivasan, VP (Tech),
Lake chemical Pvt. Ltd., Bangalore, for gifting the
Loperamide drug, Management Dr. Abdul Mujeeb,
Chairman, College Governing Council, Luqman College of
Pharmacy, Gulbarga for providing me all facilities,
throughout the research work.
REFERENCES
1. Farthing M J. Diarrhoea: a significantworldwide problem. International
Journal of Antimicrobial Agents 2000;14: 65–9.
2. Fauci AS, Bravnwold E, Isselpacker K, Wilson JD, Kasper DL, Hauser
SL et al. Harrison's Principles of Internal Medicine New York: McGraw
Hill Company;1993;1: 236.
3. Webpage by Lynh-Diem Bui- Mimosa pudica.
rd
4. Nadkarni KM. Indian Materia Medica. 3 Edition 2002;1: 1280-3.
5. India herbs – Ancient Remedies for Modern Times: Ayur State, Doctor
approved formula for prostate care.
th
6. Kokate CK, Purohit AP and Gokhale SB. pharmacognosy. 14 ed.
Nirali prakashan; 2007: 297.
7. Khandelwal, K.R. Practical Pharmacognosy, 11th ed. Nirali
Prakashan, Pune, 2004:149-56.
8. Veeraraghavan, Prema. Expert Consultant, CPCSEA, OECD
Guideline No.420, 2000.
9. Awouters F, Niemegeers CJE, Lenaerts FM, Janssen PAJ. J of Pharm
Pharmacol 1978;30: 41.
10. Mukherjee PK, Das J, Balasubramanian R, Sahakakali, Pal M, Saha
BP. Indian J Pharmacol 1995;27:262.
11. Pazhani GP, Subramanian N, Arunchalam G, Hemalatha S,
Ravichandran V. Indian Drugs 2001;38:269.
12. Williamson EM, Okpako DT, Evans FJ. Pharmacological Methods in
Phytotherapy Research: Selection, Preparation and Pharmacological
Evaluation of Plant Material 1996; 1: 25–28.
13. Altman DF. Drugs used in gastrointestinal diseases. In: Katzung, B.G.
(Ed.),Basic and Clinical Pharmacology, 18th ed. McGraw-Hill, San
Francisco; 2001:1070-1.
14. Capasso F, Mascolo N, Izzo AA, Gaginella TS. Dissociation of castor
oilinduced diarrhoea and intestinal mucosal injury in rat: effect of NGnitro-Larginine
methyl ester. British J of Pharmacol 1994;113: 1127-
30.
15. Jaffe BM. Prostaglandins and serotonin: Nonpeptide diarrhoeogenic
hormones. World J Surg 1979; 3: 565-78.
Address for Correspondence
Md. Saifuddin Khalid, Assistant Professor, Department of Pharmacology,
Luqman College of Pharmacy, Old Jewargi Road, Gulbarga-585 102,
Karnataka, India.
Email: khalid2568@yahoo.com

A B S T R A C T
RGUHS Journal of Pharmaceutical Sciences
Assessment of Various Combination of Drugs Used in Treatment of Lower
Respiratory Tract Infection
3 1 2 1
Imran Ahmad Khan* , Shobha Rani RH , Geeta S and Mahvash Iram
1
Department of Pharmacy practice, Al-Ameen college of Pharmacy, Bangalore-560027
2
Department of medicine, St. Martha's Hospital, Bangalore – 560001
3
Biocon India Ltd, Bangalore - 560100
Both macrolides as well as cephalosporins are widely used in the treatment of various lower respiratory tract infections either alone or in
combination. The most commonly prescribed macrolide is azithromycin, generally in combination with different cephalosporins. Thus
arises the need to evaluate different combinations of azithromycin and cephalosporins generally prescribed and to compare their
efficacy, safety (adverse drug reactions) as well as cost. A prospective study was conducted in the medicine ward at St. Martha's
Hospital, Bangalore. Efficacy was determined based upon the clinical response (reduction in symptoms) and length of hospital stay.
Safety was assessed by the occurrence of ADR and their severity. Cost of treatment was calculated by cost effective analysis. Data of 88
patients was analyzed and it was observed that different combinations prescribed were azithromycin + cefotaxime, azithromycin +
ceftriaxone and azithromycin + cefuroxime. The most commonly prescribed combination was found to be azithromycin with cefotaxime
and it showed statistically significant difference in the reduction of clinical symptoms thereby indicating greater efficacy. 18% of the
patients experienced ADRs which were mild in nature with none severe representing that all the combinations were safe. The cost
effective analysis revealed that combination of azithromycin and cefotaxime was most economical.
Keywords: Lower respiratory tract infection, Azithromycin, Cephalosporins, Combination
INTRODUCTION
Infections of the respiratory tract are very common due to the
air pollution; especially those affecting the upper tract. Upper
respiratory tract infections (URTIs) are usually caused by
viruses and are rarely serious. They include common cold,
tonsillitis, sore throat, sinusitis, laryngitis and croup. Lower
respiratory tract infections (LRTIs) are usually more serious
since they affect the breathing tubes (trachea and bronchi) and
the lungs. Bronchitis, acute bronchiolitis and pneumonia are
the various types of LRTI.
Most upper respiratory tract infections are caused by viruses.
Each time when a person gets cold it is caused by a slightly
different virus, and once it is fought off, body acquires
immunity (resistance) to that particular virus. Lower
respiratory tract infections are caused by both viruses and
bacteria. Infection may begin with a virus, but if bacteria get
into the lower respiratory tract, then it can cause more serious
problems. LRTIs are more common in people with a weak
immune system, such as geriatrics and those receiving
1
immunosuppressive treatment; for example, cancer patients .
RGUHS Journal of Pharmaceutical Sciences
Received: 21/6/2011, Modified: 12/7/2011, Accepted: 1/8/2011
141
Original Research Article
Respiratory tract infections (RTI) are very common in the
community and are one of the major reasons for
2
appointments to primary care physicians. The broad
diagnosis of RTI includes the two principal sub-diagnoses of
lower respiratory tract infection (LRTI) and upper respiratory
3
tract infection (URTI) . Community-acquired lower
respiratory tract infection is a common cause of acute illness
in adults. The spectrum of disease ranges from mild mucosal
colonization or infection, to acute bronchitis or acute
exacerbation of chronic bronchitis (AECB) or chronic
obstructive pulmonary disease (COPD), to overwhelming
parenchymal infection in patients with community-acquired
4
pneumonia (CAP) . The term LRTI includes a wide range of
diseases which have different underlying pathologies and
5,6
etiologies, e.g. acute bronchitis and pneumonia . In the outpatient
setting, LRTI accounts for the majority of all
antibiotics prescribed, burdening healthcare budgets. In most
of the adults with LRTI, the illness is self-limiting and its
course will not be modified by antibiotic therapy, representing
viral or clinically non-relevant bacterial diseases. However,
failure to initiate an antibiotic therapy within four hours in
cases of community acquired pneumonia (CAP) is associated
7
with an increased mortality rate. The major problem in the
management of the LRTI is the inability to determine the
8
causative micro-organism in the majority of patients.
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

There are enormous logical differences in the prescription of
antibiotics for LRTI, between countries and between different
9
healthcare providers in the same country. The "first
generation" of guidelines was mostly consensus-based,
whereas those published in 2000/2001 are partly evidencebased.
However, there is still lack of evidence in many areas of
the LRTI, also interpretation of the available evidence is
10
variable.
MATERIALS AND METHODS
Study Location
The present study was conducted at medicine wards of St.
Martha's Hospital, Bangalore which is a tertiary care teaching
hospital providing specialized health care services. Ethical
clearance was obtained from the Institutional review board of
St. Martha's Hospital, and an Informed consent was taken
from the patients before initiating the study. The study was
conducted for a period of 8 months.
Study Population
All adult and geriatric hospitalized patients of medicine
department who were diagnosed with lower respiratory tract
infection being prescribed with a combination of
azithromycin and cephalosporin during the study period and
who were willing to participate in the study were included. A
total of 88 patients were enrolled. Out patients,
Pregnant/lactating patients, Pediatric patients, Non
consenting patients were categorized under exclusion group.
Study Design
Imran Ahmad Khan et al./ Assessment of Various Combination of Drugs Used In Treatment of Lower Respiratory Tract Infection
It was a prospective study where in the data was collected from
the case sheets of inpatients diagnosed with LRTI. A detailed
description of demographic details, Presenting complaints,
Past History, Personal History, Family History, Drug history,
Laboratory parameters was taken. Patient follow up was
carried out until discharge.
Efficacy was determined based upon the clinical response i.e.
reduction in the symptoms such as sputum production, cough,
wheezing, dyspnea, fever, discolored sputum and length of
hospital stay. The patients were monitored throughout till
discharge and the symptoms were noted at a regular interval
of every three days. The patients were also monitored for any
adverse drug reactions during the treatment.
Cost of treatment was calculated by “Cost Effective Analysis”.
It is an economic evaluation method of pharmacoeconomics
where cost is measured in monetary terms and consequences
are measured in non-monetary units. Cost effective analysis is
used when there is single measurable dimension of
effectiveness for both treatments. This method is used when it
142
is necessary to measure both cost and clinical outcomes of
drugs.
The cost effective ratio for each treatment option is calculated.
This ratio is total cost of the drug divided by the number of
units of output (benefit). In this case, the output is reduction in
the symptoms on the seventh day of the treatment. Preferred
drug is the one with lower cost per unit of output or health
improvement. The difference in the reduction of symptoms in
different treatment groups was statistically analyzed by Chi-
square test.
RESULTS
After appropriate scrutiny 88 patients met the inclusion
criteria and were enrolled after taking informed consent.
Demographic details, diagnosis, co-morbidities and habits are
depicted in (Table 1). Different LRTI's were diagnosed of
which pneumonia was the form of illness in 40% of patients
making it highest among others.
The various laboratory parameters evaluated were, White
Blood Corpuscles (WBC), Erythrocyte Sedimentation Rate
(ESR), Platelet count, Partial Pressure of Oxygen and Carbon
dioxide (PaO2, PaCO2), Bicarbonates (HCO3) and Oxygen
Saturation (SaO2). 84% of the patients got admitted with the
complaint of cough and 83% with the complaint of excessive
sputum production. Other complaints included, wheezing,
dyspnea, myalgia, nausea, fever and discolored sputum.
The combinations of macrolide and cephalosporin therapy
prescribed to the patients according to the diagnosis for the
treatment of their relevant conditions are positioned in (Table
2).
The length of hospital stay of patients ranged from 1 to 12
days and minimum stay was observed in azithromycin +
ceftriaxone combination, whereas patients on combination of
azithromycin + cefuroxime experienced maximum hospital
stay.
Further evaluation of each symptom was done individually to
assess the severity. The results are presented in (Table 3) and
Table 1: Demographic details of the study subjects
Demographic Details n* %
Males 52 59
Females 36 41
Age (50yrs-59yrs) 29 33
Co-Morbidities (Hypertension) 44 50
Smoking 52 59
Alcohol 26 29.5
Tobacco
n*- No of patients
30 34
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

Table 2: Combinations of Macrolide and Cephalosporin therapy as per diagnosis
No. of Patients (%)
Diagnosis Azithromycin + Azithromycin Azithromycin Total
Ceftriaxone + Cefotaxime + Cefuroxime
PNEUMONIA 37 43 20 40
AECOPD* 40 45 15 23
AEBA# 38 54 8 14
BRONCHITIS 30 60 10 23
*Acute Exacerbation of Chronic Obstructive Pulmonary Disease (AECOPD)
#Acute Exacerbation of Bronchial Asthma (AEBA)
(Table 4) depicts the Cost Effective Ratio of the different
combinations of drugs used for the treatment of LRTI.
DISCUSSION
Imran Ahmad Khan et al./ Assessment of Various Combination of Drugs Used In Treatment of Lower Respiratory Tract Infection
Azithromycin was the common antibiotic prescribed along
with the cephalosporin to the enrolled patients at a dose of
500 mg O.D. The minimum dose of cefotaxime prescribed to
the patients was 1g B.I.D and the maximum dose was 2g
Q.I.D. In case of ceftriaxone, the minimum dose was 1g B.I.D
and the maximum dose was 2g T.I.D. In case of cefuroxime,
the minimum dose prescribed to the patients was 1g B.D and
the maximum dose prescribed was 2g Q.I.D.
The efficacy of medications was evaluated mainly by
observing the reduction of symptoms from the time of
th
admission up to the 7 day of treatment. According to the
(Table 3), it was found that reduction in symptoms was greater
in case of the combination of azithromycin with cefotaxime
group compared to the other two groups. As the percentage
of reduction in severe symptoms was greater in combination
of Azithromycin with cefotaxime (58%), compared to
ceftriaxone (40.6%) and cefuroxime (36.9%) group of
patients, cefotaxime combination seems more effective in
reducing the symptoms.
Statistically there was a significant difference found in the
reduction of sputum production and dyspnea between the
treatment groups. However there was no statistically
significant difference in the symptoms namely cough,
wheezing and fever with different combinations.
The length of hospital stay ranged from 1 days to 12 days,
maximum patients (56.82%) got discharge between 5 – 8 days.
In case of cefotaxime and ceftriaxone group maximum
patients ie 69.77% and 56.25% respectively got discharge
between 5 – 8 days whereas in case of cefuroxime group
maximum patients (76.92%) got discharged between 9 – 12
days. Based on the number of days for discharge, the patients
of cefotaxime group were found to have improved and got
discharged earlier compared to the other two groups. Thus
the combination of azithromycin and cefotaxime seemed
most effective.
143
Safety of the treatment was evaluated by monitoring the
adverse drug reactions of the treatment groups throughout
the study period. 21.59% of patients had complaints of
ADRs. Cefotaxime group of patient experienced lesser
number of ADRs compared to the ceftriaxone and
cefuroxime group. In case of patients prescribed with the
combination of azithromycin + cefotaxime, there was no
complaint of arthralgia, gingivitis, abdominal pain and heart
burn, but CNS side effects such as agitation and dizziness were
found. However, none of the ADRs were severe and life
threatening. Hence, we can say that all the three combinations
were safe.
The cost of the therapy was calculated by cost effective
analysis. As per (Table 4), cefotaxime combination was found
to be more economical in comparison with ceftriaxone and
cefuroxime combinations. The average cost effective ratio of
the cefotaxime combination was found to be Rs. 1650,
whereas in case of ceftriaxone and cefuroxime combination
the average cost per treatment for 100 % reduction in
symptoms was found to be Rs. 2230 and Rs. 2631 respectively.
CONCLUSION
The various cephalosporins used along with azithromycin for
the treatment of LRTI in the medicine wards of the hospital
were cefotaxime (3rd generation), ceftriaxone (3rd
generation) and cefuroxime axetil (2nd generation). The
combinations prescribed were appropriate with respect to the
diagnosis. All the three combinations showed a decrease in the
clinical symptoms of the patients, but cefotaxime group of
patients exhibited a faster decrease compared to the other two
groups. Length of the hospital stay was also less in the patients
treated with cefotaxime and azithromycin combination.
Combination of azithromycin with cefotaxime proved to be
more efficacious clinically than azithromycin with ceftriaxone
and azithromycin with cefuroxime axetil.
Azithromycin with cefotaxime showed a lesser number of
adverse drug reactions than the other two combinations.
However the ADRs observed in patients taking all the three
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

Table 3: Comparison of severity of symptoms
Change in Symptoms Treatment
Azithromycin + Azithromycin + Azithromycin + P value
Ceftriaxone Cefotaxime Cefuroxime
Baseline Sputum Production
Severe 84.4 83.7 84.6
Mild/Moderate 12.5 9.3 0.0 0.458
Absent
Day 7
3.1 7.0 15.4
Severe 43.8 25.6 46.2
Mild/Moderate 9.4 0.0 30.8 0.003
Absent 25.0 39.5 15.4
Baseline Cough
Severe 81.2 88.4 84.6
Mild/Moderate 6.2 7.0 0.0 0.574
Absent
Day 7
12.5 4.7 15.4
Severe 40.6 18.6 53.8
Mild/Moderate 12.5 23.3 23.1 0.129
Absent 25.0 23.3 15.4
Wheezing Baseline
Severe 46.9 62.8 61.5
Mild/Moderate 12.5 11.6 23.1 0.364
Absent
Day 7
40.6 25.6 15.4
Severe 9.4 0.0 23.1
Mild/Moderate 18.8 14.0 23.1 0.060
Absent 50.0 51.2 46.2
Dyspnea Baseline
Severe 25.0 30.2 53.8
Mild/Moderate 50.0 39.5 30.8 0.368
Absent
Day 7
25.0 30.2 15.4
Severe 6.2 0.0 21.3
Mild/Moderate 3.12 16.3 46.2 0.006
Absent 40.6 48.8 23.1
Fever Baseline
Observed 37.5 37.2 61.5
Not observed
Day 7
62.5 62.8 38.5 0.261
Observed 21.9 11.6 38.5 0.131
Not observed 56.2 53.5 53.8
Color of Sputum Baseline
Observed 75.0 79.1 76.9 0.917
Not observed
Day 7
25.0 20.9 23.1
Observed 37.5 18.6 61.5 0.039
Not observed 40.6 46.5 30.8
Patients
Discharged (%)
21.9 34.9 7.7 –
P a t i e n t s %
Imran Ahmad Khan et al./ Assessment of Various Combination of Drugs Used In Treatment of Lower Respiratory Tract Infection
144
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

different combination were mild in nature and none were
serious and life threatening. Also, cost effective analysis
endorsed the fact that azithromycin with cefotaxime
combination was more cost effective.
Hence, it is concluded that combination of azithromycin with
cefotaxime was best among the three in treating LRTI.
LIMITATIONS
Several limitations of the present study should be addressed.
1. Microbiological studies were not routinely carried out on
sputum samples before initiation of the antibiotic therapy, to
determine the causative organism and to follow the
appropriate treatment guidelines.
2. Subsequently microbiological tests should also be carried
out on sputum samples at the end of the therapy in order to
determine the efficacy of the antibiotics used and complete
removal of causative micro-organism.
3. The prolongation of stay in the hospital of many of the
patients could have been indirectly due to the underlying
concomitant illness.
ACKNOWLEDGMENT
We thank Principal and Management of Al-Ameen college of
Pharmacy, Bangalore, India for providing the necessary
funding and support and The Medical Superintendent of
St.Martha's Hospital for permitting us to carry out the study
in their hospital.
REFERENCES
Imran Ahmad Khan et al./ Assessment of Various Combination of Drugs Used In Treatment of Lower Respiratory Tract Infection
Table. 4 Cost Effectiveness Ratio, Cost Effective ratio = Cost of treatment for 7 days / Reduction of symptoms by 100%
Cost of Treatment = Cost of Drug + Other associated costs (Syringe)
TREATMENT SPUTUM COUGH WHEEZING DYSPNOEA FEVER DISCOLO AVERAGE
PRODUCTION URED
SPUTUM
Azithromycin +
Ceftriaxone
1497 1497 1621 3234 3897 1621 2230
Azithromycin +
Cefotaxime
1251 1042 1158 2408 2841 1202 1650
Azithromycin +
Ceftriaxone
1822 2272 1822 2280 3043 4545 2631
*in Rs for 100% decrease in symptoms
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9. PlouffeJ, Schwartz DB, et., al. Clinical efficacy of Intravenous followed
by oral azithromycin monotherpy in hospitalized patients with
community-aquired pneumonia. Anti Microb Agents Chemother
200;44:1796-802.
10. Ortqvist A. treatment of community acquired lower respiratory tract
infections in adults. Eur Respir J 2002; 20:40-50.
Address for Correspondence
th
Imran Ahmad Khan, M. Pharm, Biocon India Ltd, 20 KM, Hosur Road,
Electronic City, Bangalore- 560100, Karnataka, India
E-Mail: iam_khan2000@yahoo.co.in

A B S T R A C T
RGUHS Journal of Pharmaceutical Sciences
Formulation and Evaluation of Mucoadhesive Buccal Drug Delivery System of
Metoprolol Tartrate by Using Central Composite Design
1 2
Prakash Rao B* and Gandhi Purvesh
Original Research Article
1 Department of Pharmaceutical Technology, Karnataka College of Pharmacy, Bangalore, Karnataka, India.
2. Department of Pharmaceutics, Visveswarapura Institute of Pharmaceutical Sciences, Bangalore, Karnataka, India.
The central composite design was used to develop the controlled release buccoadhesive tablets containing metoprolol tartrate as drug
candidate. Carbopol 934P and hydroxy propyl cellulose (HPC) were taken as formulation factors (independent variables). Bioadhesive
strength, drug release after 8 hours, T and release exponent (n) were taken as responses (dependent variables). The polymers had
50%
significant effect on bioadhesive strength and in vitro drug release. It was found that carbopol gives higher bioadhesive strength than
HPC. Comparatively HPC controls the drug release greater than carbopol. The optimized formulation follows non-Fickian release
mechanism. The FT-IR and DTA studies indicate no physico-chemical interaction. Stability studies revealed that optimized formulation
was stable. The predicted values of drug release at 8 hrs, bioadhesive strength, T release exponent, n are 64.26%, 44.84 gm, 6.10
50%,
hrs, 0.658 and actual values are 60.45%, 43.52 gm, 6.46 hrs, and 0.673 respectively.
Keywords: Metoprolol tartrate, Central composite design, Buccoadhesive, Hydroxy propyl cellulose, Carbopol 934, DTA
INTRODUCTION
In recent years, the transmucosal route like oral cavity, ocular,
nasal, rectal, vaginal offers the many advantages than the
peroral route. These include an avoidance of both hepatic
and intra-alimentary canal metabolism within GI tract. The
oral mucosal cavity for drug administration has received
much more attention because of its unique advantages over
other oral transmucosal routes. The buccal route is suitable for
sustain and controlled release administration of drug because
of its less permeability and buccal mucosa has expanse of
smooth and relatively immobile mucosa. The sublingual route
is suitable for rapid onset of action because of its high
1
permeability of the mucosa and rich blood supply . And the
problems such as hepatic first pass metabolism and
degradation of drug in GIT can be overcome by using the
buccal route. Buccal drug delivery facilitates safe and easy
2
removal of dosage form in cause of toxicity . Various buccal
3
adhesive dosage forms like discs, microspheres and bilayered
4
tablets have been prepared and reported by several research
5
groups .
Mucoadhesive polymers are essential in the development of
buccal drug delivery system. This polymer provides intimate
contact between the dosage form and the absorbing tissue and
6
increase the retention time . Increasing the retention time of
the dosage form is essential in the development of this system
RGUHS Journal of Pharmaceutical Sciences
Received: 18/1/2011, Modified: 22/5/2011, Accepted: 1/6/2011
146
and it has been reported that increase in retention time with
7, 8
an increase in the mucoadhesivity of the system . In the
literature, buccoadhesive drug delivery system for drugs like
9 10 11
carvedilol , clotrimazole and sodium fluoride are reported.
Metoprolol Tartrate (MT) is a selective beta receptor blocker
1
used in treatment of several diseases of the cardiovascular
system, especially in hypertension, angina pectoris, cardiac
arrhythmias and myocardial infraction. The half life of MT is
3 to 4 hours. Metoprolol is completely absorbed after oral
administration, but bioavailability is low (< 40%) because of
12
first pass metabolism . The short half life and severe first pass
metabolism of metoprolol tartrate make it suitable for
administration via buccal route that provides controlled drug
delivery and by passing first pass effect.
The effect of the quantity of carbopol 934 (A) and expanded
form of HPC (B) were selected as independent variables. In
vitro bioadhesive strength, drug release after 8 hours, T 50%
(Time for 50% drug release) and diffusion coefficient (n) were
selected as response variables. Computer-aided optimization
technique, using central composite design (CCD), was
employed to investigate the effect of two independent
variables (factors) on drug release parameters and bioadhesive
13
strength .
All response variables were fitted to linear, quadratic, 2FI
model and regression analysis was carried out to get a
quantitative relationship between the dependable and the
analysed independent variables.
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

OBJECTIVES
1) To formulation of controlled mucoadhesive buccal drug
delivery system by using central composite design.
2) To improve the bioavailability by passing the first pass
effect.
3) To perform the evaluation studies for thickness,
hardness, weight variation, friability and In vitro drug release.
MATERIALS AND METHOD
Materials
Prakash Rao B et al./ Formulation and Evaluation of Mucoadhesive Buccal Drug Delivery System of Metoprolol Tartrate by Using Central Composite Design
Metoprolol Tartrate was obtained as a gift sample from
Novartis pharmaceutical LTD (Mumbai, India). Carbopol
934P and magnesium stearate were purchased by S.D Fine
Chem. LTD. (Mumbai, India). HPC was received as gift
sample from Strides Arco labs LTD (Bangalore, India). All
other chemicals and reagents were of analytical grade.
14
Experimental Design
Central composite design (CCD) was selected for the
development of the formulation. CCD has three groups of
design points, two-level factorial or fractional factorial design
points, axial points (sometimes called "star" points) and
central points. Central points are usually repeated 4-6 times to
get a good estimate of experimental error. Central composite
designs have 5 levels of each factor: -Alpha, -1, 0, 1, and
+Alpha (Table 1). Effect of carbopol (A) and effect of HPC (B)
was selected as independent variables. Higher (+1) and lower
(-1) value of the independent variables were selected and
calculate the alpha value. These values were put in the
optimization software and get the different formulation given
in the (Table 2). The drug release after 8 hours (R 1),
bioadhesive strength (R 2), T 50% (time for 50% of drug release)
(R3) and diffusion coefficient (n) (R4) were selected as
responses.
Preparation of Buccal Tablet
Buccal tablets of MT were prepared by direct compression
method. All the ingredients without magnesium stearate were
accurately weighed and mixed in mortar with a pestle for 10
minutes to get the uniform powder. After sufficient mixing of
drug and polymer, magnesium stearate was added and mixed
for 3 min. The blended powder was compressed into tablets by
using the Rimek mini press –I compression machine. The
quantities of various ingredients are shown in (Table 2).
Evaluation of Buccal Tablets
Thickness
Thickness of the tablet was measured by using the digital
vernier callipers (Aerospace, china).Thickness is expressed in
15
millimeters.
Hardness
The hardness of the tablet was measured using the Monsanto
hardness tester (Scientific engineering corp., Delhi, India). It
2 16
is expressed in Kg/cm .
Weight Variation Test
20 Tablets were taken from each batch and individually
weighed by Electronic balance. Average weight of the tablets
was calculated and deviation from the actual weight was
16
determined.
16
Friability
Friability generally refers to the loss in weight of tablets in the
containers due to the removal of fines from the tablet surface.
Friability generally reflects poor cohesion of tablet
ingredients. 10 tablets were weighed, initial weight of these
tablets were recorded and placed in Roche friabilator and
rotated at the speed of 25 rpm for 100 revolutions. Then,
tablets were removed from friabilator, dusted off the fines and
Table 2: List of working formulations
Run Carbopol( X 1 ) HPC ( X 2 ) Design
(mg) (mg) point
F-1 65.00 65.00 Centre
F-2 65.00 65.00 Center
F-3 15.50 65.00 Axial
F-4 65.00 65.00 Centre
F-5 30.00 30.00 Fact
F-6 30.00 100.00 Fact
F-7 100.00 30.00 Fact
F-8 100.00 100.00 Fact
F-9 65.00 65.00 Centre
F-10 114.50 65.00 Axial
F-11 65.00 15.50 Axial
F-12 65.00 114.50 Axial
F-13 65.00 65.00 Centre
Each formulation contain 50 mg of MT
Table 1: Factors and their corresponding levels implemented for the construction of CCD
Independent
variable(Factor)
-Alpha( α)
-1 0 +1 +alpha(α)
Carbopol 15.50 30 65 100 114.50
HPC 15.50 30 65 100 114.50
147
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

again weighed and recorded. Followed by 2 trials were done.
Then the average of initial weights and final weights were
recorded.
initial weight - Final weight
% Friability = ---------------------------------------- * 100
Initial weight
In-vitro Mucoadhesive Strength Measurement
Porcine buccal mucosa was obtained from local slaughter
house and stored in Kreb's buffer solution. The experiment
was performed within the 3 hours of procurement of mucosa.
The porcine mucosa was washed with the distilled water and
carefully tied to the glass slide with the help of cyanoacrylate
adhesive and placed in petridish. Kreb's solution was added
into the petridish up to the upper surface of the buccal mucosa
to maintain buccal mucosal viability during the experiment.
During the experiment the solution was maintained at 37°C.
The tablet was stuck on to the glass stopper by using
cyanoacrylate adhesive. The preload of 20 gm was placed on
the glass stopper for 7 minutes to establish the adhesion
bonding between tablet and porcine buccal mucosa. The
preload and preload time were kept constant for all
formulations. The preload was removed from stopper and
water was added in to beaker from separating funnel at a
constant rate of 100 drops per minute (Fig 1). The addition of
the water was stopped when the tablet was removed from
porcine buccal mucosa. The weight of water collected in
5
beaker was weighed which is taken as mucoadhesive strength .
In-vitro Dissolution Studies
The dissolution studies of the buccoadhesive tablet was
performed in 900 ml of phosphate buffer (pH=6.8) using the
USP type II dissolution apparatus under sink condition at 37
0
± 0.2 C and 50 rpm. At the appropriate time interval, the
sample was withdrawn and volume made up with distilled
water. The samples were filtered through a 0.45 µm Millipore
filter and amount of MT which was released determined
spectrophotometrically at 274 nm and the release data were
17
evaluated kinetically .
Glass Stopper
Buccal Tablet
Buccal mucosa
Prakash Rao B et al./ Formulation and Evaluation of Mucoadhesive Buccal Drug Delivery System of Metoprolol Tartrate by Using Central Composite Design
Fig. 1: Schematic diagram of in-vitro bioadhesive
strength measurement device
Separating Funnel
148
Analytical Method
Accurate weight of powder equivalent to 50 mg metoprolol
tartrate was taken and transferred in to 100 ml volumetric
flask and volume was made up with distilled water. The
powder solution was transferred in 250 ml of beaker and
heated for 30 minutes for the extraction of the drug. Then, the
solution was cooled and filtered. The samples were diluted
appropriately and the absorbance was measured at 274 nm
using the Shimadzu (model 1601) UV- visible
16
spectrophotometer.
Regression Analysis
The response parameters were statistically analyzed by
applying one way ANOVA at 0.05 levels using commercially
available software Design-Expert software (Stat-Ease Inc,
Minneapolis, USA). The individual parameters were
evaluated using the F test and Linear, 2FI, Quadratic models
were generated for each response parameter using the
multiple linear regression analysis (MLRA) equation:
2 2 2 2
R = b 0 + b 1 A+ b2B + b3AB + b4A + b5B + b6AB + b7A B (1)
Where, R is the level of measured response, b 0 is the intercept
of the arithmetic mean response of the 13 runs, A and B are
the coded level of the independent variables. The AB is the
interaction term, show how response changes when two
2 2
factors are simultaneously used. A , B are quadratic terms of
the independent variables to evaluate the nonlinearity.
Kinetic release Studies
For the determination of the drug release kinetics from the
buccal tablet, the in vitro release data were analyzed by zero
order, first order, Higuchi and Korsmeyer and Peppas
17
equations .
Zero order release Kinetic
To study the zero order release kinetics the release data was
fitted into the Following equation:
dQ/dt = K (2)
0
Where, 'Q' is the amount of drug release, 'K ' is the zero order
0
release rate constant and 't' is the release time. The graph is
plotted percentage cumulative drug release (%CDR) verses
time.
First Order Release Kinetic
To study the first order release kinetics the release rate data are
fitted into the following equation:
dQ/dt = K Q (3)
1
Where, 'Q' is the fraction of drug release, 'K ' is the first order
1
release rate constant and't' is the release time. The graph is
plotted log %CDR remaining verse time.
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

Prakash Rao B et al./ Formulation and Evaluation of Mucoadhesive Buccal Drug Delivery System of Metoprolol Tartrate by Using Central Composite Design
Higuchi Release Model
To study the Higuchi release model the release rate data are
fitted into the following equation:
½
Q = K H t (4)
Where, 'Q' is the fraction of drug release, 'KH' is the release
rate constant and't' is the release time. The graph is plotted %
CDR verses square root of time.
Korsmeyer and Peppas Kinetics
To study the Korsmeyer and Peppas release kinetics the
release rate data are fitted in to following equation:
n
Mt/M∞ = K KP t (5)
Where, Mt/M∞ is the fraction of drug release, 'K ' is the
KP
release rate constant and 't' is the release time and 'n' is the
diffusion exponent related to mechanism of drug release. The
graph is plotted log %CDR verses log time.
Fourier Transform Infrared Spectroscopy (FT-IR)
IR spectroscopy was carried out for the following A) Pure
drug, B) Drug + carbopol C) Drug + HPC using Shimadzu
FTIR model 8700by taking KBr disc. The instrument was
operated under dry air purge and the scans were collected at
-1
scanning speed of 2mm\sec with resolution of 4cm over the
-1 18
region of 4000-400 cm .
Differential Thermal Analysis (DTA)
The sample of MT, carbopol, HPC and their binary mixtures
were weighed and sealed in 40 ml aluminum crucibles with a
pierced aluminum lid. The analyses were performed under
nitrogen (nitrogen flow rate 50 ml/min) in order to eliminate
oxidative and pyrrolytic effects at a standard heating rate of
15ºC/minute over a temperature range of 30ºC - 300ºC using
a Mettler-Toledo star system.
Stability studies
An accelerated stability study was carried out according to
ICH guidelines. The optimized formulation was kept in 2 ml
0
of glass vial and closed. The vials were kept at 40 ± 2 C / 75
RH ± 5% RH for six months in a dessicator. After end of
every month, tablets were evaluated for bioadhesive strength
and drug content.
RESULTS AND DISCUSSION
Development of Formulations
Evaluation of Tablets
The average thickness of the all buccal tablets ranges from
2.12 to 3.25 mm. The value of percentage variation in weight
and friability were found to be with in the limit of
149
conventional oral tablets stated in the Indian pharmacopeia.
Hardness of all buccal tablets was higher because of the
carbopol. In all the formulations, the assay for drug content
was found to be in the range from 47.56 mg to 49.65 mg.
In-vitro drug release study after 8 hour
Total amount of metoprolol tartrate released from all
formulations ranges from 60.45% to 87.90% in 8 hours (Table
3). Decreased rate of drug release was observed with increase
of the concentration of polymers. (Fig. 2-4) illustrates the
release profile of all formulations. When the tablets contact
Fig. 2: In-vitro drug release profiles of formulation F-1 to F-5
Fig. 3: In-vitro drug release profiles of formulation F-6 to F-9
Fig. 4: In- vitro drug release profiles of formulation F-10 to F-13
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Prakash Rao B et al./ Formulation and Evaluation of Mucoadhesive Buccal Drug Delivery System of Metoprolol Tartrate by Using Central Composite Design
Table 3:Result of bioadhesive strength and release parameter obtained for formulations by CCD
RUN CARBOPOL HPC BIOADHESIVE DRUG RELEASE T50%
STRENGTH AFTER 8 HOUR
F-1 65 65.0 36.62 69.41 5.02
F-2 65 65.0 33.45 64.85 4.97
F-3 15.50 65.0 32.95 68.00 4.52
F-4 65 65.00 33.25 64.25 4.95
F-5 30 30.00 32.88 87.90 4.25
F-6 30 100.00 38.02 68.09 5.15
F-7 100 30.00 35.72 67.95 5.11
F-8 100 100.00 43.52 60.45 6.46
F-9 65 65.00 36.23 65.54 5.35
F-10 114.50 65.00 47.14 65.41 5.55
F-11 65 15.50 34.55 85.78 4.17
F-12 65 114.50 45.06 68.66 5.65
F-13 65 65 33.65 65.68 5.25
with water the gel formation of polymers occurs which acts as
rate controlling matrix for the release of drug molecules
(Salamone, 1996). In this case, effect of both polymers can be
explained by mathematical equation in terms of actual
factors:
R 1 = + 115.03476 - 0.29224*A - 0.92256*B + 2.51224E-
2 2
003*A*B + 1.33265E-004*A + 4.42510E-00* B
The quadratic model is selected for this response with Model
F-value 10.79 and p value is 0.0035. Both the factor A,
carbopol and B, HPC decreases drug release from the tablets.
HPC is a semi synthetic polymeric derivative of cellulose, will
swell in an aqueous medium to form a gel like matrix that
controls release by acting as a barrier to drug dissolution and
19
diffusion (Park C. R, 2002). HPC shows the higher
controlling effect on the release of drug than the carbopol due
to formation of higher viscous solution. Whereas high
molecular weight carbopol is polymer of acrylic acid
crosslinked with polyalkenyl ethers or divinyl glycol polymer
which is hydrophilic in nature and swells faster and greater
17
extent at pH 6-7 due to carboxylate group on the polymer
backbone ionise, resulting in repulsion between negative
20
charges (pharmainfo.net). So, the carbopol promotes the
penetration of the dissolution medium into tablet matrix and
21
results in greater release of drug as it do not dissolve in water .
(Fig. 5) represents the observed response values compared to
that of predicted values. The effect of A and B can be further
elucidated with the help of response surface plot (Fig 6).
Higher release of metoprolol tartrate was found after 8 hours
in low concentrations of both polymers. At high level of B the
percentage release of MT at 8 hours was low. From the results,
it can be concluded that both the independent variables have
Fig. 5: Correlation between actual and predicted values
for In vitro drug release at 8 hours
Fig. 6: Response surface plot showing the effect of factor
A (Carbopol) and factor B (HPC) on in vitro drug release at 8 hours
150 RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

Prakash Rao B et al./ Formulation and Evaluation of Mucoadhesive Buccal Drug Delivery System of Metoprolol Tartrate by Using Central Composite Design
negative effect and factor B has more significant negative
effect than that of factor A on percentage MT release at 8
hours. In another words, at high level of A percentage release
has high value at all level of factor B which indicates factor A
helps more release of drug.
Effect of Variables on Bioadhesive Strength
Bioadhesion is generally understood to define the ability of a
biological or synthetic material to “stick” to a mucous
membrane, resulting in adhesion of the material to the tissue
20
for a protracted period of time . In general, bioadhesion is
considered to occur in three major stages: wetting,
interpenetration, and mechanical interlocking between
22
biological tissue and polymer . Several polymer and
hydrophilic macromolecules containing groups able to form
hydrogen bonds have showed good adhesion property that
seems to be enhanced by the incorporation of amine and
23
carboxylic groups . The strength of bioadhesion is affected
by various factors such as molecular weight of polymer,
contact time with mucus, swelling rate of polymer, and
22
biological membrane used in the study . Water uptake
process produces polymer swelling and improves the
consolidation step that increases the mobility of molecules
and facilitates that interpenetration with the biological tissue
layer. So, the polymer swelling is a property related to the
23
bioadhesion of the system . The constant and regression
coefficient for bioadhesive strength are as follow:
R = + 26.40149 + 0.066170 * A + 0.064012* B + 5.42857E-
2
004 * A * B
The linear model F-value of 6.54 and p value 0.0122 implies
the model is significant. Values of "Prob > F" less than 0.0500
indicate model terms are significant. In this case (Table 4) A,
cabopol and B, HPC are significant model terms . (Fig. 7) rep
resent the observed response values compared to that of
predicted values. The effect of A and B can be further
elucidated with the help of response surface plot (Fig 8). Both
the factor A and B have an synergetic effect on the
bioadhesive strength as AB factor has positive effect. At high
level of factor A gave slightly higher value of bioadhesive
strength than that of factor B. If A kept high level and at all
levels of B bioadhesive strength was observed slightly higher
values.
As increase the concentration of factor A and B increases the
bioadhesive strength. Carbopol gives slightly higher
bioadhesive strength, when compare to the HPC due to high
molecular weight, water dispersibility, polymer chain
flexibility for chain interpenetration and diffusion with mucin.
The carbopol forms hydrogen bond with mucin due to the
21
presence of many carboxylic groups and its shows higher
bioadhesive strength than HPC.
151
Effect of formulation variables on T 50%
The value of T ranges from the 4.17 to 6.46 hours (Table 4).
50%
The increased T was observed at high concentrations of
50%
polymers. The constant and regression coefficient for R3 T
50%
are as follows:
R = +3.25759 + 0.012952*A + 0.01551*B
3
The linear model was found to be significant for the time for
50% of drug release. The Model F-value of 48.90 and value
of p is less than 0.000100 indicate the model is significant.
Lack of fit for the model is not significant as F-value is 1.42
and p value is 0.3817. Both the factors have positive effect
Fig. 7: Correlation between actual and predicted values f
or bioadhesive strength
Fig. 8: Response surface plot showing the effect of factor
A (carbopol) and factor B (HPC) on bioadhesive strength
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Prakash Rao B et al./ Formulation and Evaluation of Mucoadhesive Buccal Drug Delivery System of Metoprolol Tartrate by Using Central Composite Design
Table 4: Summary of ANOVA table for dependable variables from CCD
Source d.f. Sum square Mean square F value Probability
MT release at 8 hour
A 1 122.09 122.09 9.44

Prakash Rao B et al Formulation and Evaluation of Mucoadhesive Buccal Drug Delivery System of Metoprolol Tartrate by Using Central Composite Design
of optimized formula found to be 0.673 which indicates the
mechanism of release is non Fickian. The factor A with
higher concentration shows the higher effect on value of the
release exponent(n) than the factor B. At high level of factor A
gave high value of n at all level of factor B which indicates that
factor A has significant effect.
Kinetics of Drug Release
The drug release data was fitted into the different model like
Korsmeyer Peppas , first order, zero order and Higuchi
2
eqation and shown very close and above 0.9 r values (Table 5).
It suggests that the release of drug from the formulations may
2
follow any one of these models.The r values of first order of
all the formulations shows higher which indicate the drug
release is directly proportional to the amount of drug
remaining. But n values range from 0.484 to 0.673 which
indicate non-Fickian diffusion mechanism. According to
Higuchi model, the drug release from matrix is directly
proportional to square root of time and explains the Fickian
diffusion. It may be coincident. However, n values of
Korsmeyer-Peppas strongly indicates that diffusion
mechanism is non-Fickian.
ANOVA, Pure Error, Lack of Fit
The result of ANOVA demonstrate that the model was
singnificant for all dependent variables (Table 6). Regression
analysis was carried out to determine the regression
coefficients. All the independent variables ( Factors) were
found to be significant for all R1, R2, R3 and R4 response
variables. The quadratic model was found to be significant for
R1. The linear model was found to be significant for R3 and
R4. The 2FI model was found to be significant for R2. So,
above result indicate that both the factors play an important
role in the formulation of buccal tablet containing metoprolol
tartrate. The data of pure error and lack of fit are
demonstrated in (Table 6), which can provide a mean
response and an estimate of pure experimental uncertainty.
The residuals are the difference between observed and
predicted value.
Fig 11. DTA thermograms of (A) Metoprolol tartrate
(B) HPC (C) Carbopol (D) Drug + HPC (E) Drug + Carbopol.
2
Table 5.Correlation coefficient (R ) of different models, drug release exponents(n), zero order release rate
constants(K 0 ), First order release rate constant(K), Korsmeyer Peppas release constant(K KP)
Kinetic profile of Korsmeyer Peppas Zero order First order Higuchi
formulation n K KP
2
R K 0
2
R K
2
R
2
R
F-1 0.554 2.0792 0.969 0.123 0.989 -0.0021 0.989 0.980
F-2 0.563 1.963 0.985 0.119 0.985 -0.00207 0.998 0.989
F-3 0.488 3.186 0.995 0.112 0.979 -0.00204 0.997 0.996
F-4 0.580 1.760 0.993 0.117 0.981 -0.00201 0.999 0.992
F-5 0.543 2.630 0.964 0.158 0.995 -0.00386 0.923 0.953
F-6 0.485 3.144 0.992 0.110 0.987 -0.00199 0.990 0.990
F-7 0.559 2.0393 0.995 0.118 0.987 -0.00209 0.994 0.991
F-8 0.673 0.888 0.994 0.112 0.996 -0.00176 0.991 0.980
F-9 0.556 1.879 0.9973 0.118 0.984 -0.00203 0.996 0.984
F-10 0.661 1.083 0.998 0.122 0.987 -0.00206 0.996 0.992
F-11 0.506 3.123 0.960 0.150 0.997 -0.00354 0.942 0.959
F-12 0.664 1.038 0.981 0.129 0.994 -0.00221 0.984 0.972
F-13 0.576 1.763 0.973 0.119 0.984 -0.00203 0.996 0.984
153
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Prakash Rao B et al./ Formulation and Evaluation of Mucoadhesive Buccal Drug Delivery System of Metoprolol Tartrate by Using Central Composite Design
Optimization
Table 6: Summary of ANOVA results in analysing lack of fit and pure error
Source Sum square d.f. Mean square F value Probability > F
MT release at 8 hour
Model 698.29 5 139.66 10.79

Prakash Rao B et al./ Formulation and Evaluation of Mucoadhesive Buccal Drug Delivery System of Metoprolol Tartrate by Using Central Composite Design
Table 8: FT-IR spectrum of MT alone and excipients
Interpretation IR absorption IR absorption band of metoprolol
band of Pure
Metoprolol Drug + Drug + HPC
Tartrate(cm -1 )
-1
Carbopol (cm )
-1
(cm )
-NH 2 ,-OH,aliphatic 3610.79 – 2360.95 3608.93 – 2359.89 3611.83 – 2356.13
and aromatic CH
Carboxylic acid salt 1573.97 1580.14 1580.57
Aromatic ring 1514.17 1515.14 1513.21
Isopropyl group 1179.51 1180.47 1174.69
Aliphatic ether, 1112.00 1112.00 1112.00 1,4
secondary alcohol
di-substituted Benzene 824.24 826.53 824.60
Table 9: Result of stability studies according to ICH guidelines
Parameter 0 Days 6 months
Physical apperence White White
Drug content 49.06 49.05
Bioadhesive strength 43.52 43.12
physical changes of the substance are recorded as a function
of temperature or time as substance is heated at a liner rate.
The DTA of the pure drug shows two endothermic peaks(Fig
0
11). The peak at 123.19 C indicates its melting point and the
0
other peak at 223.12 C may be the degredation peak. In the
DTA studies there was no shift in the melting peak. So, the
selected excipients for the formulation were found to be
compatible with the active ingredients and having no physical
interaction with the active pharmaceutical ingredient.
Stabality Studies
The optimized formula was evaluated for physical
appearance, drug content and bioadhesive strength for every
month. The values given were at end of the six months. It
was found that optimized formula was stable.
CONCLUSION
The CCD was used to find out the effect of independent
varibles on the dependable variables. The result of CCD
revealed that the carbopol 934P and HPC have significant
effect on the drug release at 8 hour, bioadhesive strength,
T50% and release exponent(n). The observed independent
variables were found to be very close to predicted values of
optimized formulation which demonstrates the feasibility of
the optimization procedure in successful development of
buccal tablets containing MT by using carbopol 934P and
HPC.
155
REFERENCES
1. Shojaei AH. Buccal mucosa as a route for systemic drug delivery: A
review. J Pharm Sci 1998;1:15-30.
2. Patel VM, Prajapati BG, Patel MM. Formulation, evaluation and
composition of bilayer and multilayered mucoadhesive buccal device
of Propranolol hydrochloride. AAPS Pharm Sci Tech 2006;8:1-15.
3. Giunchedi P, Juliano C, Gavini E. Formulation and In-vivo evaluation
of Chlorhexidine buccal tablets prepared using a drug loaded
chitosan microspheres. Eur J Pharm Biopharm 2002; 53: 233-9.
4. Perioli L, Ambrogi V, Giovagnoli S, Ricci M, Blasi P, Rossi C.
Mucoadhesive bilayered tablets for buccal sustained release
Flurbiprofen. AAPS Pharm Sci Tech 2007; 8:1-9.
5. Desai KGH, Pramod kumar TM. Preparation and evaluation of a novel
buccal adhesive system. AAPS Pharm Sci Tech 2004; 5:1-9.
6. Munasur AP, Pillay V, Chetty DJ, Govender T. Statistical optimization
of the mucoadhesivity and characterisation of multipolymeric
Propranolol matrices for buccal therapy. Int J Pharm 2006; 323:43-51.
7. Yong CS, Jung JH, Rhee JP, Kim CK, Choi HG. Physicochemical
characterization and evaluation of buccal adhesive tablets containing
Omeprazole. Drug Dev Ind Pharm 2001;27: 447-55.
8. Yan MO, Choi HG, Jung JK, Kim CK. Development of
thermoreversible Insulin liquide suppository with sodium salicylate.
Int J Pharm 1999; 189:137-45.
9. Yamsani VV, Gannu R, Kolli C, Rao ME, Yamsani MR. Development
and In-vitro evaluation of buccoadhesive Carvedilol tablets. Acta
Pharm 2007;57:185-97.
10. Khanna R, Agrawal SP, Ahuja A. Preparation and evaluation of
bioerodible buccal tablets containing Clotrimazole. Int J Pharm 1996;
138:67-73.
11. Owens TS, Dansereau RJ, Sakr A. Development and evaluation of
extended release bioadhesive Sodium fluoride tablets. Int J Pharm
2005;288:109-22.
12.
th
Tripathi KD. Essentials of medical pharmacology. 5 ed. New Delhi:
Jaypee publishers. 2003;12913. Singh B, Chakkal SK,
Ahuja N. Formulation and optimization of controlled release
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Prakash Rao B et al./ Formulation and Evaluation of Mucoadhesive Buccal Drug Delivery System of Metoprolol Tartrate by Using Central Composite Design
mucoadhesive tablets of Atenolol using response surface
methodology. AAPS Pharm Sci Tech 2005;7:1-9.14.
http://www.itl.nist.gov/div898/ handbook/ pri/section3/pri3361.htm
15. Saikat P, Marina K, Jolly R.P., Ajay B.S., Gaurav H.S., Rahul T.
Buccoadhesive Tablets of losartan: Design and Characterisation. Int.
J. Pharma. Bio. Archive 2010:1;150-4.
16. Marget C., Sachin, Debjit D.,Jayakar B. Formulation and eavaluation
of Mucoadhessive oral tablet of Clorithromycin. T. Pharm. Res.
2009:2;30-42.
17. Emami J, Varshosaz J, Saljioughian N. Development and evaluation
of controlled release buccoadhesive Verapamil hydrochloride. Daru
2008; 16: 60-9.
18. Hirlekar R S, Kadam V J. Design of buccal drug delivery system for a
poorly soluble drug. Asin. J. Pharm. Clinic. Reas., 2009:2;49-53.
19. Park CR, Munday DL. Development and evaluation of a biphasic
buccal adhesive tablet for Nicotine replacement therapy. Int J Pharm
2002; 237: 215-26.
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20. http://www.pharmainfo.net/pharmacy-student-articles/carbopol-andits-applications-pharmaceutical-dosage-forms.
21. Salamone, C. J. Polymeric materials encyclopedia. New York, USA:
CRC press 1996.
22. Patel, V. M., Prajapati, B. G., & Patel, M. M.Formulation, evaluation
and composition of bilayer and multilayered mucoadhesive buccal
device of propranolol hydrochloride. AAPS PharmSciTech., 2006:8;1-
15.
23. Liabot, J. M., Manzo, R. H., & Allemandi, D. A. Double layered
mucoadhesive tablets containing nystain. AAPS. Pharm Sci Tech.,
2002:3;1-5.
Address for Correspondence
Dr. B. Prakash Rao, M. Pharm, Ph. D, Professor and HOD, Department of
Pharmaceutical Technology, Karnataka College of Pharmacy 33/2,
Thirumenahally, Hegde Nagar Main Road, Bangalore-560064
E-mail: bprao_1111@rediffmail.com
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

A B S T R A C T
RGUHS Journal of Pharmaceutical Sciences
Development and Evaluation of Mucoadhesive Buccal Films of Nebivolol
Bushetti S.S*, Mane Prashant P and Kardame S.S
HKES's College of Pharmacy, M R Medical College Campus, Sedam Road, Gulbarga- 585105
INTRODUCTION
The buccal mucosa, along with other mucosal tissues, has
been investigated as a potential site for controlled delivery of
macromolecular therapeutic agents, such as peptides,
proteins and polysaccharides because of its accessibility and
low enzymatic activity compared to the gastro-intestinal tract.
Another interesting advantage is its tolerance (in comparison
1
with the nasal mucosa and skin) to potential sensitizers.
The potential of the buccal mucosa as an alternative site for
the delivery of drugs into the systemic circulation has recently
received much attention. There are many reasons why the
buccal mucosa might be an attractive site for the delivery of
therapeutic agents into the systemic circulation. Due to the
direct drainage of blood from the buccal epithelium into the
internal jugular vein, the first-pass metabolism in the liver and
intestine may be avoided. This first-pass effect is a major
reason for the poor bioavailability of some compounds when
administered orally. Additionally, the mucosa lining the oral
cavity is easily accessible, which ensures that a dosage form
can be applied to the required site and can be removed easily
2
in case of emergency.
The oral cavity is a moist environment; the membranes that
line the oral cavity are covered with mucus which is derived
mainly from minor salivary glands and are constantly bathed
in saliva, an aqueous substance rich in inorganic salts, proteins
and bacteria. Saliva has a variety of functions and is
RGUHS Journal of Pharmaceutical Sciences
Received: 28/1/2011, Modified: 21/2/2011, Accepted: 3/3/2011
Original Research Article
Nebivolol a third-generation cardio selective β1-blocker that is approved for the treatment of hypertension. Nebivolol undergoes
extensive metabolism in the liver after its oral administration and resulting in to a very poor (approximately 10-12%) bioavailability. Oral
administration of nebivolol can also cause gastrointestinal disturbance and abdominal or stomach pain etc. In order to improve the
bioavailability, efficacy and to minimize the side effects associated with oral administration, mucoadhesive buccal films of nebivolol using
hydroxy propyl methyl cellulose and methyl cellulose were prepared by solvent casting technique. The films of nebivolol using hydroxy
propyl methyl cellulose and methyl cellulose were smooth, elegant and uniform in thickness and weight. Among the two polymers used
hydroxyl propyl methyl cellulose showed an increased in-vitro residence time due to mucoadhesion nature of the hydroxyl propyl methyl
cellulose. Drug content uniformity study showed uniform dispersion of the drug throughout the film in the range of 96.208±1.0705 to
th
98.887±0.2558 %. In- vitro drug release study showed that more than 90% of drug was released at the end of 8 hr. The release profile of
all the formulations was subjected to various kinetic equations and the results suggested that the drug was released by diffusion
mechanism following super case-II transport.
Keywords: Nebivolol, Buccal films, Mucoadhesion, In vitro drug release and Diffusion.
157
continuously secreted into, distributed around and removed
3
from the oral cavity .
Based on the current understanding of biochemical and
physiological aspects of absorption and metabolism of
biotechnologically- produced drugs, they cannot be delivered
effectively through the conventional oral route. Because after
oral administration many drugs are subjected to presystemic
clearance extensive in liver, which often leads to a lack of
significant correlation between membrane permeability,
absorption, and bioavailability. Difficulties associated with
parenteral delivery and poor oral availability provided the
impetus for exploring alternative routes for the delivery of
4
such drugs.
Nebivolol is a third-generation β1selective β-blocker used in
5
the treatment of hypertension , it works by relaxing blood
vessels and slowing heart rate to improve blood flow and
6
decrease blood pressure . Nebivolol on oral administration
undergoes extensive metabolism in the liver resulting into very
7
poor (approximately 10- 12%) bioavailability . It can also
cause gastrointestinal disturbance and abdominal or stomach
pain, etc. In order to improve its bioavailability, efficacy and to
minimize the side effects associated with oral administration,
mucoadhesive buccal films of nebivolol using hydroxy propyl
methyl cellulose and methyl cellulose were prepared by
solvent casting technique in the present investigation.
MATERIALS AND METHODS
Nebivolol was a gift sample from Ajanta Pharma Ltd.,
Mumbai. HPMC (G-236007), and MC (040801) were
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

procured from SD Fine Chem., Mumbai and all other
chemicals used were of analytical grade.
Preparation of Nebivolol Buccal Films
Nebivolol mucoadhesive buccal films were prepared using
hydroxy propyl methyl cellulose (HPMC) and methyl cellulose
8
(MC) by solvent casting technique .
Accurately weighed quantity of hydroxy propyl methyl
cellulose was soaked ethanol (chloroform and ethanol in the
ratio of 75:25 % v/v for methyl cellulose) for 24 hrs, the
calculated amount of nebivolol was dissolved in the polymeric
solution and propylene glycol was added gradually with
continuous stirring. 5 ml resultant mixture was poured into
each fabricated glass ring placed on aluminum foil in a petri
dish, and then the petri dish was kept aside for drying at room
temperature for 24 hours. The dried polymeric films were cut
into circular films of 10 mm diameter for further evaluation.
Evaluation of Nebivolol Mucoadhesive Buccal
Films
9
Weight Uniformity
A film of 10mm diameter was weighed using Shimadzu
digital balance and the average weight was calculated (n=3).
10
Thickness Uniformity
The thickness of the film was measured using screw gauge
with a least count of 0.01mm at three different spots of the
film and the average thickness was calculated.
11
Folding Endurance
The flexibility of film can be measured quantitatively in terms
of folding endurance. The folding endurance of the film was
determined by repeatedly folding a small strip of the films at
the same place till it broke. The number of times films could
be folded at the same place without breaking indicated the
value of folding endurance and the procedure was repeated
for three times.
12
Swelling Index
A buccal film of 10 mm diameter was weighed on a preweighed
cover slip, the initial weight of the film was recorded
(W ) and then it was kept in a petri dish containing 5 ml of
0
phosphate buffer pH 6.8. The cover slip was removed at time
interval of 0.5, 1, 2, 3, 4, 5, 6, 7, 8 hr, and excess of water was
carefully removed and swollen film was re-weighed (W ). The
t
percentage swelling (%S) was calculated by following formula:
W -W
t o
%S = --------------------- x 100
W o
The mean %S was calculated (n=3).
Bushetti S.S et al./ Development and Evaluation of Mucoadhesive Buccal Films of Nebivolol
158
13
Surface pH
The film was allowed to come in contact with 1ml of
phosphate buffer pH 6.8 for 1-3 min. The surface pH was
measured using pen pH meter (n=3).
Drug Content Uniformity
This study was carried out to know the complete and uniform
dispersion of the drug throughout the film. The film of 10 mm
diameter was dissolved in methanol and the absorbance of the
14
solution (after suitable dilution) was measured at 282 nm
using UV/visible spectrophotometer (Shimadzu UV-1700).
The percentage drug content was calculated with the help of
calibration curve (n=3).
In- vitro Drug Release
The drug release from buccal film was studied by standard
cylindrical tube method using sigma dialysis membrane. The
membrane was tied to one end of open cylinder and is acted as
donor compartment, the buccal film was placed inside this
compartment and it was in contact with the receptor
compartment containing 100 ml of phosphate buffer pH 6.8.
The diffusion medium was stirred continuously using
magnetic stirrer and the temperature was maintained at
37±0.5ºC. 5 ml sample was withdrawn from the receptor
compartment at periodic intervals and the same was replaced
by equal volume of fresh buffer solution. The samples were
analyzed for drug content spectrophotometrically at 282 nm.
The amount of drug released was calculated with the help of
standard calibration curve and cumulative percentage drug
release was calculated. In-vitro release data were subjected to
release kinetic equations and were plotted for various graphs.
15
Ex vivo Mucoadhesive Strength
Sheep buccal mucosa was obtained from a local
slaughterhouse; the mucosal membrane was separated by
removing the underlining fat and loose tissues. The
membrane was washed with distilled water and subsequently
with isotonic phosphate buffer (IPB) solution of 6.8 pH at
0
37 C. The bioadhesive strength of the film was measured on
modified physical balance.
15
In vitro Residence Time
The in-vitro residence time was determined using a locally
modified USP disintegration test apparatus. A segment of
sheep buccal mucosa of 3 cm long was glued to the surface of
a glass slab, vertically attached to the apparatus. The
mucoadhesive films were hydrated from one surface using 15
µl IPB and then the hydrated surface was brought into contact
with the mucosal membrane. The glass slab was vertically
fixed to the apparatus and allowed to move up and down in
0
800 ml IPB maintained at 37 C, so that the film was
completely immersed in the buffer solution at the lowest point
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

Fig. 1: Mucoadhesive strength of patches for batch BFN1-BFN7 Fig. 4: First order plots of nebivolol buccal films
prepared using HPMC 2% (BFN1), 4% (BFN2), 6% (BFN3),
8% (BFN4) and MC 2% (BFN5), 3% (BFN6) and 4% (BFN7).
Fig. 2: In vitro residence time profile for BFN1-BFN7
Fig. 3: In vitro drug release from nebivolol buccal films
prepared using HPMC 2% (BFN1), 4% (BFN2), 6% (BFN3),
8% (BFN4) and MC 2% (BFN5), 3% (BFN6) and 4% (BFN7).
Bushetti S.S et al./ Development and Evaluation of Mucoadhesive Buccal Films of Nebivolol
159
Fig.5: Highuchi diffusion plots for nebivolol buccal films
prepared using HPMC 2% (BFN1), 4% (BFN2), 6% (BFN3),
8% (BFN4) and MC 2% (BFN5), 3% (BFN6) and 4% (BFN7).
Fig. 6: Peppas exponential plots for nebivolol buccal films
prepared using HPMC 2% (BFN1), 4% (BFN2), 6% (BFN3),
8% (BFN4) and MC 2% (BFN5), 3% (BFN6) and 4% (BFN7).
Ingredients Formulation Code
BFN BFN BFN BFN BFN BFN BFN
1 2 3 4 5 6 7
HPMC (%w/v) 2 4 6 8 - - -
MC (%w/v) - - - - 2 3 4
Propylene Glycol* (%w/w) 30 30 30 30 30 30 30
Table 1 : Formulation of Nebivolol Buccal films
* Percentage of polymer weight Each patch contains 5 mg of nebivolol
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Bushetti S.S et al./ Development and Evaluation of Mucoadhesive Buccal Films of Nebivolol
Table 2: Evaluation of nebivolol buccal films for thickness, weight variation, folding endurance, swelling index, surface pH,
mucoadhesive strength, in vitro residence time and percent drug content uniformity.
F. Weight Thickness Folding Swelling Surface pH Mucoadhesive In Vitro Drug Content
Code (mg) ±SD (mm)± SD Endurance Index (%) ±SD strength (gm) Residence Uniformity
± SD ± SD time (Hrs) ± SD (%) ± SD
BFN 1 6.333± 0.577 0.072± 0.0098 412±2.516 35.18 ± 2.188 6.82±0.105 3.100±0.1 2.10±0.050 97.176± 1.122
BFN 2 10.666± 1.527 0.10± 0.0152 367±2.516 40.956 ± 0.871 6.78±0.155 3.766±0.115 2.27±0.025 98.661± 0.127
BFN 3 14.000± 1 0.156± 0.0115 338±3.605 45.964 ± 2.406 6.94±0.227 4.333±0.115 3.23±0.036 96.393± 1.465
BFN 4 16.333± 1.154 0.223± 0.0152 298±3.605 66.359 ± 1.547 6.60±0.438 4.766±0.057 3.9±0.309 98.057± 0.204
BFN 5 6.333± 0.577 0.076± 0.0057 394±4.582 25.291 ± 1.866 6.67±0.375 4.300±0.1 4.12±0.502 98.887± 0.255
BFN 6 6.666± 0.577 0.103± 0.0057 377±2.516 30.764 ± 0.941 6.22±0.420 5.633±0.057 5.35±0.133 96.208± 1.070
BFN 7 10.333± 0.577 0.13± 0.0152 359±5.033 35.616 ± 2.808 6.56±0.526 6.260±0.115 5.52±0.072 97.494± 0.656
160
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

Formulation
2
Regression coefficient (R ) values Peppas Slope
Code Zero order First order Highuchi Peppas Values (n)
BFN 0.995 0.854 0.885 0.790 1.594
and was out at highest point. The time taken for the complete
erosion or detachment of the films from the mucosal surface
was recorded (mean of triplicate determinations).
RESULTS AND DISCUSSION
1
Bushetti S.S et al./ Development and Evaluation of Mucoadhesive Buccal Films of Nebivolol
Table 3: Drug release Kinetics of Nebivolol Buccal films
BFN 0.991 0.920 0.893 0.833 1.663
2
BFN 0.988 0.967 0.900 0.838 1.653
3
BFN 0.995 0.964 0.892 0.846 1.620
4
BFN 0.993 0.962 0.884 0.857 1.625
5
BFN 0.990 0.961 0.866 0.883 1.626
6
BFN 0.989 0.962 0.862 0.884 1.590
7
In the present research work, mucoadhesive buccal films of
nebivolol (Table 1) were prepared using HPMC and MC by
solvent casting technique to increase the efficacy of drug by
improving its bioavailability. The prepared films were
evaluated for various parameters.
The thickness of the film prepared measured in the range of
0.072 to 0.23 mm, the results (Table 2) suggested that the films
were thin enough and they did not cause any inconvenience
after their application into the buccal cavity. The surface pH
of the film was in the range of 6.22 to 6.94, the pH of films
was nearer to the salivary pH, hence any irritation was not
observed to the mucus membrane of the buccal cavity.
The films were also evaluated for folding endurance and
mucoadhesive strength (Fig. 1), the higher values of these
parameters indicated that the films were flexible enough and
they were not detached easily. These helped in retaining the
films for longer period of time at the site of application and it
was well supported by longer in-vitro residence time values
(Fig. 2). The buccal films were also evaluated for drug content
uniformity test; the results showed that the drug was
uniformly dispersed in the range of 96.208 to 98.887 %.
Finally, the films were evaluated for drug release kinetics for a
period of 8 hours, the release profiles were subjected to
various kinetic equations like Higuchi diffusion equation (Q =
1/2 n
Kt ) and Peppas exponential equation (Q = Kt ) to ascertain
the drug release mechanism (Table 3). In both the cases, the
plots (Fig. 3-6) were found to be fairly linear and the linearity
was well supported by higher regression coefficient values ('r'
values were nearer to one) and slope values of the Peppas
equation are more than one (>1) in all the cases which
suggested that the drug was released by diffusion mechanism
following super case-II transport.
161
CONCLUSION
In the present research work, nebivolol mucoadhesive buccal
film were prepared using varying concentration of HPMC
and MC by solvent casting technique with an objective of
improved bioavailability.
All the formulations possessed the good mucoadhesion, and
they were free from irritation and released the drug
completely by diffusion mechanism following super case –II
transport.
ACKNOWLEDGEMENT
Authors are thankful to Ajanta Pharma Ltd., Mumbai for
providing gift samples of nebivolol. Authors are also thankful
to the Principal of HKES's college of Pharmacy, Gulbarga for
providing lab facilities to carryout the research work.
REFERENCES
1. Hans EJ, Janet AH, CoosJV. Recent advances in buccal drug delivery
and absorption - in vitro and in vivo studies. J. Controlled Rel 1999;62:
149-59.
2. Joseph AN, Barry LR, Barrie CF. Buccal penetration enhancers-How
do they really work? J Controlled Rel 2005:105:1 – 15.
3. Michael JR, Bernadette KD, Ian GT. The oral cavity as a site for
systemic drug delivery. Advanced Drug Del Rev 1994;13:1-22.
4. Yajaman S, Ketousetuo K, Bandyopadhyay AK. Buccal bioadhesive
drug delivery - A promising option for orally less efficient drugs. J
Controlled Rel 2006;114:15-40.
5. Judy WMC. Nebivolol: A third-generation β-blocker for hypertension.
Cli Therapeutics 2009;31(3):447-62.
6. http://www.drug.com/nebivolol:medline plus druginformation.html.
th
7. Sweetman SC. Martindale: The Complete Drug Reference. 35 ed.
Pharmaceutical press: Londen; 2007;1211.
8. Raghuraman S, Velrajan G, Ravi R, Geyabalan B, Johnson DB,
Sankar V. Design and evaluation of Propranolol Hydrochloride buccal
films. Indian J Pharm Sci 2002; 64 (1): 32-36.
9. Rasool BKA, Khan S. In vitro evaluation of miconazole mucoadhesive
buccal films. Int J Applied Pharm 2010; 2(4):23-26.
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Bushetti S.S et al./ Development and Evaluation of Mucoadhesive Buccal Films of Nebivolol
10. Singh S, Gangwar S, Garg G, Garg V, Sharma PK. Formulation and
evaluation of rapidly disintegrating film of Levocetrizine
Hydrochloride. Der Pharmacia Letter 2010; 2(2): 434-9.
11. Koland M, Sandeep VP, Charyulu NR. Fast dissolving sublingual films
of Ondansetron Hydrochloride: Effect of additives on in vitro drug
release and mucosal permeation. J Young Pharm 2010;2(3):216-22.
12. Noha AN, Fatma AI, Nabila AB, Lobna MM. Mucoadhesive buccal
patches of miconazole nitrate: in vitro/in vivo performance and effect
of ageing. Int J Pharm 2003; 264:1–14.
13. Vinod R, Ashok KP, Rao SB, Kulkarni SV, Shankar MS. Design and
evaluation of miconazole nitrate buccal mucoadhesive patches. J
Pharm Res 2010;3(6):1338-41.
162
14. Mishra P, Shah K, Gupta A. Spectrophotometric methods for
simultaneous estimation of nebivolol hydrochloride and amlodipine
besylate in tablets. Int J Pharm and Pharmaceutical Sci 2009;1(2):55-
61.
15. Giradkar KP, Channawar MA, Kajale AD, Sridhar E, Kamble RS,
Bakde BV et al. Design, development and in vitro evaluation of
bioadhesive dosage form for buccal route. Int J Pharm Res Dev 2010;
2(6):1-20.
Address for Correspondence
Bushetti S.S, HKES's College of Pharmacy, M R Medical College Campus,
Sedam Road, Gulbarga- 585105
E-mail: sharansb69@rediffmail.com
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

A B S T R A C T
RGUHS Journal of Pharmaceutical Sciences
Chitosan Loaded Mucoadhesive Microspheres of Gliclazide: In vitro and In vivo
Evaluation
Senthil A*, Thakkar Hardik R, Ravikumar and Narayanaswamy V.B
Department of pharmaceutics, Karavali College of Pharmacy, Mangalore, Karnataka.
The objective of the present investigation was to design chitosan loaded mucoadhesive microspheres of gliclazide: in vitro and in vivo
evaluation. Type 2 diabetes mellitus is a heterogeneous disease of polygenic origin and involves both defective insulin secretion and
peripheral insulin resistance. Despite the availability of new agents for treatment of type 2 diabetes mellitus, oral sulfonylureas remain a
cornerstone of therapy, because they are relatively inexpensive and are well tolerated. Since, the site of absorption of gliclazide is from
stomach thus dosage forms that are retained in stomach by mucoadhesion; would increase absorption, improve drug efficiency and
decrease dose requirements. Microspheres were prepared by simple emulsification phase separation technique. On the basis of the
2
preliminary trials 3 full factorial designs were employed, to study the effect of independent variable X polymer-to-drug and the stirring
1
speed X on dependent variables percentage mucoadhesion, drug entrapment efficiency and particle size. The optimized formulation
2
exhibited a high drug entrapment efficiency of 60%, swelling index 0.42, Percentage of mucoadhesive after 1 hour 62% and the drug
release was also sustained for more than 10 hours. In vivo testing of the mucoadhesive microspheres to albino Wistar rats demonstrated
significant hypoglycemic effect of gliclazide.
Keywords: Mucoadhesive, Gliclazide, Chitosan, Glutaraldehyde.
INTRODUCTION
A primary object of using mucoadhesive formulations orally
would be to achieve a substantial increase in length of stay of
the drug in the GI tract. Stability problem in the intestinal
fluid can be overcome. Therapeutic effect of drugs insoluble
in the intestinal fluids can be improved. Recently, dosage
forms that can precisely control the release rates and target
drugs to a specific body site have made an enormous impact in
the formulation and development of novel drug delivery
1-3
systems . Microspheres form an important part of such novel
drug delivery systems. They have carried applications and are
1
prepared using assorted polymers . However, the success of
these microspheres is limited owing to their short residence
5
time at the site of absorption . It would therefore be
advantageous to have means for providing an intimate contact
6-9
of the drug delivery system with the absorbing membranes .
Bioadhesive microspheres have advantages such as efficient
absorption and enhanced bioavailability of drugs owing to a
high surface-to-volume ratio a much more intimate contact
with the mucus layer and specific targeting of drugs to the
10-13
absorption site . Gliclazide, a second generation
sulphonylurea derivative and is preferred in therapy because
of its selective inhibitory activity towards pancreatic K+ ATP
RGUHS Journal of Pharmaceutical Sciences
Received: 15/7/2011, Modified: 1/8/2011, Accepted: 12/8/2011
163
Original Research Article
channels, antioxidant property, low incidence of producing
severe hypoglycemia and other haemobiological effects. The
daily dose, which is given in two fractions, is generally between
40 and 80 mg at the beginning of treatment, but the dose can
be increased also at severe conditions. Gliclazide is well
absorbed from GIT, approximately 80% is absorbed. One
dose of gliclazide has a half-life less than 10 hours with the
peak absorbance occurring at about 4-6 hours. Like most
sulphonylureas, gliclazide binds primarily to plasma albumin
(85-99%), allowing it to be distributed uniformly throughout
18-21
the body . Thus, an attempt was made in this investigation
to use chitosan as a natural mucoadhesive polymer and
prepare microspheres. The microspheres were characterized
by in vitro and in vivo tests, and factorial design was used to
optimize the variables.
MATERIALS AND METHOD
Gliclazide was obtained as gift sample from Aurobindo
pharmaceuticals, Hyderabad, India. Chitosan (Purified,
Viscosity grade 50) was obtained from Fourt's India Limited,
Chennai. Dioctyl sodium sulfosuccinate, Heavy and Light
liquid paraffin, Glutaraldehyde and Petroleum ether (80:20)
was procured from Will son Lab, Mumbai.
Preparation of Microspheres
Microspheres were prepared by simple emulsification phase
separation technique. The different volume of cross-linking
agent glutaraldehyde was used as per method described in
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

14
Thanoo et al . Twenty preliminary trial batches (F1 to F20)
were prepared, 1.5 gm of Polymer was dissolved in 150 mL of
1% v/v aqueous acetic acid solution and 500 mg of drug was
dispersed in the polymer solution. The resulting mixture was
extruded through a syringe (No. 20) in 1 L of liquid paraffin
heavy and light (1:1 ratio) containing 0.2% dioctyl sodium
sulfosuccinate. The stirring was performed using a propeller
stirrer 1000 rpm at constant for all the batches. After 15
minutes, glutaraldehyde was added and stirring was
continued. The amount of glutaraldehyde and cross linking
time was found varying for all the batches. On the basis of the
2
preliminary trials 3 full factorial designs were employed. The
polymer-to-drug ratio (1:1, 3:1 and 6:1) and stirring speed
(500, 1000 and 1500 rpm) were varied in all the nine factorial
design batches. All other variables were used as mentioned in
preliminary trial batches. Microspheres thus obtained were
filtered and washed several time with petroleum ether (80:20)
to remove traces of oil. They were finally washed with water to
remove excess of glutaraldehyde. The microspheres were
then dried at 25°C and 60% RH for 24 hours.
Evaluation of Microspheres
Drug Content
According to literature review the assay for gliclazide was
estimated by uv spectrophotometric method. Aqueous
solution of drug was prepared in phosphate buffer (pH 6.8)
and absorbance was measured on uv spectrophotometer at
229 nm the method is validated for linearity, accuracy and
22
precision . The method obeys Beer's law in the concentration
range of 5 to 50 mµg/mL, a standard drug solution was
analyzed repeatedly, the accuracy and precision were
determined.
Drug Entrapment Efficiency
50 mg of microspheres were crushed in a glass mortar and
pestle, and the powdered microspheres was suspend in 10 mL
of phosphate buffer solution (pH 6.8). After 24 hours, the
solution filtered and the filtrate was analyzed for the drug
content. The drug entrapment efficiency was calculated using
the following formula; Practical drug content / Theoretical
drug content x 100.
Surface Morphology
Surface morphology of the microspheres was determined by
using scanning electron photomicrograph.
Particle Size
A Senthil et al./ Chitosan Loaded Mucoadhesive Microspheres of Gliclazide: In vtro and In vivo Evaluation
The particle size of the microspheres was determined by using
23
optical microscopy method .
164
Swelling Index
The swelling ability of microspheres in physiological media
was determined by optical microscopy method. The 100
microspheres were suspended in 5 mL of stimulated gastric
fluid USP (pH 1.2). The particle size was monitored by
microscopy technique every 1 hour up to 8 hours using an
25
optical microscope .
In vitro Wash-off Test for Microspheres
A rat stomach mucosa was tied onto a glass slide (3 inch by 1
inch) using thread. Microspheres were spread onto the wet
rinsed tissue specimen, and the prepared slide was hung onto
one of the groves of a USP tablet disintegrating test apparatus
containing the simulated gastric fluid (pH 1.2). The
disintegrating test apparatus were operated such that the
tissue specimen was given regular up and down movements.
At the end of 30 minutes, 1 hour, and at hourly intervals up to
10 hours, the number of microspheres still adhering onto the
26
tissue was counted .
Drug release Study
The drug release study will perform using USP XXIV basket
apparatus at 37°C±0.5°C and 100 rpm using 900 mL of
phosphate buffer (pH 6.8) as dissolution medium.
Microspheres equivalent to 10 mg of gliclazide were used for
the test. Sample 5 mL was withdrawn at predetermined time
intervals and filtered through a 0.45 micron membrane filter,
diluted suitably and analyzed. Percentage drug dissolved at
different time intervals was calculated using the Lamberts-
27
Beer's law equation .
Release Kinetics and Mechanism
To know the release mechanism and kinetics of gliclazide,
optimized formulation was attempted to fit in to
2
mathematical models and n, r values for zero order, first
order, higuchi and peppas models.
Factorial Design
A statistical model incorporating interactive and polynomial
terms was utilized to evaluate the responses.
2 2
Y=b 0+b1X 1+b2X 2+b12X1X 2+b11X 1 +b22X2 Where, Y is the dependent variable, b 0 is the arithmetic mean
response of the nine runs, and b 1 is the estimated coefficient
for the factor X 1. The main effects (X 1 and X 2)
represent the
average result of changing one factor at a time from its low to
high value. The interaction terms (X 1 X 2)
show how the
response changes when two factors are simultaneously
2 2
changed. The polynomial terms (X 1 and X 2 ) are included to
investigate non-linearity. On the basis of the preliminary trials
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

2
a 3 full factorial design was employed to study the effect of
independent variables i.e. polymer-to-drug ratio (X 1)
and the
stirring speed at rpm (X 2)
on dependent variables %
mucoadhesion, drug entrapment efficiency and particle size.
In-vivo Anti-diabetic Study
In-vivo evaluation studies for gliclazide mucoadhesive
microspheres were performed on normal healthy wistar rats
weighing 250 to 300 g each. The approval of the Institutional
Animal Ethics Committee was obtained before starting of the
study. Two groups of Wistar rats (5 in each group) that were
fasted with water at least 12 hours before the experiments
were used for the study. Before drug administration, a blood
sample as a control was taken for each rat from behind the
eyeball through the angle of ocular cavity using small
capillary tubes. The blood glucose level for the control and test
sample was determined using the glucose measuring
instrument. The instrument was self calibrated, and the
samples were allowed to dry before the results were read to
avoid contamination of the lens. Pure gliclazide and
mucoadhesive microspheres of gliclazide were administered
orally to each group using stomach intubations. A dose of 800
g/kg of gliclazide was administered in suspension form for
each rat. Blood samples were collected at predetermined time
at 1 hour intervals up to 24 hours, and the blood glucose level
was performed as per method described earlier. The
31-32
percentage reduction in blood glucose level was measured .
Stability Testing
Optimized formulations of microspheres were tested for
stability studies. Both the formulations were divided into 3
sample sets and stored at 4±1°C, 25±2°C and 60±5% RH
and 37±2°C and 65±5% RH. After 30 days, in vitro drug
release studies and percentage entrapment efficiency were
determined.
RESULT AND DISCUSSION
A Senthil et al./ Chitosan Loaded Mucoadhesive Microspheres of Gliclazide: In vtro and In vivo Evaluation
The gliclazide mucoadhesive microspheres were prepared by
simple emulsification phase separation technique using
chitosan as natural polymer. Acetic acid from 1% to 6% v/v
was used to prepare polymer solution. But there was no effect
in concentration of acetic acid was observed on percentage
mucoadhesion or drug entrapment efficiency, therefore 1%
v/v of acetic acid was used. Based on viscosity of polymers
solution three different concentrations 0.5, 1 and 2% v/v were
selected for trial batches, from this 1% concentration showed
a maximum sphericity was observed so we select 1% w/v of
polymer in 1% v/v acetic acid was found to be the optimum
concentration and 1:1 heavy and light paraffin was used as
dispersion medium and 0.2% dioctyl sodium sulfosuccinate
165
surfactant to dispersion medium was found to be essential to
minimize aggregation of microspheres. Cross-linking agent
glutaraldehyde was selected due to its high rate of crosslinking
and increased in glutaraldehyde concentration caused
highly cross-linked spheres and become dense by hardening
process. The long term exposure to 100 ppb glutaraldehyde
vapour cause respiratory tract lesions including hyperplasia
of squamous epithelium therefore, it is important to remove
excess of glutaraldehyde from the microspheres to avoid any
toxic reactions. The chitosan microspheres are a useful tool to
improve the uptake of hydrophilic substance across epithelial
layer. The glutaraldehyde was deposited on the surface of
microspheres so easy removal of the unreacted free
33
glutaraldehyde as reported by Sahin et al .
Preliminary trail batches of microspheres were prepared by
using chitosan as polymers, the volume of cross-linking agent
10 to 70 mL and stirring speed were varied from 500, 1000
and 1500 rpm. From these batches F13, 60 mL of crosslinking
agent and 1 hour cross-linking time was the optimum
amount and time used for the preparation of mucoadhesive
microspheres. Increase in the cross-linking time (1 to 4 hours)
was inversely affected the percentage mucoadhesion. The
cross-linking chitosan mucoadhesive polymer probably
becomes more rigid and thus mucoadhesiveness decreases.
The cross-linking time did not have a significant effect on the
percentage drug entrapment efficiency were shown in (Table
1).
2
On the basis of the preliminary trials 3 full factorial design
were employed, to study the effect of independent variable X 1
(polymer-to-drug ratio 1:1, 3:1 and 6:1) and the stirring speed
X 2 (500, 1000 and 1500 rpm) on dependent variables
percentage mucoadhesion, drug entrapment efficiency and
particle size. The results depicted in (Table 2) clearly indicate
that all the dependent variables are strongly dependent on the
selected independent variable as they show a wide variation
among the nine batches.
Factorial Equation for Drug Entrapment Efficiency
and Particle Size
The drug entrapment efficiency was an important variable for
assessing the drug loading capacity of microspheres and their
drug release profile, thus suggesting the amount of drug
availability at site. The following polynomial equation was
derived by multiple regression analyses of the data.
2 2
Y = 69.11 + 10.01 X1 - 2.91 X2 - 0.484 X1 -7.03 X2 - 0.38 X1 X2
The drug entrapment efficiency of chitosan loaded
mucoadhesive microspheres varied from 49% to 54%, 66% to
72%, and 70% to 77% at lower, medium and higher levels of
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A Senthil et al./ Chitosan Loaded Mucoadhesive Microspheres of Gliclazide: In vtro and In vivo Evaluation
Table 1: Preliminary Trial Batches of Gliclazide Mucoadhesive Microspheres Loaded Chitosan
Batch Vol. of Cross- % Drug Sphericity of
code Glutaraldehyde linking Mucoadhesion Entrapment Microsphere
(mL) time(hr) after 1 hr. Efficiency (%)
F1 10 1 84 40 Very Irregular
F2 10 2 82 42
F3 10 3 79 44
F4 10 4 77 46
F5 20 1 86 52 Slightly Irregular
F6 20 2 78 55
F7 20 3 72 58
F8 20 4 68 60
F9 40 1 78 57 Spherical free flowing
F10 40 2 69 59
F11 40 3 64 60
F12 40 4 60 62
F13 60 1 78 60
F14 60 2 68 62
F15 60 3 64 64
F16 60 4 58 64
F17 70 1 59 64
F18 70 2 52 69
F19 70 3 45 70
F20 70 4 39 71
Note: All batches were prepared by polymer-to-drug ratio of 3:1 at 1000 rpm stirring speed
Table 2: Formulations of Chitosan Loaded Gliclazide Mucoadhesive Microspheres by 3 2 Full Factorial Design Layout
Variable levels in
% Drug Swelling Particle
Batch coded from
Mucoadhesion Entrapment Index Size
Code X 1 X 2
After1 hours Efficiency (%)
A1 -1 -1 52 54.25 0.888 60.6
A2 -1 0 46 52.68 0.824 58.2
A3 -1 1 43 49.12 0.812 50.2
A4 0 -1 78 72.00 1.182 67.1
A5 0 0 69 70.84 1.123 64.0
A6 0 1 62 66.96 1.082 60.8
A7 1 -1 80 77.12 1.412 98.0
A8 1 0 73 73.54 1.298 89.8
A9 1 1 67 70.67 1.242 74.4
Note: All batches were prepared using 60 mL of glutaraldehyde and cross-linking time of 1 hours
Translation of coded levels in actual units
Variables level Low (-1) Medium (0) High (+1)
Polymer-to-drug ratio (X 1 ) 1:1 3:1 6:1
Stirring speed (X 2 ) rpm 500 1000 1500
166
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

polymer-to-drug ratio respectively, have shown good
correlation coefficient 0.9984. Results of the equation
indicate that the effect of (X ) polymer-to-drug ratio is more
1
significant than (X ) stirring speed. However stirring speed has
2
a negative effect on drug entrapment efficiency, hence the
stirring speed increased, the particle size decreased, and the
drug entrapment efficiency has also decreased. The particle
size of the mucoadhesive microspheres of chitosan varied
from 50 to 98 µm and has shown good correlation coefficient
0.9878. However stirring speed has a negative effect on
particle size, thus the stirring speed increased, the particle
sizes decreased.
2
Y= 67.1 + 4.69 X1 - 4.47 X2 - 0.98 X2 - 0.98 X1 X2
Factorial equation for percentage mucoadhesion
and swelling index
The in vitro wash-off test for percentage mucoadhesion of
chitosan loaded mucoadhesive microspheres after 1 hour
varied from 43% to 52%, 62% to 78% and 67% to 80% at
lower, medium and higher levels of polymer-to-drug ratio and
has shown good correlation coefficient 0.9803. However
stirring speed has a negative effect on percentage
mucoadhesion. As the polymer-to-drug ratio increases, the
percentage mucoadhesion also increases; because more
amounts of polymer results in higher amount of free-COOH
groups, which are responsible for binding with sialic acid
groups in mucus membrane and thus results in increase in
mucoadhesive properties of microspheres. In vitro
mucoadhesive test has shown that gliclazide mucoadhesive
microspheres adhered more strongly to gastric mucous layer
and would retain in gastrointestinal tract for an extended
period of time were shown in (Fig.1).
2
Y= 77.81 + 15.78 X1 - 6.91 X2 - 9.98 X2 - 2.1X1 X2
A Senthil et al./ Chitosan Loaded Mucoadhesive Microspheres of Gliclazide: In vtro and In vivo Evaluation
The microspheres (100) were suspended in 5 mL of simulated
gastric fluid USP (pH 1.2). The particle size would be
monitored by microscopy technique every 1 hour using an
optical microscope. The increase in particle size of the
microspheres would be noted up to 8 hours. The swelling
index of chitosan varied from 0.812 to 1.412. Surface graphs
showing effect of variables on % mucoadhesion, drug
entrapment efficiency, swelling index and particle size for
optimized batch were shown in (Fig. 2). Chitosan could be
covalently cross-linked with glutaraldehyde through its amino
groups. The aldehyde groups of the glutaraldehyde formed
covalent imine bonds with the amino groups of chitosan, due
to the resonance established with the adjacent double
ethylenic bonds via a Schiff reaction. It is reflects that
polymeric chains during cross-linking procedure, the extent
of the swelling index depends on the cross-linking. Therefore,
167
Fig. 1: In vitro Wash-Off Test Carried out on Gliclazide
Loaded Chitosan Mucoadhesive Microspheres (batch A4),
using Rat Stomach
Figure 2: Surface Graphs Showing Effect of Variables on
(1) % Mucoadhesion (2) Drug Entrapment Efficiency
(3) Swelling Index (4) Particle Size for Optimized Batch (A4).
1 2
3 4
the denser the cross-linking bridges between the chitosan
molecules, the more packed is the structure. Such structure
can be characterized by lower and slower penetration of the
solvent through the chain structure of the polymer, suggesting
that the swelling ratio and hence the release characteristics of
the microspheres can be controlled by varying the content of
the cross-linking agent used during the manufacturing
process. Since glutaraldehyde is responsible for the formation
of cross-links, increasing the amount of glutaraldehyde and
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A Senthil et al./ Chitosan Loaded Mucoadhesive Microspheres of Gliclazide: In vtro and In vivo Evaluation
cross-linking time will increase the polymer density, resulting
in reduction of the macromolecular chain mobility, and
formation of more stable and rigid spheres that shows a lower
tendency to swell. The finding of this investigation is in
agreement with an earlier study performed by many group of
researchers report. The plots of cumulative percentage drug
release vs. time, cumulative percent drug retained vs. time, log
cumulative percent drug retained vs. time and cumulative
percent drug release in (mg) vs. time and result of curve fitting
of best batch were drawn and represented graphically. The
maximum incorporation efficiency was 78.73% for chitosan
and the in vitro drug release for eight hours was 87.86%.
Among these A4 batch has shown the good percentage of
mucoadhesion 78%, 87.86% of drug release for eight hours,
and 72% of drug entrapment efficiency while comparing with
all the polymers and they were spherical in shape and the drug
remained dispersed in the polymer matrix in amorphous
state. A4 batch seem promising candidates for achieving drug
release up to 10 hours were shown in (Table 3). The drug
release mechanism from the mucoadhesive microspheres was
found to be controlled release because plots of percentage
cumulative drug release vs. square root of time were found to
be linear with the regression coefficient (r). The release profile
fitted to higuchi-matrix equation, 'r' correlation coefficient
value was found to be 0.9440 for the best batch (A4). The
release profile fitted to korsmeyer-peppas equation, the 'r'
value was found to be 0.9550 and 'n' value was 0.5920 for the
best batch (A4), where n = slope (n ≤ 0.5 - fickian diffusion;
0.5 < n 1-
super case- II transport). The release profile fitted to hixoncrowell
models equation, the 'r' value was found to be 0.9300
for the best batch (A4). The release profile fitted to zero order
and first order equation, the 'r' value was found to be 0.9890
and 0.8890 for the best batch (A4) were shown in (Table 4).
The curve fitting, simulation and plotting was performed in
Excel (Microsoft Software Inc., USA) and Sigma plot®
version 10.0 (Sigma plot soft ware, Jangel Scientific Software,
San Rafael, CA). The effects of independent variables on the
response parameters were visualized from the contour plots.
Numerical optimization using the desirability approach was
employed to locate the optimal settings of the formulation
variables so as to obtain the desired response. An optimized
formulation was developed by setting constraints on the
dependent and independent variables. The formulation
developed was evaluated for the responses and the
experimental values obtained were compared with those
predicted by the mathematical models generated. Counter
plot showing the effect of polymer-to-drug ratio (X ) and
1
stirring speed (X ) on: % mucoadhesion, entrapment
2
efficiency, swelling index and particle size were shown in (Fig.
3).
In vivo anti-diabetic study
Based on the results of other associated parameters present
formulations A4 were chosen for evaluation of in vivo antidiabetic
study in standard animal models. The drug glypizide
was administered at a dose equivalent to 800 µg/kg.
Gliclazide pure drug was administered in a suspension form at
Table 3: In vitro Release Profile of Gliclazide Mucoadhesive Microspheres (A4)
Time Root Log Abs CDR CDR% Log% % Drug Log% %
Time Time CDR retained retained retained
1 1 0 0.0294 5.094 25.47 0.707 74.53 1.872 4.208
2 1.414 0.3010 0.0342 6.502 32.51 0.813 67.49 1.829 4.071
3 1.752 0.4771 0.0387 7.886 39.43 0.896 60.57 1.782 3.927
4 2 0.6020 0.0433 9.334 46.67 0.970 53.33 1.726 3.764
5 2.236 0.6989 0.0493 11.164 55.82 1.047 44.18 1.645 3.535
6 2.441 0.7781 0.055 12.988 64.94 1.113 35.06 1.544 3.272
7 2.645 0.8450 0.0621 15.23 76.15 1.182 23.85 1.377 2.878
8 2.828 0.9030 0.0693 17.572 87.86 1.244 12.14 1.084 2.298
Table 4: Model Fitting for the Release Profile of Optimized Microspheres
Zero First Higuchi Hixon-
Formulation Order Order Matrix Crowell Best Fit
Code R R R R N R Model
Chitosan 0.9890 0.881 0.944 0.955 0.592 0.930 Zero
R = correlation coefficient; N= slope (≤ 0.5 – fickian diffusion; 0.5 < n < 1 – non fickian diffusion;
1 – Case – II transport; > 1 – super case –II transport
168
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

A Senthil et al./ Chitosan Loaded Mucoadhesive Microspheres of Gliclazide: In vtro and In vivo Evaluation
Fig.3: Counter Plot showing the Effect of Polymer-to-
Drug Ratio (X1) and Stirring Speed (X2) on: %
Mucoadhesion (a), Swelling Index (b), Drug Entrapment
Efficiency (c), Particle Size (d) for Optimized Batch (A4).
a b
c d
Fig. 4: Percentage Reduction in Blood Glucose Levels
following Oral Administration of Pure Drug and Formulation
in Normal Rats (n = 5)
the same dose. Gliclazide pure drug was administered, a rapid
reduction of 42.83% was observed within 2 hours after oral
administration. Blood glucose levels were recovered rapidly to
the normal level within 8 hours. In the chitosan loaded
gliclazide mucoadhesive microspheres, the reduction in blood
glucose levels was slow and reached maximum reductions
within 4 hours after oral administration were shown in (Fig. 4).
This reduction in blood glucose levels was sustained over a
longer periods of time 10 hours. Kahn and Shechter have
suggested that a 25% reduction in blood glucose level is
34
considered a significant hypoglycemic effect . 25% of
169
hypoglycemic effect was maintained for a period of 0.5 to 2
hours period after oral administration of pure gliclazide. In
the case of gliclazide mucoadhesive microspheres shows
significant hypoglycemic effects was maintained for a period
of 1 to 9 hours. The sustained hypoglycemic effect observed
over long period of time because of the mucoadhesive
microspheres is due to slow release of drug and absorption of
gliclazide over longer periods of time. Gliclazide sustained
release formulation is significantly more effective than the
immediate release formulation of gliclazide in reducing
fasting plasma glucose levels and side effects as per Berelowitz
35
et al . The optimized batch A4 was studied its potential and
associated to control blood glucose level in animal. In this
study sustained release mucoadhesive microspheres of
gliclazide exhibited significant important in diabetic
parameters like glucose as compared to immediate release
formulation of sustained drug gliclazide. It may be the other
polysaccharides such as starch and other additive also contain
the precursors of glucose in the formulation of oral dosage
forms administrated available in market. The result reflects
that mucoadhesive microspheres were sustain regimen
maintain the vivo significant effect in animal models were
shown in (Fig. 4).
Stability studies revealed that, there was a reduction in
entrapment efficiency after storage for one month at 4±1°C,
25±2°C 25±2°C 60±5% RH and 37±2°C have shown
maximum entrapment followed by the storage at 25±2°C at
60±5% RH and 37±2°C at 65 ± 5% RH conditions. A4
batch stored at 4±1°C has shown 91.12 % drug release, at
25±2°C with 60±5% RH has shown 95.32% and at 37±2°C
with 65±5% RH has shown 98.76%, and the percentage of
drug entrapment efficiency at 4±1°C, 25±°C with 60±5%
RH and 37±2°C with 65±5% RH for one month was found
to be 72%, 72% and 69%.
The IR spectra of the pure drug gliclazide as well as the
combination spectra of the drug and polymers. In all the
combinations the wave numbers related to aliphatic
secondary amine, carbonyl, sulphur-oxy groups were nearly
same, which indicates no interaction between gliclazide and
polymers.
CONCLUSION
2
The results of a 3 full factorial design revealed that the
polymer-to-drug ratio and stirring speed significantly affected
the dependent variables percentage mucoadhesion, drug
entrapment efficiency, particle size and swelling index. As the
concentration of glutaraldehyde increases, the
mucoadhesiveness decreases and there was no significant
effect in time. Stirring speed has negetive effect on drug
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

elease. Chitosan microspheres A4 batch exhibited a high
percentage mucoadhesion of 78%, drug entrapment
efficiency of 72%, mean particles size of 67.10μm, swelling
index of 1.18 and 87.86% of drug release for eight hours
indicates the mucoadhesive microspheres of gliclazide could
sustain the release of the drug for more than 10 hours.
Biodegradable microspheres are one of the most useful
devices to deliver materials in an effective, prolonged and safe
manner. The in-vivo study demonstrated significant
hypoglycemic activity of the mucoadhesive microspheres of
gliclazide from chitosan shown significant activity.
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of mucoadhesive controlled release oral tablets of glipizide.
Ind J Pharm Sci 2003; 65:591-4.
22. The United States Pharmacopeial Convention. XXVI In: The
United States Pharmacopeia. Rockville, MD: The United
States Pharmacopeial Convention Inc; 2003:859.
23. Milling Eugene L, Lachman L, Liberman HA. Theory and
Practice of Industrial Pharmacy. 2nd ed. India.
24. The United States Pharmacopeial Convention. XXVI In: The
United States Pharmacopeia. Rockville, MD: The United
States Pharmacopeial Convention Inc; 2003:2528.
25. Ibrahim El-Gibaly I. Development and in vivo evaluation of
novel floating chitosan microcapsules for oral use: comparison
with non-floating chitosan microspheres. Int J Pharm 2002;
249:7-21.
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A Senthil et al./ Chitosan Loaded Mucoadhesive Microspheres Of Gliclazide: In Vitro And In Vivo Evaluation
26. Lehr CM, Bowstra JA, Tukker JJ, Junginger HE. Intestinal
transit of bioadhesive microspheres in an in situ loop in the rat.
J Control Release 1990;13:51-62.
27. Nelson KG, Wang LY. Determination of time course of
tabletdisintegration II: method using continuous functions. J
Pharm Sci 1961; 67:86-9.
28. Achar.L, Peppas N. Preparation, Characterization and
mucoadhesive interactions of poly (methacrylic acid)
copolymers with rat mucosa. J Controlled Release1994;
13:71-8.
29. Hand book of pharmaceutical controlled release technology
edited by Donald L. wise.
30. Biodegradable polymers as Drug delivery system. Edited by
mark chasim. Robert langer.
31. Sridhar GR, Yarabati VR. Diabetes in India, Indian J Endocrinal
Metab 2000; 4:70-80.
32. John H, Mcnill F. Experimental Models of Diabetes, Jaypee
Brothers. 1996;219-26.
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33. Sahin S, Selek H, Ponchel G, Sargon M. Preparation and
characterization and in-vivo distribution of terbutalin sulphate
loaded albumin microspheres. J Control Res 2002;82:345-58.
34. Kahn CR, Shechter Y. Oral hypoglycemic agents and the
pharmacology of the endocrine pancreas, In: Theodore WR,
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The pharmacology Basis of Therapeutics. 8th ed. New York,
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efficacy of a once-daily controlled release formulation of
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Address for Correspondence
A. Senthil, Department of Pharmaceutics, Karavali College of Pharmacy,
Mangalore-575028, Karnataka, India
E-mail: senthilac12@gmail.com
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

ABSTRACT
RGUHS Journal of Pharmaceutical Sciences
Effect of Different Acids on the Formation of E and Z Isomers of Dothiepin
1 2 1 3
Gopal Krishna Rao* , Ramesha A.R , Amit Kumar Jain and Sanjay Pai P.N
1
Department of Pharmaceutical Chemistry, Al-Ameen College of Pharmacy, Bangalore, Karnataka, India
3 Dept. of Quality Assurance, Al-Ameen College of Pharmacy, Bangalore, Karnataka, India
2 R.L.Fine Chemicals, Yelahanka, Bangalore, Karnataka, India
Dothiepin is an antidepressant drug useful in the treatment of mild to moderate endogenous depression. The synthesis of dothiepin
involves dehydration of alcohol using acid catalysis leading to the formation of E- and Z- isomers. So herein, we report the use and effect
of various acid catalysts on the formation E- and Z- isomers. Characterization of both the isomers was achieved by using HPLC and
NMR. Both NMR and HPLC analysis showed the formation of E-isomer as the major component.
Keywords: Dothiepin, E and Z isomer, NMR and HPLC
INTRODUCTION
Many important and widely used drugs are marketed as
mixture of optical isomers that often differ in
pharmacological, toxicological and pharmacokinetic
1
properties . Qualitatively and quantitatively enantiomers may
have similar or different pharmacological effects. This may be
related to stereo selective pharmacokinetics or
pharmacodynamics. The terms 'eutomer' for the more potent
isomer and 'distomer' for the less potent one have been often
used. No generalizations can be made concerning
enantiomers since they exhibit varied effects, e.g.,
cyclophosphamide and flecainide for equipotent
enantiomers; one enantiomer with all or most of the activity
as exhibited by NSAID's and β-blockers; both enantiomers
active with similar therapeutic and toxic effects but different
magnitude as shown by warfarin and both enantiomers active
but with quantitatively different therapeutic and toxic effects
2
as evidenced by verapamil . Hence efforts in formation of one
of the isomers of high pharmacological importance during
the synthesis itself can be of immense value.
Dothiepin, chemically is N,N-dimethyl-3-(dibenz[b,e]
thiepin-11(6H)-ylidene) propylamine. It also differs in
structure from amitriptyline by the presence of sulphur atom
in the central ring, which leads to the formation of E and Z
isomer. Of the two isomers, E isomer is predominant in its
3
composition . Dothiepin, a tricyclic antidepressant possesses
marked anticholinergic and sedative properties and is also
known to prevent reuptake of noradrenaline and serotonin at
nerve terminals. The tricyclic antidepressants are particularly
4
useful in the treatment of endogenous depression .
RGUHS Journal of Pharmaceutical Sciences
Received: 6/4/2011, Modified: 12/5/2011, Accepted: 21/5/2011
172
Original Research Article
In continuation of our efforts in understanding the catalytic
effects of various acids on the formation of E and Z isomers of
5
doxepin and encouraging results obtained therein, we hereby
report the effect of various acids on the formation of E and Z
isomers of Dothiepin.
Dothiepin was synthesized using different acid catalysts on
(11RS)-11-[3-(Dimethyl amino) propyl]-6,11dihydrodibenzo[b,e]
thiepin-11-ol (Compound A). Using
NMR technique we were able to identify the isomers and
confirm the E- and Z- isomer formation in the experiments
involving use of various acids. This study was confirmed by
HPLC to know the percentage of both the isomers in
6
dothiepin .
MATERIAL AND METHOD
The chemicals and reagents used in the present project were
of AR grade and LR grade and were purchased from
Lancaster, Sigma, NR Chem etc. Melting points of the
synthesized compounds were determined in open capillary
1
tubes and are uncorrected. H NMR (400 MHz) spectra were
recorded in deuterated chloroform in Amx-200 liquid state
NMR spectrometer (Astra Zeneca, Bangalore) using TMS as
internal standard. HPLC chromatograms were recorded on
Shimadzu SPD10 A UV-visible detector, Ray Chemicals Pvt
Ltd,Yelahanka,Bangalore.
STEP-1: General procedure for preparation E- & Z-
isomers of N,N-dimethyl-3- (dibenzo[b,e]thiepin-
11(6H)ylidene) propylamine (1a-g)
( 1 1 R S ) - 1 1 - [ 3 - ( D i m e t h y l a m i n o ) p r o p y l ] - 6 , 1 1 -
9
dihydrodibenzo[b,e]thiepin-11-ol [Compound A] (10 g,
0.0311 mol) was taken in a 500 ml round bottom flask, to
which acid (0.0377 mol) and toluene (100 ml) was added with
stirring at 110 °C. The reaction mixture was stirred for a
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

period of 6-8 h. After the reaction, the reaction mixture was
extracted with toluene and poured in to 150 ml of water and
made basic to pH-9. The aqueous and toluene layers were
separated. Toluene was distilled out to form a semisolid
product. Acetone was added and made acidic to pH-2 by the
addition of acid to obtain the final product.
-1
IR (KBR) cm :3068(ArC-H str), 2951(aliph C-Hstr),
1629(C=C, str), 1469(C-S-C str), 1081(C-N str) (Compound
GKRA-13).
1
HNMR (TMS) δ: 6.96-7.33{m, 8H,Ar-H};5.90 {t,1H,
Unsat. C=CH-E-isomer};5.83{t,1H, Unsat. C=CH-Zi
s o m e r } ; 4 . 7 8 - 4 . 8 5 { t , 2 H , N C H 2 } ; 3 . 3 8 - 3 . 4 5
{t,1H,CH 2};3.08{m, 2H, CH2-S-C }; 2.53-2.70 {s, 6H, 2 х
CH 3 }[Compound GKRA-13 obtained from p-Toluyl
sulphonic acid] (Fig. 1).
Compounds GKRA-07 to GKRA-12 were obtained in the
similar manner by using various acids (Table 1).
Determination of percentage of (E- and Z-) N,Ndimethyl-3-
(dibenzo[b,e]thiepin-11(6H)ylidene)
propylamine hydrochloride by High performance
liquid chromatography
Test solution: Dissolved 0.02 g of the N,N-dimethyl-3-
(dibenzo[b,e]thiepin-11(6H)ylidene) propylamine
hydrochloride in the mobile phase and dilute to 20 ml with the
mobile phase. 1 ml of this solution was diluted to 10 ml with
the mobile phase.
Chromatographic Procedure
Separation was carried out on Wakosil II (SGE) C-18
column, length 10cm and internal diameter 0.5 cm, particle
o
size 5µ. Column oven was set at 50 C and detection was
carried out at 254 nm. Mobile phase comprised of a mixture
1
Fig 1: H-NMR spectrum of GKRA-13
Gopal Krishna Rao et al./ Effect of Different Acids on the Formation of E and Z Isomers of Dothiepin
1
HNMR (TMS) δ: 6.96-7.33{m, 8H,Ar-H};5.90 {t,1H, Unsat.
C=CH-E-isomer};5.83 {t,1H, Unsat. C=CH-Z-isomer};4.78-
4.85{t,2H,NCH 2};3.38-3.45 {t,1H,CH 2};3.08{m, 2H, CH2-S-C };
2.53-2.70 {s, 6H, 2 х CH 3 }[Compound GKRA-13 obtained from
p-Toluyl sulphonic acid] Scheme - 1
173
of 30 volumes of methanol and 70 volumes of a 30 g/l
solution of sodium dihydrogen phosphate, previously
adjusted to pH 2.5 with phosphoric acid.
Procedure
20 µl of the test solution was injected. The system sensitivity
was adjusted so that the height of the principal peak is at least
50 % of the full scale of the recorder. The test is not valid
unless the resolution between the first peak (E-isomer) and the
second peak (Z-isomer) is at least 1.5 (Fig. 2).
The percentages of both the isomers formed are tabulated in
(Table 2).
RESULT AND DISCUSSION
The percentage of E-isomer is found to be higher than Zisomer
in all the experiments. The conversion of (11RS)-11-
[3-(dimethylamino) propyl]-6,11-dihydrodibenzo[b,e]
thiepin-11-ol (Compound A) to E- & Z- N,N-dimethyl-3-
(dibenzo[b,e]oxepin-11(6H) ylidene) propylamine
(Compounds 1 and 2) was complete as shown by IR spectra
-1
with appearance of C=CH peak at 1602 cm .
SCHEME 1
E-isomer
1
CH 3
S
OH
(A)
N
CH 3
CH 3
Acid catalyst
toluene
S S
N
CH3 H3C H3C N
Z-isomer
2
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

Fig. No-2: HPLC chromatogram of [GKRA-13]
E-isomer= 94.07 % ; Z-isomer= 5.84 %
Z- isomer
Gopal Krishna Rao et al./ Effect of Different Acids on the Formation of E and Z Isomers of Dothiepin
E- isomer
Fig 3: General method for Calculation of percentage
1
of isomers using H-NMR spectrum
1
The H-NMR spectral analysis also revealed the formation
of the E- and Z- isomers, N,N-dimethyl-3-
(dibenzo[b,e]thiepin-11(6H) ylidene)propylamine at δ value
5.90 and 5.83 respectively in all the experiments. General
method of calculation of E and Z isomers using NMR
technique is depicted in( Fig. 3).
Further confirmation was done by HPLC, which showed
formation of E-isomer in the range of 89-94 % whereas Zisomer
formation was in the range of 6-10 % for dothiepin
Table 2.
Table 1: Data of Percentage Yield, Melting Point of Dothiepin with various acids
Sl. No. Compound Acid Prac. Yield % Yield M.P. (ºC)
1 GKRA-07 Maleic acid 7.5 g 80 220-222
2 GKRA-08 Oxalic acid 7.8 g 83 218-220
3 GKRA-09 Tartaric acid L(+) 8.2 g 88 218-220
4 GKRA-10 Phosphoric acid 5.4 g 58 189-191 *
5 GKRA-11 Tartaric acid DL 5.5 g 60 198-200 *
6 GKRA-12 Indian Resin-220 7.4 g 79 186-188 *
7 GKRA-13 p- Toluyl sulphonic acid 8.7 g 92 218-220
* Formation of isomers and separation was not possible in these cases.
Table 2: Percentage of Dothiepin isomers evaluated by 1 H-NMR and High Performance Liquid
Chromatography.
Sl. No. Compound Acid used E:Z ( 1 H-NMR) E:Z (HPLC)
1 GKRA-07 Maleic acid 85:15 92:8
2 GKRA-08 Oxalic acid 85.3:14.7 92:8
3 GKRA-09 Tartaric acid L(+) 84:16 89:11
4 GKRA-10 Phosphoric acid *** ***
5 GKRA-11 T Tartaric acid DL *** ***
6 GKRA-12 Indian resin-220 *** ***
7 GKRA-13 p-Toluyl sulphonic acid 93:7 94.07:5.84
*** Formation of isomers and separation was not possible in these cases
174
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

Best results were obtained with p-toluyl sulphonic acid for Eisomer
of dothiepin (94%) while other acids also yielded more
than 84%.
CONCLUSION
The main focus of this research work was to understand the
effect of different acid catalysts on the formation of E- and Z-
isomers of Dothiepin and to characterize the isomers by
1 HNMR and HPLC methods. The yield of E-isomer of
dothiepin was achieved in the range of 89-94 %. From the
experiments conducted, use of p-toluyl sulphonic acid
(GKRA-13) yielded maximum of 94% E-isomer and was
found to be ideal reagent for the dehydration of the alcohol
[Compound-A] to form E- and Z- isomers of dothiepin.
Other acids namely L(+) tartaric acid, oxalic acid and maleic
acid led to the formation of E isomer in the range of 89-92%.
Quantification of E- and Z-isomers of dothiepin obtained by
NMR showed the range of 85:15 and 94:06 and HPLC was
found to be in the range of 89:11 and 94:6.
ACKNOWLEDGEMENT
The authors would like to thank Prof. B. G. Shivananda
Principal, Al-Ameen College of Pharmacy, Bangalore and
Mr Anjan K. Roy, Managing Director, R.L.Fine Chemicals,
Bangalore for providing support and necessary facilities,
Department of Inorganic and Physical Chemistry, Indian
Institute of Science, Bangalore, for providing help in
obtaining the various spectra.
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Address for Correspondence
Gopal Krishna Rao, Department of Pharmaceutical Chemistry, Al-Ameen
College of Pharmacy, Bangalore, Karnataka, India
E-mail: gkfadnis@gmail.com
RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2

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listed followed by et al.)
You CH, Lee KY, Chey WY, Menguy R. Electrogastrographic study of
patients with unexplained nausea, bloating and vomiting.
Gastroenterology 1980;79:311-4.
Books and other monographs
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Eisen HN. Immunology: an introduction to molecular and cellular
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1974.
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Dausser J, Colombani J, editors. Histocompatibility testing 1972.
Copenhagen: Munksgaard; 1973.
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Institute of Medicine (US). Looking at the future of the Medicaid program.
Washington: The Institute; 1992
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Morse SS. Factors in the emergence of infectious diseases. Emerg Infec
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Rajiv Gandhi University of Health Sciences Journal of Pharmaceutical Sciences (RJPS)
American Journal of Pharmacy- (Amer J Pharm) Analytical Chemistry-
(Anal Chem)
British Journal of Pharmacology and Chemotherapy- (Brit J Pharmacol)
Canadian Journal of Pharmaceutical Sciences- (Can J Pharm Sci)
Clinical Pharmacokinetics- (Clin Pharmacokinet) Drug Development
and Industrial Pharmacy- (Drug Develop Ind Pharm) Helvitica Chimica
Acta- (Helv Chim Acta) Indian Journal of Medical Sciences- (Indian J
Med Sci) Indian Journal of Pharmaceutical Sciences- (Indian J Pharm
Sci).
Journal of the American Chemical Society, The- (J Amer Chem Soc)
Journal of Biological Chemistry- (J Biol Chem) Journal of Organic
Chemistry, The- (J Org Chem) Journal of Pharmacology and
Experimental Therapeutics- (J Pharmacol Exp Ther) New England
Journal of Medicine- (N Engl J Med) Pharmaceutical Journal, The
(Pharm J)Pharmacological Research Communications-(Pharmacol Res
Commun)
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Journal of Pharmaceutical Sciences (RJPS)
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Dr. Tilak R. Bhardwaj, India
Dr. Yadav M.R., India
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Dr. Veerapur V.P, Tumkur
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Scientific Tools
Preamble
Gowraganahalli Jagadeesh..................................................................................................................................................................................... 96
Creative, Critical Thinking and Logic in Research
Fredricka Reisman ..................................................................................................................................................................................... .. 97 - 102
Review Article
Need for Inclusion of Scientific Writing Skill Subjects in Indian Post Graduate Pharmacy Course
Patil J.S, Kotnal R.B, Birajdar R.P, Marapur S.C and Kadam D.V .............................................................................................................. 103 - 106
Research Article
A Novel Spectrophotometric Estimation of Pramipexole in Bulk Drug and Formulations
Shobha Manjunath, Satish Middi and Venkatesh Chouhan ........................................................................................................................ 107 - 110
Validated UV-Spectrophotometric Estimation of Entecavir in Bulk and Formulations
Malipatil S.M, Bharath S Athanikar and Mogal Dipali. ..................................................................................................................................111 - 116
Antihyperlipidemic Effect of Ethanolic Extract of Hibiscus rosa sinensis Flowers in Hyperlipidemic Rats
Sikarwar Mukesh S. and Patil M.B ...............................................................................................................................................................117 - 122
A Study on Drug-Drug Interaction of Diltiazem with Nateglinide in Diabetic Animals
Suresh D.K, Raza Hasan, Hamza Sheth, Md. Saifuddin Khalid and Mohiuddin M ..................................................................................... 123- 126
Influence of Vitamin C with Lansoprazole in Pylorus Ligation Induced Ulcer Model in Rats
Nitin M, Prasad K, Girish M, Ather Javed, Chetan M and Krunal S. ............................................................................................................127 - 130
Assessment of Safety and Efficacy of Doxycycline and Azithromycin Preparations in Patients with Acne Vulgaris
Mahendra Kumar B.J, Ramakrishna S, Kranti Basavant Patil, Sandeep A, Bhimaray S Krishnagoudar and Katti Ravi Venkappa .......... 131 - 135
Antidiarrhoeal Activity of Aqueous Extract of Mimosa pudica Leaves
Md. Saifuddin Khalid, Shah Jinesh Kumar, Suresh D.K., Rajnish Kumar Singh, Reddy Narasimha I.V. and Shaikh Azhar Hussain ........ 136 - 140
Assessment of Various Combination of Drugs Used in Treatment of Lower Respiratory Tract Infection
Imran Ahmad Khan, Shobha Rani R.H, Geeta S, Mahvash Iram ............................................................................................................... 141 - 145
Formulation and Evaluation of Mucoadhesive Buccal Drug Delivery System of Metoprolol Tartrate by Using Central
Composite Design
Prakash Rao B and Gandhi Purvesh ........................................................................................................................................................ 146 - 156
Development and Evaluation of Mucoadhesive Buccal Films of Nebivolol
Bushetti S.S, Mane Prashant P and Kardame S.S ..................................................................................................................................... 157 - 162
Chitosan Loaded Mucoadhesive Microspheres of Gliclazide: In vitro And In vivo Evaluation
Senthil A, Thakkar Hardik R, Ravikumar and Narayanaswamy V.B ...........................................................................................................163 - 171
Effect of Different Acids on the Formation of E and Z Isomers of Dothiepin
Gopal Krishna Rao, Ramesha A.R, Amit Kumar Jain and Sanjay Pai P.N ................................................................................................ 172 - 175
Instructions to Authors