Book 1.indb - Journal

Scientifi c Tools
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
CONTENTS
RGUHS Journal of Pharmaceutical Sciences
Vol 2, Issue 1, Jan–Mar, 2012
• Knowing the known to understand the unknown: a systematic approach to reviewing
the scientifi c literature 1
Yamuna V. Kuberappa, Arun H.S. Kumar 10.5530/rjps.2012.1.1
Meeting Report
• Pharmaceutical discovery and development 8
(A report on the symposium held in 63rd annual conference of the Indian Pharmaceutical Congress-2011)
Pitchai Balakumar, Gowraganahalli Jagadeesh 10.5530/rjps.2012.1.2
Review Articles
• Nanobiotechnology: An overview of drug discovery, delivery and development 14
Bhupinder Singh Sekhon 10.5530/rjps.2012.1.3
• Nasal drug delivery–a review 24
Twarita Deshpande, Rajashree Masareddy, Archana Patil 10.5530/rjps.2012.1.4
Research Articles
• Design and optimization of levofl oxacin gastroretentive tablets 38
D. Nagendrakumar, S.B. Shirsand, M.S. Para, A.D. Chauhan 10.5530/rjps.2012.1.5
• Study of the binding properties of hydroxypropyl guar and its utilization in the formulation
and evaluation of metoprolol tartarate tablets 45
Swamy N.G.N., Dharmarajan T.S. Paranjothi K.L.K. 10.5530/rjps.2012.1.6
• Phytochemical investigation of root extract of the plant Carissa spinarum 55
Karunakar Hegde, D. Satyanarayana, Arun B. Joshi 10.5530/rjps.2012.1.7
• Synthesis and antimicrobial activity of 4-hydroxy-1-methyl/phenyl-3- (substituted anilinoacetyl)
quinolin-2(1H)-one 60
Girish Bolakatti, Manjunatha S. Katagi, S.N. Mamledesai, Sujatha M.L., Prakash Dabadi, Narayana Miskin.
10.5530/rjps.2012.1.8
• Evaluation of gastroprotective ability of Amorphophallus paeoniifolius corms against indomethacin
induced gastric ulcers 67
H.N. Nataraj, R.L.N. Murthy, Ramachandra Setty 10.5530/rjps.2012.1.9
• Immunomodulatory activity of methanolic extracts of Pongamia glabra Vent. seeds and bark
in cyclophosphamide induced mice 74
Sanjeev Heroor, Arunkumar Beknal, Nitin Mahurkar 10.5530/rjps.2012.1.10
• Simultaneous fi rst derivative UV spectrophotometric estimation of ramipril and olmesartan 78
Santosh R. Karajgi, C.C. Simpi, Kalyane N.V. 10.5530/rjps.2012.1.11
• Purifi cation and characterization of thermostable amylase from a strain of
thermoactinomyces thalpophilus KSV 17 83
K. Sreenivasa Rao, P. Ellaiah, Karnakumar V. Biradar 10.5530/rjps.2012.1.12

Knowing the known to understand the unknown
A systematic approach to reviewing the scientifi c literature
Yamuna V Kuberappa 1 , Arun HS Kumar 2
1 Freelance MEMS Engineer, Cork, Ireland
2 Department of Medicine (Cardiovascular), University College Cork, College Road, Cork, Ireland
ABSTRACT
Every work must have a starting point, and scientifi c research is no exception. Reviewing literature is an initial
step in undertaking scientifi c research. Reviewing literature, as an ongoing process, provides a critical overview
on the topic of interest and keeps the researcher up-to-date to pursue novel research plans. While reviewing
literature helps early career investigators identify their research niche, established researchers review the literature
to propagate the state of the art progress/opinions in their fi eld of expertise. Most of these reviews often guide
the general research community. Various formats are often adopted in writing a review depending on the context,
audience, depth/complexity of subject and extent of commercial value. The primary goal is to provide a critical
and simplifi ed analysis of the facts. Essentially, every researcher should adapt to these formats in a unique way
and should try to develop their own style of reviewing the literature. In this review, we will describe some of the
essential steps to approaching various formats of reviewing the literature.
Key words: Reviewing literature, scientifi c research, business reviews, book reviews
INTRODUCTION
Reviewing literature is not only a skill but
also an art, wherein scientifi c knowledge
and opinions are disseminated in a critical,
concise and simplifi ed language to scientifi c
and non-scientifi c communities. 1–16 The
purpose of literature review is to provide
critical opinions on established scientifi c
facts and in the process evolve the strengths,
weakness, opportunities and limitations on
the topic of research interests (Figure 1).
Hence, the emphasis is always on providing
critical opinions on a topic rather than
compiling scientifi c facts. 4,10,11,17–19 When one
attempts to critically compile an opinion,
it helps to identify gaps in the existing
literature, and develop research questions
for further investigation. Thus, the seed of
research is sown. A researcher nurtures this
seed over the course of his/her career, to
grow into a fruitful tree. Before starting a
review, clearly defi ne the objectives of the
study and identify the targeted audience.
Based on these two factors, decide the portal
of publication for which the review would
best fi t. The next step is to understand
the procedure for searching and collecting
materials for the intended work. This will
be described in subsequent issues of this
journal. This article focuses on general
steps for systematically reviewing scientifi c
literature.
What necessitates the review
of literature?
Seeking information to gain understanding
of any concept is a fundamental feature of
learning. This is especially true in scientifi c
research, where one needs to understand
an established concept on a relevant topic
to progress further, to evolve innovative
ideas and thoughts, and to avoid any
duplication. 2,6,10,18,22 The famous quote of ‘life
begets life’ in the scientifi c research arena can
Scien fi c Tools
Received Date : 11-02-2012
Revised Date : 29-02-2012
Accepted Date : 03-03-2012
DOI: 10.5530/rjps.2012.1.1
Address for
correspondence
Arun HS Kumar, DVM, PhD.
Senior Scientist and Head
(Functional Biology)
Department of Medicine
(Cardiovascular)
University College Cork
Cork, Ireland
E-mail: a.kumar@ucc.ie
www.rjps.in
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 1

Figure 1: Six major reasons for reviewing the literature.
be represented as ‘research begets research’ and ‘innovations’
i.e., every research work contributes to further research
bringing in scientifi c advancement and innovations.
Therefore, the fundamental reason to proceed with
reviewing the literature is to gain clear understanding on
the topic of interest, so as to set the vision for future
research and to provide the latest source of information
on the topic to a wider scientifi c and non-scientifi c
audience 10,16,25 (Figure 1). The overall objectives of
writing a literature review are summarized in Figure
2. There are several benefi ts of conducting scientifi c
literature review (Box 1).
The following questions are helpful before initiating a
literature review.
Why? Ask yourself, why do you want to review the
literature on a specifi c topic? Come up with rational
thinking on why a review of the literature is required on
the topic at this time.
Why not? This aspect is specifi cally relevant to
young researchers, which emphasizes on curiosity and
inquisitiveness. Hence, start reviewing the literature as
a self-learning step to gain more insight on a specifi c
scientifi c topic and ask yourself if it provokes interests
or motivates you to proceed further.
Box 1: Benefi ts in conducting a methodical literature
review
1. Gives a general and critical overview on the research topic
2. Helps to improve the experimental methods and avoid
duplication of research work
3. Assists in identifying gaps in current research work
4. Helps to build and defi ne research questions and
hypotheses
Yamuna V Kuberappa et al.: Knowing the known to understand the unknown
Why not me? Question yourself on how well you are
suited to reviewing the literature. This is more about
setting the objectives based on strengths and weaknesses
and defi ning targeted audience or scientifi c area of
research.
Why not me now? This question is more relevant to
established researchers, wherein they initiate literature
review to suite the requirement of the scientifi c
community.
The four why’s described above in the context of
literature review might be applicable to any type of
literature review and thus, readers are encouraged to
follow them.
How to start writing a review of literature?
As stated previously, master the skills to search the
literature. A major challenge is how to be effective in
critically judging the published literature? With a recent
surge in the number of scientifi c journals, the number
of scientifi c manuscript/books published on any topic/s
is on an exponential rise. Thus, vast and diversifi ed
resources are available for researchers. It is often diffi cult
to maintain the quality and standards uniform among all
these resources; hence, the researchers should develop
skills to critically evaluate the available literature. 6,9,10,26–28
One of the ways to critique is by avoiding a researcher’s
own inference based on the reported results and rather
looking into the techniques by which the reported
results were derived. Hence, it is emphasised that
while reviewing any literature, one must be critical in
interpretation whether the reported results are based on
rigorous experimental designs or, are merely based on
protocols, which are subjective, controversial and not
rigorous. 10,23,27,28
It is said ‘well begun is half done’; hence, it is important to
start writing with a clear concept and focus by clearly
identifying the objectives of the review. The best way
Figure 2: The basic objectives of any literature review process.
2 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

to do this is, by writing an abstract and clearly outlining
headings and subheadings. 1,4,9,10,16,22,28,29 Once an outline
is framed, start compiling the review by critically
providing an opinion on the topic rather than merely
summarizing the established facts. Attempt to identify
various gaps in existing literature, such as an overworked
area. Based on the existence of such gaps, write research
questions. 10,28,29
It is important to be creative in thoughts and to bring
the researcher’s own signature style of formulating the
literature review. Researchers should not hesitate to
rationally challenge assumptions of existing scientifi c
theory. The review should not be just a free fl ow of
words with stagnant thoughts; rather it should be a
free fl ow of critical thoughts and concepts which are
innovatively constructed/reconstructed. 2,10,27,28,30 An
important factor to innovation is being divergent in
thoughts and attempting to think critically. A detailed
review on critical thinking has recently been published
in this journal, and readers are encouraged to read the
article. 27 In order to draw critical insights from existing
literature, it is necessary to follow an approach of slicing
and dicing of the existing literature to highlight differing
arguments, theories and methodologies. It often
helps to summarize the existing research in the form
of tables, key points on the box and/or fi gures rather
than being extensively textual in literature review. Finally,
it often helps to pre-identify the journal to which you
intend to submit your literature review. This helps you
to structure your review to fi t the aims and scope of
the journal and plan your writing to meet the needs
of specifi c audience.
Defi ning the scope and formulating
the literature review
Composing of literature review begins with clearly
defi ning the objectives based on nature of its intended
audience and the degree of novelty (Figure 3). A wellcomposed
literature review should comprehensively
highlight the current state of the art on the topic,
identify the areas of controversial and/or confl icting
opinions and should hypothesize the topics that need
future research focus. 10,29,30 Since several publications
with either similar and/or contradictory opinions are
being referenced, it is appropriate to use linker words
and phrases such as similarly, in addition to, moreover, likewise,
again (for studies with similar opinions) and albeit, on the
contrary, however, on the other hand, conversely, nevertheless (for
articles with contradictory opinions) while compiling
a literature review. 10,31 It is also essential to include a
summary paragraph at the end to highlight briefl y the
major focus and opinions expressed in a literature review
that often might link to a research hypothesis and future
research directions. 10,22,31
Yamuna V Kuberappa et al.: Knowing the known to understand the unknown
Figure 3: Factors contributing to structuring literature review.
The outline of any literature review is based on nature of audience
and degree of novelty of the contents, which accordingly reflects
its primary goal (creating awareness, educational or scientific
advancement).
Having familiarized with the general formula for
composing a literature review, we now look into the
practical recipes of literature review as it is very much
context dependent. Every scientifi c literature review
begins with the acquaintance of an existing literature and
it is vital to identify type of materials to read. 2,10,18,23,28–30,32
It often helps to categorize the existing literature based
on the degree of novelty and similar versus contradictory
opinions. Although these aspects are more relevant to
established investigators, the novices are often directed
by their guides/supervisors toward a list of introductory
reference materials. While reading these introductory
references, it is important to make notes and write
critical comments as bullet points, which would come
handy at the time of composing the literature review.
The basic elements of a literature review include
an introduction/background section highlighting
the central theme and related context of the review.
This is followed by the body of the literature review
consisting of bulk of the text discussing the origin of
the sources, theories and methods. Finally, the icing on
the cake is the conclusions/recommendations section,
a short paragraph consisting of critical opinion of the
researcher on the subject matter and the scope for further
research. Recently, a ‘Grounded Theory’ based approach
(systematically adopting 5 stage processes) has been
proposed for composing rigorous literature reviews 26,29
(Figure 4), which is a signifi cant advancement of the
matrix-based approach. 33 Although these approaches are
primarily emphasized for information systems, general
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 3

Figure 4: The Grounded theory of composing literature review
involves 5 sequential steps starting from defining to composing the
literature review.
concepts can be adopted for biological/life/medical
sciences as well.
Adopting a language style in composing
literature review
The major purpose of any language style is effective
communication; hence, style is secondary, as long as
writing is formal, clear and concise. 2,9,10,22,28,31 We highly
encourage researchers to develop their own signature
style, which is distinct from others. It is important to
be objective rather than subjective to others opinions
and always use present tense while referring to general
opinions/theories and past tense while addressing specifi c
study results. 10,31 Do not plagiarize any statements. One
of the ways to avoid plagiarism is to make bullet points
and reconstruct sentences to convey your opinion(s) in
simple language. Remember, the essence of science is
to make things as simple as possible. 10,31 Once a draft
is done, it should be read several times and request a
colleague to proofread. Incorporate all corrections and
suggestions in the revised document.
A literature review can take various formats depending
on the purpose for which it is compiled. The following
sections describe each of these formats.
Composing the review of literature
chapter for a thesis
The major purpose of reviewing the literature for a
thesis is to build the context on a research hypothesis
on which the project is based. Thus, the subject matter
should be organized around a pre-defi ned objectives and
research parameters. 10,28,29,34 It often helps to organize
Yamuna V Kuberappa et al.: Knowing the known to understand the unknown
the structure of the literature review by compiling a
list of clearly defi ned research questions/objectives
and selectively reading the relevant literature to fi nd
answers to these questions and in the process identify
the gap in the knowledge base. 10,28,29,34 It is often easy
to get side tracked; hence, keep focused on objectives
and work toward formulating a clear thesis (research
topic) statement and later propose a scientifi cally valid
hypothesis. 7,10,16,20,22,25,26,28,29,34 Box 2 provides a checklist
for compiling a literature review for a thesis.
Organize literature review into a general outline
consisting of introduction, body (major and minor
topics) and conclusion sections. Follow the approach
of composing rather then compiling, and wherever
possible, summarize the data in the form of tables and/or
fi gures. 7,10,16,20,22,25,26,28,29,34 Emphasise on why the intended
work is important for the advancement of scientifi c fi eld
and the likely benefi ts it intended to contribute. 7,10,28,29,34
In doing so, comprehensively cover the existing
literature within and closely related to the scientifi c area
of investigation. Never avoid or ignore any scientifi c
reports that are contradictory or controversial, rather
acknowledge them with a comment why the reported
views are different from those of the researcher. 10,28,34
As noted previously, the review should be proofread
by colleagues and friends who are from outside the
scientifi c domain of the researcher as this clears blind
spots and improves readability. 10,28,34 It is important
that the literature review should lead the reader toward
a conclusion that a rational and realistic research
hypothesis is being proposed.
For most thesis work, the sources are organized in a
chronological pattern. For example, 10 if a researcher
has decided to work on a novel stent-based therapeutics
to achieve myocardial regeneration (MR) following
myocardial infarction, he/she should start with background
information on pathology of myocardial infarction and
epidemiological data on the incidence of myocardial infarction
world-wide and its socio-economic implications. The body of the
review consists of, earlier therapeutic approaches, limitations of
the existing therapeutic approaches and the concept of myocardial
regeneration as a therapeutic option to treat myocardial infarction.
BOX 2: Check list for the compilation of literature
review for a thesis
1. Does the literature review substantiate the research
hypothesis?
2. What is the key message delivered?
3. Do you adequately cover the methodologies and techniques
you intend to adopt?
4. Have you adequately identifi ed the gaps and limitations in
the knowledge base?
5. Have you covered the existing literature up-to-date?
4 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

Follow this with a theory on why stents would be appropriate to
deliver the therapeutics for myocardial regeneration. This should
be followed by a discussion on the developments in design of stents
and the era of introduction of drug eluting stents with its pros
and cons. Furthermore, describe the limitations of current stent
designs, the scope for advancement in the design of stents to deliver
myocardial regenerative therapeutics and its futures perspectives.
Finally, conclude with a statement why the reviewer approach
is better than the other options available. (This paragraph is
reproduced from our previous publication). 10
Instructions for reviewing the literature for
research or review articles
Every research article consists of an introduction with
a brief review of literature, which builds the context
of the research work/hypothesis investigated. The
purpose of literature review in a research article is to
gain support for the scientifi c argument of a researcher.
The purpose in the context of a review article, is to
generate views by composing and synthesizing the
arguments and ideas of others. 1,9,10,18,28,29,35,36 Both
research and review articles have the same elements
of literature but differ in terms of elaboration and
discussion. In research articles, the literature review is
integrated into the introduction and discussion section
and the emphasis is on supporting a researcher’s
argument. On the other hand, in a review article,
the literature review is emphasised on background
information and identifying gaps in knowledge base to
build a critical opinion on the subject topic and future
research directions. 2,9,10,29,36
For example, if one discovers a novel stent design for
treating coronary artery disease (CAD) and report the
same in a research article, the following pattern may
be used.
‘Stents are a widely used medical device for treating CAD…. The
bare metal and drug-eluting stents, which are extensively used,
have serious drawbacks with respect to developing late stenosis
and thrombosis complications … Several studies … report the
complications associated with the currently available stents. Critical
evaluation of the pathology …has revealed that complications are
a consequence of the unique design of the stents. To overcome this
limitation we have designed a novel stent, which we foresee to be
devoid of the side effects observed with the currently available stent
designs,…and tested it for performance in the current study with
the following objectives’. (This paragraph is reproduced from
our previous publication). 10
While all the general guidelines for composing a
literature review are applicable to research as well as
review articles, it is acceptable and essential to include
the reviewer’s opinions in the review articles, especially
on the methodologies adopted and the robustness of
the conclusions reported. Several categories of review
articles exist (comprehensive review, meta-analysis, brief
Yamuna V Kuberappa et al.: Knowing the known to understand the unknown
review, hypothesis-based review, opinion article, stateof-the-art-review,
etc.) depending on the scientifi c insight
and extent of focus on the subject by the reviewer. For
instance, the above paragraph in the context of a review
article can be redesigned as follows.
Start with a brief introduction on CAD, its incidence, its impact
on human health and its socio-economic consequences. Follow this
with a discussion on why are stents the obvious choice for CAD,
what was the approach adopted before the introduction of stents
with its pros and cons, how has the use of stents infl uenced the
CAD events. Include a few paragraphs on the nature/design of
stents used initially and the developments over time on the stent
design and coatings. Follow this with one or two paragraphs on
the types of stents currently in use, their limitations and commonly
encountered side effects (critically evaluate these studies as they
form the basis for further investigations in stent designing and
coatings). Include a paragraph on the reviewer’s opinions for the
limitations encountered with the use of currently available stents
for CAD and the future strategies to overcome these limitations.
Finally, conclude with the room available for future advancement/
development. (This paragraph is reproduced from our
another publication). 10
Art of literature review for books/book chapters
With the growth in digital media and the rising
popularity of tablet devices/computers, the demand
for eBooks has surged. Books/book chapters remain a
vital source of established knowledge to both scientifi c
and non-scientifi c audiences. 2,10,18,29 Unlike research
articles, the language used in books must be simple
(avoiding scientifi c jargons wherever possible) and
comprehensible to a wide audience. 10,29 In reviewing a
topic for a book, one should limit to including established
facts and present it with adequate diagrams, tables and
fl ow charts to simplify concepts. It is often essential to
adopt a narrative approach rather than validate scientifi c
facts. Additionally, summarize each subsections, as this
greatly improves readability and comprehension of
the topic. 10,29
Literature review for grant applications and
research proposals
The general outline and focus of the literature review
for grant applications and research proposals are similar
to the one outlined above. However, the emphasis is
more on novelty and applicability aspects of research
proposed and it is important to be precise and to the
point. 10,22,28,29,37 More importantly, the applicant’s own
research to the progress of the proposed project
should be included in the application. This would instill
confi dence in grant reviewers on the abilities and skills
of the researcher to perform the work as described in
the research proposal. 10,28,37 Such confi dence build-up
measure among reviewers helps achieving high rating and
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 5

hence, the success of the grant application. As subject
experts often review the grants/research proposals, it
is necessary to highlight scientifi c advancement rather
than basic information and scientifi c novelty. Needless
to mention that the literature review supporting the
scientifi c advancement must always be tailored to meet
the scope and objectives of the funding agency.
Writing literature review for business proposals
Increasing trends in the academic-industrial partnerships
and academic spin-offs have lead to many academicians
adopting business and entrepreneurship style in running
their research labs. 10,28 Such an integration of corporate
atmosphere in academic environment has progressively
introduced the concept of academic business proposals,
which often involve academic partnerships with
industries to capitalize on the basic/applied science
research to achieve mutual benefi ts. 10,28 Business
proposals by academic researchers should focus on the
following elements.
1. Commercial benefi ts and the economics of the
research work in question.
2. Critical reviews of the applicant’s research work
within the context of commercialization, novelty
and patentability over related research competitors.
Hence, unlike literature reviews for scientifi c manuscripts,
grant proposals, and books, the write-up for business
proposals should highlight on the economic and
commercialization impact of the proposed work with
direct links to novelty and patentability.
SUMMARY
Literature reviews are a valuable knowledge source and a
measure of consolidating the current research progress
in any scientifi c fi eld. There are several categories of
review articles, e.g., comprehensive reviews, metaanalysis,
brief reviews, opinion articles, etc. All literature
reviews adopt the same broad outline/structure, but
vary in detail depending on the objectives. In general,
these objectives identify gaps in existing work, avoid
duplication of work, and propose a scientifi cally valid
hypothesis. Depending on the context in which it is
composed, a literature review has many benefi ts. It may
be a source of self-education or educating others and
publication. In addition, reviews attract successful grant
funding and might build a commercialization case for a
body of research. We emphasise that literature reviewing
is an art of scientifi c communication, which can, if
properly routed, lead to intellectual questions and grants
or business opportunities. Hence, it is necessary to invest
considerable amounts of time, effort and dedication to
this vital scientifi c activity.
Yamuna V Kuberappa et al.: Knowing the known to understand the unknown
CONFLICT OF INTEREST: None.
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RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 7

Pharmaceutical discovery and development
(A report on the symposium held in 63rd annual conference
of the Indian Pharmaceutical Congress-2011)
Pitchai Balakumar 1 , Gowraganahalli Jagadeesh 2,§
1 Department of Pharmacology, Institute of Pharmacy, Rajendra Institute of Technology and Sciences, Sirsa 125055,
Haryana, India
2 Division of Cardiovascular and Renal Products, Center for Drug Evaluation and Research, US Food and Drug Administration,
Silver Spring, MD 20993, USA
ABSTRACT
The 63rd Annual conference of the Indian Pharmaceutical Congress (IPC) held at Bangalore International Exhibition
Centre, Bengaluru, Karnataka, India, from 16 to 18 December, 2011, was organized by ‘Indian Pharmaceutical
Congress Association’ and hosted by ‘The Indian Pharmaceutical Association’, Karnataka State Branch. The
conference included a 3-hour symposium on “Pharmaceutical Discovery and Development”. The objectives of this
symposium were to enlighten the delegates on a few key aspects of drug discovery and development. To that
end, the symposium addressed on drugs and drug targets, early evaluation of pharmacokinetics and metabolism
in drug discovery, toxicity tests in drug development, regulatory review process, and biomarkers.
INTRODUCTION
Drugs are miracles of modern science. They
are used to prevent, treat, or cure diseases
and medical conditions. Disease processes
are often complex and involve a defi ned
sequence of events. Drugs are designed to
intervene in the disease process. They can
simply alleviate symptoms or even control
subsequent development of a chronic
disease condition. Except for antimicrobials
and to some extent anti-infl ammatory and
anti-neoplastic drugs, most other drugs
infrequently cure diseases. Yet, these
drugs improve the quality of life and, in
general, a sense of well-being. Billions of
prescriptions are fi lled worldwide every year.
The process of discovering, developing
and testing new drugs encompasses some
of the most inspiring areas of scientifi c
discovery. 1 Drug development involves
multiple disciplines encompassing a large
number of skilled scientists at different
levels, interdependent science managers
involved in important decision-making,
huge volumes of generated data, and
ultimately the drug regulatory agencies (e.g.,
US FDA, European Medicines Agency,
Japanese Ministry of Health and Welfare). 1
It is a complicated, time-consuming (often
>10 years), and costly process (over US $1
billion) whose end result is seldom known
at the outset. 2
The initial stages of drug discovery
research are characterized by target discovery
(identifi cation and validation) and lead
discovery (generation and optimization).
Integration of combinatorial chemistry,
high throughput screening, and molecular
§ The opinions expressed herein are those of GJ and do not necessarily refl ect those of the US Food and Drug
Administration.
Mee ng Report
Received Date : 15-02-2012
Revised Date : 27-02-2012
Accepted Date : 02-03-2012
DOI: 10.5530/rjps.2012.1.2
Address for
correspondence
Gowraganahalli
Jagadeesh, PhD.
Senior Expert Pharmacologist
Division of Cardiovascular and
Renal Products
Food and Drug
Administration (FDA)
10903 New Hampshire Ave
Bldg 22, Rm 4128
Silver Spring, MD
20993-0002, USA.
Phone: 001-301 796 1093
Fax: 301-796-9838
E-mail: gowra.jagadeesh@fda.
hhs.gov (G Jagadeesh)
pbala2006@gmail.com
(P Balakumar)
www.rjps.in
8 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

modeling generates an abundance of candidate molecules
with drug-like properties. The lead compounds
are selected based on a high degree of selectivity for
the target. Such compounds are also optimized in terms
of potency, physicochemical properties, pharmacokinetics
and drug metabolism, and toxic profi le. 3,4 This
information helps to reduce attrition of compounds
and contributes markedly to success rates in identifying
lead candidates. With only additional minor changes in
structure or other physico-chemical properties of the
lead compound, a potentially viable drug candidate is
launched (Box 1).
The quality of the lead compound is crucial to the
success of the refi nement and development process. The
lead drug candidate that enters the initial developmental
stage (see Box 1) must have a strong chance of meeting
the target profi le in terms of effi cacy, safety, route of
administration, and treatment regimen. An early step of
drug development includes pharmacologic, toxicologic,
pharmacokinetic, and metabolism/biotransformation
studies both in vitro and in animals. These studies
should reveal potentially useful (intended target) and
harmful (off target) properties of the drug candidate
in a qualitative and quantitative manner, thus permitting
an assessment of therapeutic risk. An investigational
new drug (IND in the USA) application is then fi led
with the drug regulatory agency demonstrating that
the CMC (chemistry and manufacturing controls),
nonclinical pharmacologic and toxicologic properties
of the drug have been well characterized. The next
step in drug development (clinical phase) is to
demonstrate that the benefi t-to-risk ratio as observed
in pharmacology and toxicology studies is confi rmed
Box 1: The Discovery to Development Scheme
1. Drug discovery (Early pharmaceutical research)
A. Target discovery (Exploratory phase)
1. Target identifi cation
2. Target validation
B. Hits discovery
1. Hits identifi cation
2. Hits validation (after hits reproducibility and dose effect)
C. Lead discovery
1. Lead generation/identifi cation
2. Lead refi nement/optimization
2. Drug development (Late Pharmaceutical R & D; drug
candidate launched)
A. Drug candidate selection
B. Preclinical development (short and long term studies)
C. IND application fi led
D. Clinical development (Phase 1, 2, 3)
E. NDA fi led
F. Drug regulatory agency approval for marketing
Balakumar, Jagadeesh: Pharmaceutical Discovery and Development
in patients. Standard endpoints in animal safety studies
focus upon morphologic assessments of tissues,
whereas the end points in initial human safety studies
(Phase 1) are largely physiologic, including assessments
of the cardiovascular system, central nervous system,
respiratory, and renal functions. At the completion of
phase 2 and phase 3 trials, if conclusive evidence of the
new molecule’s safety and effectiveness for a specifi c
indication exists, a new drug application (NDA in the
USA) is submitted to the drug regulatory agency. If
benefi ts of a drug for the intended patient population
outweigh the risks, the drug will receive approval from
the regulatory agency and thus, can be marketed (Box 1).
An overview of the symposium: This symposium addressed
selected key topics in drug discovery and development,
such as potential drug targets for therapeutic interventions,
early evaluation of pharmacokinetics and drug
metabolism, preclinical toxicity studies, and regulatory
guidelines in submission of investigative and new
drug applications. It concluded with the role of novel
and traditional biomarkers in monitoring disease or its
treatment during the course of clinical trials. The purpose
of this report is to highlight and disseminate key
information presented at the symposium as a means of
educating the scientifi c community so that they can stay
current with this rapidly developing fi eld.
The symposium had fi ve distinguished speakers affi liated
to drug regulatory affairs, industries and academia
from India and the USA. The symposium was arranged
by Dr. G. Jagadeesh of the US Food and Drug Administration
(FDA), and chaired by Prof. P. Balakumar of the
Institute of Pharmacy, Rajendra Institute of Technology
and Sciences, Sirsa, Haryana, India.
Drug Targets
The symposium began with a welcome address by
Dr. H.G. Shivakumar of J.S.S College of Pharmacy,
Mysore, Karnataka. Prof. P. Balakumar, the chairman
of the symposium, briefl y overviewed the topics for
presentation and profi led the speakers. Dr. G. Jagadeesh
(US FDA) then delivered the fi rst lecture on ‘Drugs and
Drug Targets’. A main element of early pharmaceutical
research is to reveal potential drug targets for therapeutic
intervention in the intended patient population.
Determination of the optimal molecular targets for
drug intervention provides the basis for the discovery
of new medicines. Disease-relevant target identifi cation
is considered an important criterion for determining
the correlation between drug and indication.
An important focus of drug discovery has been on
protein drug targets as a means of treating various
disorders. The advent of genomic sciences has
advanced our knowledge in the development of new
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 9

pharmaceuticals, as we now have a better understanding
of how genes are linked to diseases. It is estimated that
approximately 10% of the 25,000–30,000 known human
genes are linked to diseases. Identifying individual genes
considered potential targets (i.e., drugome) is not an ideal
task, because the functional product of each gene or
proteome is under multiple regulatory controls, including
splice variants and post-translational modifi cations.
Thus, targeting the body’s proteins or proteome is ideal
because druggability, or the ability of a drug to affect
a protein’s function, may be an inherent property of a
given protein. However, this does not constitute a useful
drug target unless the protein is also linked to a disease.
Nearly half of the disease-associated proteins are not
druggable as they lack a hydrophobic binding site for
a potential orally-delivered, cell membrane-permeable
drug molecule. This restricts the number of potential
drug targets to less than 1500. 5 Further, protein drug
targets should be viewed in the context of various
interrelated networks involved in the disease process.
Various factors such as genetic mutations, epigenetic
changes and pathogens can cause perturbations in the
function of networks, disrupting signaling pathways and
resulting in diseases. According to the Online Mendelian
Inheritance in Man, more than 1284 distinct disorders
have been reported. These are classifi ed into 22 disorder
classes based on the physiology of the system and 1777
disease-related genes. 6 Although the majority of disease
genes and the molecular basis for these diseases are not
known, knowledge of the human genome sequence has
increased potential new targets. The effective number of
novel drug targets has been calculated by the intersection
of the number of druggable proteins with the number
of disease-associated proteins, or 600–1500 novel drug
targets (2–5% of the genome). 5,7
Noting the report of Rask-Andersen et al., 8
Dr. Jagadeesh said that for the past thirty years (1982
to 2010) of drug target innovation, on average, about
18 new drugs targeting human proteins have been
approved by the FDA every year, of which about 4
are fi rst-in-class that act on novel target structures that
are encoded by the human genome, and the remaining are
follower or ‘me-too’ drugs. 8 According to these authors,
this translates to 435 drug targets over that time period.
Enzymes are the largest target class (47%) followed by
receptors (G-protein coupled receptors account for 30%,
the second largest), transporters (voltage-gated calcium
channels, the largest) and others. The most common
pharmacologic action of the approved drugs is antihypertensive
followed by anti-cancer, anti-infl ammatory,
sedative and hypnotic, anti-allergic, anti-convulsant, antiarrhythmic,
anti-psychotic, anti-depressant and analgesic.
Most drugs target only a select number of proteins
but some can target as many as 19 (e.g., bromazepam).
Balakumar, Jagadeesh: Pharmaceutical Discovery and Development
The average number of target proteins reported per drug
is 1.8 surpassing the number of single target drugs, 6 and
thus, are called target multipliers (polypharmacology).
Multi-target drugs are useful in cases where a
specifi c disease-associated protein is not druggable.
Dr. Jagadeesh also discussed various interactions, such as
between proteins (interactome), drugs (drug network),
drug and target (drug-target network) and disease and
disease genes (diseasome). It is anticipated that analysis
of these complex networks may also lead to new avenues
for drug target prediction.
Early Pharmacokinetics and Drug Metabolism
After lead molecules are identifi ed, they are optimized
in terms of potency, selectivity, ADME (absorption,
distribution, metabolism, and excretion) and toxicology
before they can become candidates for further drug
development. 3,9 It is important to emphasize the
integral role of drug metabolism and pharmacokinetics
(DMPK) in early phases of drug discovery, so as
to avoid failure due to poor pharmacokinetics and
bioavailability that largely result from suboptimal
physico-chemical properties. 10 DMPK scientists play
a multifaceted role in various functional areas, such as
drug biotransformation, pharmacology and toxicology
that help provide valuable information related to effi cacy
and safety of a new molecule. Dr. Sandhya Mandlekar
(Bristol-Myers Squibb India Ltd., India) spoke on the
topic of ‘Pharmacokinetics and Metabolism in Early
Drug Discovery’. While defi ning the evolving role
of DMPK studies in early pharmaceutical research,
Dr. Mandlekar described traditional ADME properties,
such as characterizing bioavailability and half-life in
animals and man. She also discussed advances in the fi elds
of pharmacogenetics, pharmacogenomics and drug
transporters; the shift in drug metabolism paradigms,
such as enzyme regulation, drug-drug interaction and
inter-individual variability; and predictive biomarkers
from animals to man. She also described the availability
of new technologies and using a tiered approach to
DMPK studies. Her presentation also focused on in vitro
models to predict hepatic clearance, CYP inhibition and
induction assays, Caco-2 permeability determination
assay for poorly soluble compounds, generating
in vivo EC50s by modeling nonclinical PK/PD data, and
investigating unusual metabolic reactions. Dr. Mandlekar
also addressed necessary steps in early pharmaceutical
research ADME studies (Box 2).
Finally, Dr. Mandlekar suggested that the mission of
discovery ADME studies is to ensure no development
candidate fails in the clinic due to unforeseen ADME
properties, and to improve developability characteristics
and risk assessment, in parallel with effi cacy, to get better
success rates during clinical development.
10 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

Box 2: A Focused Application of Discovery ADME
Studies
1. Active involvement earlier in the Discovery Process
2. Timely guidance to Chemistry to select chemotypes with
desirable ADME properties
3. Maximize informed decision making during Lead
Optimization
4. Improved ability to predict human DMPK
5. Stronger partnership with all areas of Drug Discovery and
Development
Toxicology Studies
A remaining critical issue in drug development is the
failure to identify, suffi ciently, toxicological problems
pertaining to new chemical entities early enough in the
drug research and development process. Better success
in developing early toxicologic screens will certainly
enhance the number of successful new chemical entities
entering to the clinical developmental phases and, in
fact, avoid the expense of terminating drugs in latestage
clinical development. Toxicity is a leading cause
of attrition at both clinical and nonclinical stages of
drug development. 4,10 The primary objective of nonclinical
toxicology studies in the drug discovery process
is to evaluate the safety of potential drug candidates for
human use. The ultimate goal is to translate the in vitro
and animal responses into an understanding of the risk
for human subjects under therapeutic conditions. Thus,
non-clinical toxicology testing is utilized at all stages of
the drug discovery and development processes.
Dr. K.S. Rao (Rao Toxicology Foundation, Bengaluru,
India, and Editor-in-Chief of Toxicology International)
spoke at length on ‘Toxicology Issues in Drug
Development’. Dr. Rao addressed various toxicological
issues encountered during the early phases of research
and drug development. The objectives of toxicity
tests, whether conducted in the whole animal or in
cell systems, should be to predict the adverse effects
of the tested compound in human beings both in a
qualitative and quantitative manner. While in silico and
in vitro models will continually be developed and refi ned,
in vivo preclinical safety models remain as the gold
standard for assessing human risk. Various cell culture
systems are an in vitro model of a target tissue in the
human body and mimic the response of human cells
to drug exposure. Several differentiated cell types have
been used in toxicology investigations. Among them,
the more widely used are cells derived from liver, lung,
heart, kidney, muscle, nervous and reticulo-endothelial
systems. Primary cultures or isolated cells are widely
used because the cells retain their specialized functions.
Dr. Rao also discussed the rapid development of
Balakumar, Jagadeesh: Pharmaceutical Discovery and Development
‘Functional genomics’, which includes proteomics and
transcriptomics, as an emerging discipline that represents
a global and systematic approach to identifying biological
pathways and processes in both normal and abnormal
physiological states. Although numerous tests have been
validated in the recent past, they not yet gained wide
spread use in discovery toxicology.
Dr. Rao noted that the fi rst of two stages in new drug
development is comprised of non-clinical (pharmacology
and toxicology) studies. The second stage is initiation
of clinical studies and completion of long-term animal
toxicity studies. The objectives of non-clinical studies
are to demonstrate the biologic activity (effi cacy and
selectivity) against the targeted disease and to provide
data for toxicity and safety evaluation (risk assessment).
Characterization of toxic effects with respect to target
organs, dependence of dose to effect, relationship to
systemic drug exposure, and potential reversibility of
any toxicities are all helpful in human dose escalation
strategies. The toxicity studies are limited at the beginning
of clinical development, but additional long-term toxicity
studies are conducted in tandem with on-going clinical
studies to help support the intended dose and duration
of the clinical trials. Serious adverse effects noted in
ongoing clinical trials may call for additional toxicologic
studies or even result in the clinical trials being halted
(clinical hold).
International regulatory guidelines require that new drugs
be tested for safety and effi cacy before marketing. The
recommendations for the extent of non-clinical safety
studies to support various stages of clinical development
differ only slightly among the regions of Europe, USA
and Japan. International Conference on Harmonization
(ICH) guidelines (ICH-M3) describe the consensus that
exists regarding the scope and duration of non-clinical
safety studies to support the conduct of human clinical
trials for medicinal products. The major toxicology
studies that are conducted to demonstrate the safety of a
Box 3: Major Categories of Toxicology Studies
Conducted During Drug Development (in Tandem
with Clinical Phases)
(http://www.fda.gov/Drugs/GuidanceCompliance
RegulatoryInformation/Guidances/ucm065007.htm)
1. Type, duration, and timing of non-clinical studies in support
of clinical trials (ICH M3(R2))
2. Safety Pharmacology studies (ICH S7A and S7B)
3. Toxicokinetics (ICH S3A)
4. Single and Repeat dose toxicity studies (ICH S4A)
5. Genotoxicity studies (ICH S2(R1))
6. Carcinogenicity studies (ICH S1A, S1B, S1C(R2))
7. Reproductive and development toxicity (ICH S5A, S5B)
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 11

new drug candidate are outlined below (Box 3). Besides
ICH, three other guidelines are available. Each of these
describes methodology and assay criteria for most of the
toxicology studies. They are: i) CFSAN Redbook 2000:
Toxicological Principles for the Safety Assessment of
Food ingredients; 11 ii) OECD guidelines; 12 and iii) EPA
guidelines for toxicity testing. 13
Regulatory Review Process in New Drug
Development
The long road from original concept to fi nal drug approval
has multifaceted phases that include pharmacologic,
pharmacokinetic and drug metabolism, toxicologic and
clinical studies. Dr. Gopi Vudathala (Global Regulatory
Affairs-Interface CMC Sanofi -Aventis, USA) spoke on
‘Overview of the Drug Development Process: The
IND and NDA Process’. His talk focused on the drug
development process describing the regulations and
guidances specifi c to the IND and NDA process. These
included pre-IND, IND application and types of reviews,
various phases of clinical trials, timing and duration of
non-clinical studies necessary to support clinical trials,
NDA registration process and necessary submission
requirements, NDA review process, and fi nally, NDA
actions. Dr. Vudathala integrated many of the issues
with strategic preclinical and clinical drug development
to the regulatory decision involved in licensing the
product for marketing. He also spoke on ‘relevant Laws’
(Federal Food, Drug, and Cosmetic Act; Public Health
Service Act-Part F Licensing of Biological Products and
Clinical Laboratories) and ‘relevant regulations’ (IND
regulations (both drugs and biologics) 21 CFR 312.23;
NDA (drugs) regulations-21 CFR 314.50; Product
licensing (biologics)-21 CFR 601; Protection of human
subjects and informed consent regulations-21 CFR 50;
IRB regulations-21 CFR 56) for INDs and NDAs. In
the presentation, Dr. Vuduthala listed requirements for
a new IND with respect to chemistry/manufacturing/
controls (CMC or Quality) data and pharmacologic and
toxicologic data.
On the subject of clinical trials, Dr. Vuduthala noted that
the key to initiating clinical trials in the US is to ensure
that a good safety and preclinical toxicology package is
available for review in the initial IND stage along with
a good clinical plan to evaluate proof of concept in
healthy volunteers (Phase 1). Subsequently, performing
dose ranging studies and confi rming effi cacy in a small
population of relevant patients will need to be done in
early Phase 2 studies. Based on the results, a detailed
clinical plan is developed for Phase 2b and 3 pivotal
clinical trials in a larger patient population. Throughout
the drug development process, there are a number of
opportunities for the industry to meet with members
of the regulatory agency to discuss preclinical, clinical
Balakumar, Jagadeesh: Pharmaceutical Discovery and Development
and CMC plans and any issues requiring resolution.
A pre-IND meeting, end of phase 2 meeting and pre-
NDA meetings are keys to discuss the path forward
and ensure the sponsor’s development plans are vetted
by the Agency. Development of the product label
(or package insert) occurs in parallel to the above, and
labeling discussions are an important part of the NDA
registration process.
Biomarkers
A fi nal topic discussed, which poses a great challenge
to drug developers, regulators and physicians, is
identifi cation of a ‘biomarker’, a biologic indicator,
parameter or marker that can be used to measure
the disease progress or the effects of treatment.
The parameter can be physical or chemical as well as
biological. A biomarker is defi ned as a characteristic
that is objectively measured and evaluated as an
indicator of normal biologic processes, pathogenic
processes, or biological responses to a therapeutic
intervention. 14 The last speaker of the symposium,
Dr. Samba Reddy (Texas A&M University Health
Science Center, USA) spoke on ‘Biomarkers in
Preclinical and Clinical Drug Development’. Dr. Reddy
focused on current knowledge on biomarkers in drug
development (Box 4).
Dr. Reddy discussed differences between diseaserelated
and drug-related biomarkers. Disease-related
biomarkers give an indication of the disease pathways
or disease mechanism; whereas drug-related biomarkers
indicate whether a drug will be effective in a specifi c
patient and how the patient’s body will process it. The
speaker covered the various aspects of biomarkers,
including ‘Research Breaking in Classifi ed Biomarkers’,
‘Biomarker Discovery Technology’, ‘Biomarker
Quantifi cation and Assay’, ‘Biomarkers in Drug
Discovery and Development’, ‘Disease Biomarkers
and Healthcare’ and ‘Cancer Biomarkers’. The rapid
emergence and evolution of translational medicine have
Box 4: Traits of Biomarkers
1. A biomarker can be a substance that is introduced into an
organism to examine organ function or other aspects of
health
2. Biomarkers can be used in early diagnosis, disease
prevention, drug treatment, drug response, drug target
identifi cation, etc.
3. Biomarkers represent key tools and parameters for the
accelerated new drug discovery and clinical development
4. Biomarkers can be classifi ed into three types: Natural
history markers; Drug activity markers; and Surrogate
markers
5. In clinical trials, the biomarker serves as a surrogate
endpoint for evaluating clinical benefi ts
12 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

pushed biomarkers into the spotlight for successful drug
development. He said that established pharmaceutical
companies are integrating the biomarker strategies into
their research product pipelines. In concluding remarks,
Dr. Reddy said that the path from biomarker discovery
to commercialization is fraught with numerous obstacles.
Novel technologies are needed for biomarker discovery,
and such approaches would be helpful in streamlining
the drug development process by reducing time and
costs, as well as minimizing drug attrition associated
with safety issues.
ACKNOWLEDGEMENTS
We sincerely thank Drs. Sandhya Mandelkar (Head,
Department of Pharmaceutical Candidate Optimization
(PCO), Bristol-Myers Squibb India Ltd., Biocon BMS
R&D Center, Bengaluru, India), K.S. Rao (Chief Scientifi c
Offi cer, Rao Toxicology Foundation, Bengaluru, and
Editor-in-Chief of Toxicology International), Gopi
Vudathala (Associate Vice President, Global Regulatory
Affairs-Interface CMC Sanofi -Aventis, New Jersey, USA)
and Samba Reddy (Associate Professor, & Principal
Investigator, Texas A&M University Health Science
Center, College of Medicine, College Station, Texas,
USA) for providing us their presentation materials
from which this meeting report was prepared. We also
express our gratitude to Dr. B.R. Jagashetty (Drugs
Controller for the State of Karnataka, and Chairman
of the Local Organizing Committee of 63rd IPC),
Dr. N. Udupa (Convener, Scientifi c Services of IPCA),
and Mr. S.M. Mudda (Chairman, Local Scientifi c
Balakumar, Jagadeesh: Pharmaceutical Discovery and Development
Committee of 63rd IPC) for their inspiration and
support for this symposium.
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RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 13

Nanobiotechnology: An overview of drug
discovery, delivery and development
Bhupinder Singh Sekhon
PCTE Institute of Pharmacy, Jhande, Near Baddowal Cantt (Ludhiana)-142 021, Punjab, India
ABSTRACT
Nanobiotechnology is a new technology concerned specifi cally with the functionalism and modifi cation of
chemical-physical structures on a biomolecular scale, and also is the application of nanotechnology to the life
sciences. Nanotechnology for biotechnology and pharmaceutical applications has progressed from the concept
stage to commercialization. Nanobiotechnology represents the future of medicine and healthcare. Various physical,
chemical, electrical tools and methods used to investigate biological nanoobjects include optical tools, nanoforce
and imaging, surface methods, mass spectrometry and microfl udics. Its application has an impact on diagnostics,
drug delivery as well as drug discovery. Nanobiotechnology focuses on various areas such as nanobiotechnology
and cancer, drug discovery and tools, and nanobiotechnology and medicine. Applications are emerging from
all branches of nanobiotechnology in medicine and pharmacy. Several technologies including nanoparticles
and nanodevices such as nanobiosensors and nanobiochips have been used to improve drug discovery and
development. Some nanosubstances such as fullerenes and dendrimers/biodendrimers could be potential drugs
for the future. Moreover, nanobiotechnology has the potential for combining drug design and drug delivery.
However, limitations of the available nanoparticles still to be resolved for their application in the drug-discovery
studies exist. The benefi ts of nanotechnology are enormous and so these benefi ts should be maximized while
efforts are made to reduce the risks.
Keywords: Nanobiotechnology, nanobiomaterials, nanobiostructures, nanocarriers
INTRODUCTION
Nanotechnology is a broader term applicable
to small things in nanometer range (roughly
in the 1−100 nm size regime in at least
one dimension). Nanobiotechnology is a
big word made up of three parts: NANO
is really, really tiny, BIO is living things,
and TECHNOLOGY is about tools.
Nanobiotechnology is an emerging area
of science which is concerned with the
application of tools and processes in order
to assemble devices for the study of objects
in biological systems. Nanobiotechnology
is that branch of nanotechnology that deals
with biological and biochemical applications
or uses. Nanobiotechnology often studies
existing elements of living organisms and
nature to fabricate new nano-devices.
Generally, nanobiotechnology refers to
the use of nanotechnology to further
the goals of biotechnology. Relationship
of nanotechnology, biotechnology and
nanomedicine is shown in Figure 1.
Further, nanobiotechnology (an integration
of physical sciences, molecular engineering,
biology, chemistry and biotechnology) has
yielded healthcare discoveries that have
been used for drug delivery and diagnostic
purposes. 1
In medicine and pharmacology, nanobiotechnology
opens up new perspectives in
analytics and therapy. Medical applications
of nanobiotechnology resulted in appearance
of new fi eld i.e. nanomedicine. An
increasing use of nanobiotechnology by
the pharmaceutical industries includes
(i) drug delivery, and (ii) disease therapy.
The potential topics in nanobitechnology
Review Ar cle
Received Date : 17-11-2011
Revised Date : 04-01-2012
Accepted Date : 17-02-2012
DOI: 10.5530/rjps.2012.1.3
Address for
correspondence
Bhupinder Singh Sekhon
PCTE Institute of Pharmacy
Jhande, Near Baddowal Cantt)
(Ludhiana)-142 021
Punjab, India
E-mail: sekhon224@yahoo.co
Mobile: 91-161-9876242299
www.rjps.in
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Bhupinder Singh Sekhon et al.: Nanobiotechnology: An overview of drug discovery, delivery and development
Figure 1: Relationship of nanotechnology, biotechnology and nanomedicine.
include molecular bioprobes, nanoparticles and nanobiosystems,
nanobiomaterials, biomolecular assemblies
and supra-biomolecules, nanobiosensors and nanobiochips,
BioNEMS and nano-biofl uidics, nanobiophotonics,
single-molecule detection and manipulation
and molecular motors. Experts are of the opinion that
nanobiotechnology has the potential to yield a scientifi
c and industrial revolution, as envisioned by the
numerous programs on nanotechnology/nanobiotechnology
launched over the last decade by councils and
governments worldwide.
The potential uses and benefi ts of nanobiotechnology
are enormous. 2 Nano-systems in biology, the most
complex and highly functional nanoscale materials and
machines have been invented by nature. Proteins and
nucleic acids, and other naturally occurring molecules
(polymers) regulate and control biological systems with
incredible precision. Many nanotechnologists are in fact
drawing inspiration from biology to device new materials
and devices. Moreover, nanotechnology/nanobiotechnology
is expected to rule tomorrow’s world.
NANOBIOSTRUCTURES
The use of biological principles is becoming widespread in
the design of nanomaterials. Representative examples of
nanobiostructures include DNA nanostructure, peptide
structure and biomimetics. 3D-DNA nanostructures
have emerged as promising tools for biology and materials
science. In this regard, DNA cages, nanotubes, dendritic
networks, and crystals with deliberate variation of their
size, shape, persistence length, and porosities exhibited
dynamic character, allowing their selective switching
with external stimuli. Short peptides can spontaneously
associate to form nanotubes, nanospheres, nanofi brils,
nanotapes, and other ordered structures at the nanoscale.
Further, peptides can also form macroscopic assemblies
such as hydrogels with nanoscale order. The theory and
the mechanisms behind peptide self-assembly process
and their bionanotechnology applications have been
reported. 3 In general, nanobiotechnology requires
the organization of atoms and molecules in a two- or
three-dimensional space. Self-assembly properties of
biomolecules have ability to spontaneously organize
into nanostructures, which allows mimicking the living
cell membranes.
Nanoparticle usually forms the core of nanobiomaterial.
Nanobiomaterial is made of nanoparticles and
nanobiomaterials are emerging as the most hopeful
area of research within the area of biological materials
science and engineering. They have an increased number
of atoms and crystal grains at their surfaces and possess
a higher surface area to volume ratio than conventional
microscale biomaterials. These differences in surface
topography alter the corresponding surface energy
for protein adsorption. Nanobiomaterials can be used
for human implant, orthopedics, drug delivery, gene
therapy, antimicrobial treatments, array technologies,
and diagnostics. Moreover, nanobiomaterials help
with targeting, measuring, sensing, and imaging. The
approaches used in constructing nanobiomaterials are
given in Figure 2.
NANOBIOTECHNOLOGY BASED DRUG DELIVERY/
DEVELOPMENT
Nanomedical approaches to drug delivery center
on developing nanoscale particles or molecules to
improve the bioavailability of a drug. Nanomaterials
and nanoparticles are likely to be cornerstone of
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Bhupinder Singh Sekhon et al.: Nanobiotechnology: An overview of drug discovery, delivery and development
Figure 2: Specific configuration of nanobiomaterial when applied to biological or medical applications.
innovative nanomedical devices employed for drug
discovery and delivery, discovery of biomarkers
and molecular diagnostics. 4 Preparation methodsproperties
of nanoscale systems including liposomes,
micelles, emulsions, nanoparticulates, and dendrimer
nanocomposites, and clinical indications are known
for imaging in vivo, sustained and targeted delivery of
drugs, genes, and proteins. The recent development
in drug delivery system has opened up new potential
& possibility for production of pharmaceutical drugs,
oral peptides, gene therapy and nanocosmeceuticals. 5
Nanobiotechnology exploits nanotechnology and
biotechnology to analyze and create nanobiosystems
to meet a wide variety of challenges and develops a
wide range of applications. Biomaterials like DNA and
proteins combined with electronic systems consequently
results in the formation of new devices, sensors, and
systems. Examples include (i) a carbon nanotube
with single-stranded DNA wrapping around it. (ii) a
graphene sheet with duplex DNA molecules. (iii) a
nanoparticle attachment with antibodies, (iv) a nanorod
after enzyme immobilization and (v) Streptavidin
(protein) attachment to nanoparticles along with biotin
(protein). 6 Several technologies, including nanoparticles
and nanodevices such as nanobiosensors, nanobiochips,
nanosubstances were used to improve drug discovery
and development. 7
Gold nano rod-DARPP-32siRNA complexes (nanoplexes)
have been reported suitable for brain-specifi c
delivery of appropriate siRNA for therapy of drug
addiction and other brain diseases. 8 Boron nitride nanotubes
are structural analogues of carbon nanotubes in
nature: alternating B and N atoms entirely substitute for
C atoms in a graphitic like sheet with almost no change
in atomic spacing. By virtue of their magnetic properties,
boron nitride nanotubes could be exploited for
magnetic, physically guided, drug targeting. The interactions
between boron nitride nanotubes and living cells
were reported and the piezoelectric properties of boron
nitride nanotubes make them attractive candidates as
bionanotransducers for cell sensing and stimulation,
a use which still has to be exploited. 9
DENDRIWORMS
Dendriworms (magnetic nanoworm + dendrimer) are
synthetic polymers that can be used to carry a large range
of molecules such as siRNA (made up of magnetic
nanoparticles as well as a fl uorescent nanoparticle)
which allow the nanoworm to be traced as to where
it is. Tremendous progress has been achieved in the
recent years in our understanding of the ability of small
interfering RNAs to silence gene expression in mammalian
cells. This has provided us with a revolutionary new tool
to modulate the expression of disease-causing genes.
Short interfering RNAs (siRNAs) have emerged as a
potent new class of therapeutics, which regulate gene
expression through sequence-specifi c inhibition of
mRNA translation. Lipid-, polymer-, and nanoparticlebased
siRNA delivery vehicles have proven effective
in improving the stability, bioavailability, and target
specifi city of siRNAs following systemic administration
in vivo. Additionally, these methods provided a platform
to modify siRNAs with a variety of contrast agents and
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Bhupinder Singh Sekhon et al.: Nanobiotechnology: An overview of drug discovery, delivery and development
have enabled nuclear and magnetic resonance imaging
of siRNA delivery in preclinical studies. Such imageguided
delivery approaches represent a crucial step in
the transition of siRNA therapeutics to the clinic. 10
Scientists reported the development of dendriworms
as a modular platform for siRNA delivery in vivo.
Researchers have demonstrated that siRNA-carrying
dendriworms can be readily internalized by cells and
enabled endosomal escape across a wide range of loading
doses, whereas dendrimers or nanoworms alone were
ineffi cient. In addition, dendriworms carrying siRNA
against the epidermal growth factor receptor reduced
protein levels of epidermal growth factor receptor in
human glioblastoma cells by 70−80%, 2.5-fold more
effi ciently than commercial cationic lipids. Reported
data established dendriworms as a multimodal platform
that enabled fl uorescent tracking of siRNA delivery
in vivo, cellular entry, endosomal escape, and knockdown
of target proteins. 11
INORGANIC NANOCRYSTALS
Researchers reported the fi rst use of inorganic
fl uorescent lanthanide (europium and terbium) ortho
phosphate [LnPO ·H O, Ln = Eu and Tb] nanorods as
4 2
a novel fl uorescent label in cell biology. These nanorods,
synthesized by the microwave technique, retained their
fl uorescent properties after internalization into human
umbilical vein endothelial cells (HUVEC), 786-O cells,
or renal carcinoma cells (RCC). At concentrations up to
50 μg/ml, the use of [ 3H]-thymidine incorporation assays,
apoptosis assays (TUNEL), and trypan blue exclusion
illustrated the non-toxic nature of these nanorods,
a major advantage over traditional organic dyes. 12
DESIGN OF NEW-AGE DELIVERY SYSTEMS
Scientists have shown a modifi ed version of the bacteriophage
phi29 DNA-packaging motor, when reconstituted
into liposomes and inserted into planar lipid bilayers,
allowed the translocation of double-stranded DNA.
Moreover, this engineered and membrane-adapted
phage connector is expected to have applications in
microelectromechanical sensing, microreactors, gene
delivery, drug loading and DNA sequencing. 13
Aptamers
Aptamers, single stranded DNA or RNA molecules,
generated by a method called SELEX (systematic
evolution of ligands by exponential enrichment) have
been widely used in various biomedical applications.
Technology combination of nanobiotechnology
with aptamers opened the way to more sophisticated
applications in molecular diagnosis. Recent developments
in SELEX technologies and new applications of
aptamers have the ability to discriminate between two
closely related targets, for example a cancerous cell and
an untransformed cell of the same tissue type, makes
aptamers suitable as imaging reagents for non-invasive
diagnostic procedures. 14
Implantable biomedical devices
A bilayer structure comprising a thin gold layer and
a polypyrrole fi lm has been developed as a valve and
holds promise for implantable biomedical devices. 15
Implantable medical devices that comprise a substrate
and a porous layer comprising close packed spherical
pores disposed over the substrate have been reported
and the porous layer may also comprise a therapeutic
agent. 16
Nanoprobes
Tiny nanoprobes have shown to be effective in delivering
cancer drugs more directly to tumor cells - mitigating
the damage to nearby healthy cells - and research has
shown that the nanoprobes are getting the drugs to
right cellular compartments. 17 Carbon nanotubes’
unique properties including low cytotoxicity and good
biocompatibility attract their use as vector system in
target delivery of drugs, proteins and genes. 18
Researchers have focused on the integration of biological
molecules (DNA, antibodies, and enzymes)
into micro- and nanostructures, with state-of-theart
bioelectronic read-out systems, extracting useful
analytical signals with interest for various fi elds. In
this direction, nanotechnology based biosensors are
the product of this integration with great interest for
several applications that aim at a signifi cant improve
of the quality and security of citizen’s life. 19 Rapamune
was the fi rst product based on approved NanoCrystal
Technology. Nanodrugs and nanodevices approved by
the FDA, and emerging nanoproducts that may pose
a challenge for current regulatory schemes both in the
U.S. and internationally, have been reported. 20
Scientists have designed a novel drug delivery vehicle
by hybridizing macrophages with nanoparticles through
cell surface modifi cation. Nanoparticles immobilized
on the cell surface provided numerous new sites for
anticancer drug loading, hence potentially minimizing
the toxic effect of anticancer drugs on the viability and
hypoxia-targeting ability of the macrophage vehicles.
In particular, quantum dots and 5-(aminoacetamido)
fl uoresceinlabeled polyamidoamine dendrimer G4.5,
both of which were coated with amine-derivatized
polyethylene glycol, were immobilized to the sodium
periodate-treated surface of RAW264.7 macrophages
through a transient Schiff base linkage. Further, a
reducing agent, sodium cyanoborohydride, was applied
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Bhupinder Singh Sekhon et al.: Nanobiotechnology: An overview of drug discovery, delivery and development
to reduce Schiff bases to stable secondary amine linkages.
The distribution of nanoparticles on the cell surface was
confi rmed by fl uorescence imaging, and it was found to
be dependent on the stability of the linkages coupling
nanoparticles to the cell surface. 21
Protein therapy
Protein therapy, which delivers proteins into the cell
to replace the dysfunctional protein, is considered the
most direct and safe approach for treating disease and
has been looked into as a possible alternative to gene
therapy. Scientists have demonstrated a general, effective;
low-toxicity intracellular protein delivery system based
on single-protein nanocapsules, and opened a new
direction not only for protein therapy but also for
cellular imaging, tumor tracking, cosmetics and many
other applications. 22 RNAi screen of the protein kinome
identifi ed checkpoint kinase 1 (CHK1) as a therapeutic
target in neuroblastoma. 23
Stabilizing proteins at high concentration is of broad
interest in drug delivery, for treatment of cancer
and many other diseases. Scientists created highly
concentrated antibody dispersions (up to 260 mg/ml)
comprising of dense equilibrium nanoclusters of
protein [monoclonal antibody (mAb) 1B7, polyclonal
sheep Immunoglobulin G (IgG) and bovine serum
albumin (BSA)] molecules, which upon dilution in vitro or
administration in vivo, remained conformationally stable
and biologically active. The nanoclusters are formed by
adding trehalose as a co-solute which strengthened the
short-ranged attraction between protein molecules. The
protein cluster diameter was reversibly tuned from 50 to
300 nm by balancing short-ranged attraction against
long–ranged electrostatic repulsion of weakly charged
protein at a pH near the isoelectric point (pI). 24
Dendrimers/biodendrimers
Dendrimers are nanostructures produced from
macromolecules such as polyamidoamine, polypropyleneimine
and polyaryl ether; and are highly branched with
an inner core. The particle size range is between 1 to
100 nm although their sizes are mostly less than 10 nm.
Biodendrimers are dendrimers composed of repeating
units known to be biocompatible or biodegradable
in vivo to natural metabolites. Medical applications of
dendrimers as multifunctional nanosized containers are
shown in Figure 3.
Dendrimers have successfully proved themselves
as useful additives in different routes of drug
administration to be applied in routes with particular
reference to intravenous, oral, transdermal, and ocular
delivery systems. 25 Dendrimers can act as vectors, in
gene therapy and as an agent for Photodynamic Therapy
of tumorigenic keratinocytes. 26
Figure 3: Some medical applications of dendrimers/biodendrimers.
NANOSTRUCTURED DRUG DELIVERY SYSTEMS
Bionanofabrication
2D crystallization of proteins, especially engineered
proteins, is emerging as a powerful tool for bottom-up
approaches to the nanofabrication of functional
structures. Some aspects of 2D protein crystallization,
including key approaches to growing 2D protein crystals
and their potential applications ranging from biosensors,
diagnostic kits, vaccine applications, and templates for
mineral formation have been reported. 27
Nanostructured lipid carriers (NLC) that can deliver
active pharmaceutical ingredients across the skin have
emerged as novel systems composed of physiological
lipid materials suitable for topical, dermal and
transdermal administration. The design characteristics,
production and composition of semi-solid formulations
containing NLC as API carriers (for example hydrogels)
have been reported. 28 Results of characterization studies
strongly supported the potential application of these
drugs-loaded NLC as prolonged delivery systems for
lipophilic drugs by several administration routes, in
particular for intravenous administration. 29
Fullerenes for Medical Diagnostics
Scientists have created new materials from Fullerenes by
fi lling them with atoms of various metals. An important
example is a fullerene that encases a sensitive contrast
agent (gadolinium) for MRI applications, including as
a diagnostic and therapeutic agent for brain tumors.
Researchers have co-invented a hands-off process for
fi lling fullerenes with radioactive material and these fi nding
could be utilized in medical applications, such as MRIs
and diagnostic and therapeutic agents for brain tumors. 30
NANOBIO TECHNOLOGY AND TISSUE ENGINEERING
Scientists discussed the current applications of nanoscale
materials to bladder tissue engineering. 31 Researchers
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Bhupinder Singh Sekhon et al.: Nanobiotechnology: An overview of drug discovery, delivery and development
explored the bottom-up and top-down nanofabrication
technologies and their use in various drug delivery and
tissue engineering applications. 32
NANOBODIES
The structural properties of Nanobodies and their
possible therapeutic applications were discussed and
data from phase I clinical trials of the novel ‘fi rstin-class’
anti-thrombotic agent ALX-0081 (Ablynx NV)
were reported. 33 Results suggested that the generation
of Nanobody-displaying immune phage libraries and
subsequent in vivo biopanning in appropriate animal
models is a promising approach for the identifi cation of
novel vascular targeting agents. 34
NANOBOTS/NANOROBOTS
Nanorobots could be employed for the diagnostics,
targeted drug dispensation, elimination of xenogenous
particles from the body, and repair of cells and tissues,
e.g. the skin and teeth. 35 Nanorobots could also be
programmed to perform delicate surgeries or remove
obstructions in the circulatory system. 36
BIOMARKERS
Biomarkers are molecules that can be measured in blood,
other body fl uids, and tissues to assess the presence or
state of a disease. They have the potential to help us
detect cancer earlier, determine a tumor’s aggressiveness,
or predict a patient’s response to a particular treatment.
Biomarkers play a role in use of pharmacogenetics,
pharmacogenomics and pharmacoproteomics for
development of personalized medicine. Label-free
nanosensors can detect disease markers to provide
point-of-care diagnosis that is low-cost, rapid, specifi c
and sensitive. Scientists showed specifi c and quantitative
detection of two model cancer antigens from a 10 μl
sample of whole blood in less than 20 min. They used
nanowire sensors to detect and measure concentrations
of two specifi c biomarkers: one for prostate cancer
and the other for breast cancer. This novel device acted
as a fi lter, catching the biomarkers, antigens specifi c
to prostate and breast cancer on a chip while washing
away the rest of the blood. 37 The emerging trends in
the development of biomarkers for early detection and
precise evaluation of cancer disease were reported. 38
This bio-nano-info convergence holds great promise for
molecular diagnosis and individualized therapy of cancer
and other human diseases. Scientists have demonstrated
the feasibility of multiplex detection using the surfaceenhanced
Raman scattering-based molecular sentinel
(MS) technology in a homogeneous solution. Two MS
nanoprobes tagged with different Raman labels were
used to detect the presence of the erbB-2 and ki-67
breast cancer biomarkers. The multiplexing capability of
the MS technique was demonstrated by mixing the two
MS nanoprobes and tested in the presence of single or
multiple DNA targets. 39
NANOBIOTECHNOLOGY IN CANCER
Application of nanotechnology/nanobiotechnology in
biomedicine may contribute to signifi cant advances in
imaging diagnosis and treatment of cancer. Quantum
dots, gold nanoparticles, magnetic nanoparticles,
carbon nanotubes, gold nanowires and many other
materials have been developed over the years, alongside
the discovery of a wide range of biomarkers to lower
the detection limit of cancer biomarkers. Current
developments in cancer detection methods with an
emphasis on nanotechnology were reported. 40 Some
of the recent development in nanotechnologies and
their applications in diagnosing and developing cancer
therapies have been reported. 41
Scientists reported a cooperative nanosystem consisting
of two discrete nanomaterials that work in concert within
the bloodstream to locate, adhere to and kill cancerous
tumors. While one type of nanoparticle improved
detection of the tumor, the other was designed to kill the
tumor. The fi rst component consists of gold nanorod
“activators” that populate the porous tumor vessels and
act as photothermal antennas to specify tumor heating
via remote near-infrared laser irradiation. In addition,
it was found that local tumor heating accelerated the
recruitment of the second component: a targeted
nanoparticle consisting of either magnetic nanoworms
(NW) or doxorubicin-loaded liposomes (LP). Nineamino
acid peptide LyP-1 (Cys-Gly-Asn-Lys-Arg-Thr-
Arg-Gly-Cys) was employed as the targeting species that
binds to the stress-related protein, p32, which was up
regulated on the surface of tumor-associated cells upon
thermal treatment. Mice containing xenografted MDA-
MB-435 tumors that were treated with the combined
gold nanorod /LyP-1- doxorubicin-loaded liposomes
therapeutic system displayed signifi cant reductions in
tumor volume compared with individual nanoparticles
or untargeted cooperative system. 42
A new nanoparticle formulation can be magnetically
guided to deliver and silence genes in cells and tumors
in mice. This formulation, termed LipoMag, consisted
of an oleic acid-coated magnetic nanocrystal core
and a cationic lipid shell. When compared with the
commercially available PolyMag formulation, LipoMag
displayed more effi cient gene silencing in 9 of 13 cell
lines, and better anti-tumour effects when systemically
administered to mice bearing gastric tumours. By
delivering an optimized sequence of a silencing RNA
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Bhupinder Singh Sekhon et al.: Nanobiotechnology: An overview of drug discovery, delivery and development
that targets the epidermal growth factor receptor of
tumor vessels, the intended therapeutic benefi t was
achieved with no evident adverse immune reaction or
untoward side effects. 43
NANOMEDICINE
Nanomedicine has triggered the wind of revolution in
medicine and important areas that nanomedicine covers
include targeted drug delivery in cancer treatment,
biotechnology, disease diagnosis to nanostructure
implants. 44
A variety of recent research combines Heparin (HP) and
nanomaterials for a myriad of applications. HP has been
conjugated to the surface of the nanoparticles, such as
magnetic and metallic nanoparticles, or biodegradable
and nondegradable synthetic polymers. HP has also
been incorporated into the nanoparticles. The different
possibilities of HP-based nanoparticle composites and
their medicinal or biological applications were reported. 45
It was qualitatively demonstrated that enhanced
fl uorescence emission signals occurred from clustered
QDs and deduced that the band 3 membrane proteins
in erythrocytes were clustered. 46
Nanonephrology
Nanonephrology is a branch of nanomedicine and
nanotechnology that deals with (i) the study of kidney
protein structures at the atomic level (ii) nano-imaging
approaches to study cellular processes in kidney cells and
(iii) nanomedical treatments that utilize nanoparticles
and to treat various kidney diseases. Various devices
based on nanotechnology are used for the studying
the different kidney processes and detecting disorders.
Nanotechnological fi lters have the potential to provide
immediate relief for dialysis patients. 47 Scientists have
demonstrated a new artifi cial renal chip by integrating
a high effi cient biocompatible polymeric nanofi bers
membrane with the polydimethylsiloxane(PDMS)
based micro-fl uidic platform via the optimization of
PDMS micro fl uidic channel network and the multiple
packaging of nanofi bers membrane. 48
Nanobiopharmaceutics
Nanobiopharmaceutics aims at the development of
innovative multidisciplinary approaches for the design,
synthesis and evaluation of molecular, nano- and microscale
functionalities for targeted delivery of therapeutic
peptides and proteins (biopharmaceutics). Nanoscale
biomaterials are categorized by metal, non-metal, carbon,
polymer, lipid, virus and miscellaneous nanostructures
as nanobiopharmaceutical carrier systems and their
medical/biological applications as well as toxicological
issues in the fi eld of biomedical nanotechnology
were reported. 49 The basis of technologic application
in biopharmaceutics is Nanoscale Drug Carrying
System and the various units of the system consist of
liposome system which varies between 30 nm to several
micrometers in diameter with their characteristics
governed by their size, surface composition and charge.
Surface modifi ed liposomes carrying doxorubicin and
antisense oligonucleotide system have successfully
targeted multidrug resistance associated protein,
messenger RNA and bcl2 RNA. System after reaching
the cell, delivered the doxorubicin and the antisense
oligonucleotides successfully and inhibited the synthesis
of MRP1 and bc12 RNA and provoked the apoptosis
of carcinomatous cell by arousing the caspase (cysteineaspartic
proteases) dependent pathway. 50
A “nanoviricide” is a fl exible nano-scale material
approximately a few billionths of a meter in size, which
is chemically programmed to specifi cally target and
attack a particular type of virus like a guided missile.
NV-INF-1, the selected candidate drug substance of the
FluCide program, has been shown to be highly effective
in controlling infl uenza viral infection in lethal infection
mouse model. NanoViricides nanotechnology possesses
potent antiviral effi cacy by targeting the mechanisms by
which viruses attach or bind to cells. 51
Nanopharmacology
Nanopharmacology is the use of nanotechnology
for (i) discovery of new pharmacological molecular
entities; (ii) selection of pharmaceuticals for specifi c
individuals to maximize effectiveness and minimize side
effects; and (iii) delivery of pharmaceuticals to targeted
locations or tissues within the body . The potential
applications of biochips, nanosensors, bioreactors,
neural stem cells, immune nanoparticles, biodegradable
polymers, and convection-enhanced drug delivery in the
diagnostics and treatment of diseases were reported.
Numerous novel medicinal forms were reported,
including polymeric nanoparticles, nanotubes, micelles,
liposomes, dendrimers, fullerenes, and hydrogels. In
particular, highly stable glycosphingolipid nanotubes
and nanoliposomes were proposed as drug delivery
systems. For this purpose, the model of stimulation of
skin vasomotor reactions by nitroglycerin application
was developed. The effect of nitroglycerin was found to
increase 1.5 times when used in the form of dispersion
with nanotubes as carriers and almost 2.5 times in the
case of dispersion with nanoliposomes as carriers.
Nanotechnologically manufactured biologically active
substances Apiton-25 (containing apis products) and
Microhydrin (containing SiO nanoparticles, silicon
2
hydrogen bonds Si-H, and free negative charges for free
radical neutralization) results have indicated that both
Apiton-25 and Microhydrin upon peroral administration
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Bhupinder Singh Sekhon et al.: Nanobiotechnology: An overview of drug discovery, delivery and development
enhanced the cyclic trial performance during prolonged
submaximal exercise in endurance-trained cyclists. 52
Scientists have developed a combination drug that
promises a safer, more precise way for medics and fellow
soldiers in battle to give a fallen soldier both morphine
and a drug that limits morphine’s dangerous side effects.
They have used nanotechnology to devise ultra-small
polymer particles capable of carrying the drugs into
the body. The development of the combination drug
makes possible a precise feedback system that can safely
regulate release of the drugs aboard the nanoparticles. 53
NANOBIOTECHNOLOGY AND PERSONALIZED
MEDICINE
Nanobiotechnology will facilitate the integration
of diagnostics with therapeutics and facilitate the
development of personalized medicine. 54 Nanobiotechnology
is being used to refi ne discovery of biomarkers,
molecular diagnostics, drug discovery and
drug delivery, which are important basic components of
personalized medicine. 55 Personalized management of
cancer, facilitated by nanobiotechnology, is expected to
enable early detection of cancer, more effective and less
toxic treatment increasing the chances of cure. 56
Nanobiomaterials/Nanobioanalysis/
Nanobiochips
Novel opportunities and challenges offered by nanobiomaterials
in tissue engineering have been reported. 57
The current state of development of nanodiagnostic
technologies including nanobiochips and nanobiosensors
has been reported. 58 Neuronanobiotechnology
based delivery system are developing rapidly and one
example of current nanobiotechnological research
involved nanospheres coated with fl uorescent polymers.
59 An assay based on gold nanoparticles could
detect recurrences of prostate cancer sooner than is
possible with existing techniques. 60 Carbon nanotubes
have been used to probe the properties of bilayer systems
resembling living cell membranes. 61
A multifunctional one-dimensional nanostructure
incorporating both CdSe quantum dots (QDs) and Fe O 3 4
nanoparticles (NPs) within a SiO -nanotube matrix was
2
successfully synthesized based on the self-assembly of
preformed functional NPs, allowing for control over the
size and amount of NPs contained within the composite
nanostructures. This specifi c nanostructure is distinctive
because both the favorable photoluminescent and
magnetic properties of QD and NP building blocks
were incorporated and retained within the fi nal silicabased
composite, thus rendering it susceptible to both
magnetic guidance and optical tracking. Moreover, the
resulting hydrophilic nanocomposites were found to
easily enter into the interiors of HeLa cells without
damage, thereby highlighting their capability not
only as fl uorescent probes but also as possible drugdelivery
vehicles of interest in nanobiotechnology. 62
A large variety of nanobioanalysis methods have been
reported. 63 Scientists have described the construction
and use of two major classes of nano-bio-chip designs
that serve as cellular and chemical processing units.
These nanobiochips possess capabilities for measuring
such diverse analyte classes as cells, proteins, DNA and
small molecules in the same compact device. Further,
applications such as disease diagnosis and prognosis
for areas including cancer, heart disease and HIV were
reported. 64 One-dimensional nanostructures such as
nanowires are ideal for diagnosis as they can be integrated
into microfl uidic chips that provide a complete sensor
system. 65
DNA nanomachines are synthetic assemblies that
switch between defi ned molecular conformations
upon stimulation by external triggers. Researchers have
reported the construction of a DNA nanomachine called
the I-switch, which is triggered by protons and functions
as a pH sensor based on fl uorescence resonance energy
transfer inside living cells. Moreover, this was found
an effi cient reporter of pH from pH 5.5 to 6.8, with
a high dynamic range between pH 5.8 and 7, thereby,
illustrating the potential of DNA scaffolds responsive
to more complex triggers in sensing, diagnostics and
targeted therapies in living systems. 66
INTEGRATION OF RECOMBINANT TECHNOLOGY
AND NANOSCIENCE
Recombinant technology is the most important
prerequisite for the effective engineering of nanostructured
deoxyribonucleic acid and protein based
materials in nanoscience. This technology allowed the
manipulation of the properties of molecules, including
physico-chemical properties of proteins that control
electron transport and photochemical processes in
the development of molecular electronic devices and
device fabrication. Recombinant molecules, such as
recombinant ovalbumin and recombinant ovalbumin
mutants have provided a powerful means for the study
of their physico-chemical and structural characteristics,
and thereby for their use in nanoscience. The researchers
have provided an overview of the integration of
recombinant technology and nanoscience through
reported studies in areas, including food, environment,
medicine, physics and chemistry. 67
CONCLUSIONS AND PERSPECTIVES
Nanobiotechnology is an emerging fi eld that seeks new
solutions to pressing health and environmental problems
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 21

Bhupinder Singh Sekhon et al.: Nanobiotechnology: An overview of drug discovery, delivery and development
by combining physical sciences and engineering with
life sciences and medicine. This exciting frontier of
discovery is generating new therapies, devices, diagnostic
tools, and a better understanding of the relationship
between cells and disease. Nanobiotechnology also
deals with the investigation and utilization of the newly
conceived nanomaterials, as well as the construction of
functionalized nanobiosystems. Nanopharmaceuticals
offer the ability to detect diseases at much earlier
stages. Drug delivery is the most advanced tool of
nanobiotechnology. Nanomedicine combines nanotechnology
and medicine, and nanotherapeutics is the
use of nanomedicine in therapy. The ultimate goal of
nanotherapeutic is comprehensive monitoring repair
and improvement of all human biologic system.
Nanobiotechnology should provide many of the tools
necessary to enable the components that will one day be
inserted into commercialized products.
Genomics, proteomics, and metabolomics combined
with the power of nanobiotechnology have the potential
to understand the disease in a way that was previously
not possible and it is expected that the disease will
be targeted more effectively and precisely. Experts
support a comprehensive technology assessment of
nanobiotechnologies along with a full-blown risk/benefi t
analysis before their adoption. New biofriendly imaging
probes based on functionalized inorganic nanocrystals are
being developed and are available to facilitate state-of-theart
bioimaging studies. The research on the combination
of chemotherapeutic agents with gene therapy should
be further strengthened to overcome the limitations of
conventional cancer treatment. In this context, a strongly
synergistic antiproliferative effect was observed in colon
cancer cells when E gene expression was combined with
the activity of the 5-Fluorouracil–loaded biodegradable
poly(ε-caprolactone) nanoparticles, thereby indicating
the potential therapeutic value of the combined
therapy. 68 It is hoped that nanobiotechnology will extend
the limits of current molecular diagnostics and enable
point of care diagnostics, integration of diagnostics
with therapeutics, and development of personalized
medicine. Three-dimensional nanobiostructure-based
self-contained devices consisting of a glucose/oxygen
sensor, a biofuel cell and a wireless signal transmitter to
demonstrate wireless monitoring of glucose and oxygen
in biological fl uids, wounds, and cell cultures is under
development. 69 As yet there are no directives to regulate
nanobiotechnology by various regulating bodies but as
products are ready to enter market, these are expected
to be in place. The largest expansion is expected in
coming years. 70 Nanobiotechnology is likely to trigger
advances in the early detection of a variety of diseases
and improvements in biological implants. Experts are of
the opinion that efforts should be made to educate, and
increase awareness about nanobiotechnology through a
transparent public dialogue.
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RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 23

Nasal drug delivery–a review
Twarita Deshpande* 1 , Rajashree Masareddy 2 , Archana Patil 1
1 Rani Chennamma College of Pharmacy, Belgaum
2 College of Pharmacy, K.L.E. University, Belgaum
ABSTRACT
This paper discusses the theory of mucoadhesion along with various approaches to improve nasal absorption by
the use of mucoadhesive polymers and absorption enhancers. An account of various mucoadhesive polymers is
also given. A note on absorption enhancers has been included. Finally the growing market for nasal drug delivery
is discussed.
INTRODUCTION
Nasal drug delivery for systemic effects has
been practiced since ancient times. In modern
pharmaceutics, the nose had been considered
primarily as a route for local drug
delivery. The last two decades heralded a
number of advances in pharmaceutical
biotechnology resulting in possibilities for
large-scale productions of biopharmaceuticals
especially proteins and peptides. The
inability to administer these drugs by routes
other than parenteral injection motivated
scientists to explore other possibilities such
as pulmonary and nasal administration. The
initial enthusiasm was soon confronted
with disappointing in vivo results showing
poor bioavailabilitiy, typically in the order
of 5–10% for large molecules. 1 On the
other hand, very good results were obtained
with small organic molecules, which led
to the successful development of a number
of products currently on the market
list of products that is steadily increasing.
Examination of the causes of failure led
to the conclusion that the short residence
time of the formulation within the nasal
cavity coupled to the low permeability of
the latter did play signifi cant roles. Consequently,
the attention shifted to the evaluation
of mucoadhesive polymers, some
of which would even demonstrate additional
permeation-enhancing capabilities. 2,3
The encouraging results and the desire to
overcome some new challenges stimulated
the development of new generations of
polymers based on pH, thermal responsiveness,
4 modifi ed existing polymers having
improved bioadhesive or permeationenhancing
properties. 5,6,7 Even though a
number of challenges are still to be overcome,
especially with respect to toxicity, the
potential of nasal drug delivery (NDD),
including the ability to target drugs across
the blood–brain barrier (BBB), are very
high and continues to stimulate academic
and industrial research groups so that we
will keep witnessing increasing number of
advanced nasal drug delivery products. To
optimize nasal administration, bioadhesive
hydrogels, Bioadhesive microspheres (dextran,
albumin and degradable starch) and
liposomes have been studied.
ANATOMY
The nasal cavity is divided into two
halves by the nasal septum and extends
posteriorly to the nasopharynx, while the
most anterior part of the nasal cavity, the
nasal vestibule, opens to the face through
the nostril. The atrium is an intermediate
region between the vestibule and the
respiratory region. The respiratory region,
the nasal conchae or turbinates, which
Review Ar cle
Received Date : 08-08-2011
Revised Date : 11-02-2012
Accepted Date : 14-02-2012
DOI: 10.5530/rjps.2012.1.4
Address for
correspondence
Twarita D. Deshpande
Department of Pharmaceutics
Rani Chennamma College of
Pharmacy
Vaibhav Nagar, Belgaum
Pin Code-590010
E-mail: twarita.deshpande@
gmail.com
Mobile: +91 9886181824
www.rjps.in
24 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

occupies the major part of the nasal cavity, possesses
lateral walls dividing it into 3 sections: the superior,
middle and inferior nasal turbinate fi g 1. These folds
provide the nasal cavity with a very high surface area
compared to its small volume.
The epithelial cells in the nasal vestibule are
stratifi ed, squamous and keratinized with sebaceous
glands. Due to its nature, the nasal vestibule is very
resistant to dehydration and can withstand noxious
environmental substances and limits permeation
of substances. The atrium is a transitional epithelial
region with stratifi ed, squamous cells anteriorly
and pseudostratifi ed columnar cells with microvilli
posteriorly. Pseudostratifi ed columnar epithelial cells
interspersed with goblet cells, seromucus ducts, the
openings of sub epithelial seromucus glands cover
the respiratory region (the turbinates). Furthermore,
many of these cells possess actively beating cilia with
microvilli. Each ciliated cell contains about 100 cilia,
while both ciliated and nonciliated cells possess about
300 microvilli each. 8
PHYSIOLOGY
The nasal physiologic functions, such as warming and
humidifi cation, are vital for upper airway function. It has
been estimated that an adult inspires up to 10,000 liters
of air daily.
Figure 1: Anatomy of Nasal Cavity.
Twarita Deshpande et al.: Nasal Drug Delivery-A Review
Nasal breathing is healthy breathing as the air is treated
in many ways by the structures of the nose, paranasal
sinuses and the peculiarities of their lining mucosa.
1. Filtering the air
Filtration of environmental particles occurs fi rst in the
nasal cavity. Nasal mucus traps incoming particulate
matter. The largest particles are fi ltered by nasal hairs
(vibrissae).
2. Moistening the air
Humidifi cation is another important process of nasal
physiology. The nasal cavity is covered with a highly
vascular mucosa that warms and humidifi es incoming
air, increasing the relative humidity to 95% before air
reaches the nasopharynx.
3. Warming the air
Inhaled air must have a temperature between at least
33 and 35 degree Celsius to not cause pathological
reactions at the level of the alveoli. Again, by the
turbulence, the cold air is forced to make contact with
the warm surface of the mucosa and thus heated during
its passage. A number of nasal neurovascular refl exes
occur as well. If needed, underlying capillaries will dilate
and warm up the upper laying mucosa, giving more heat
to the passing air.
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 25

4. The sense of smell
Nasal aerodynamics also contributes to the olfactory
system. In addition, the active process of sniffi ng allows
environmental particles to reach the olfactory system
located at the skull base.
Even the smallest particles are detected by the olfactory
receptors, warning us about danger, food, or any other
biologically meaningful sign detectable through the
sense of smell.
5. The nasal cavity as a sound box
The nose and sinuses serve as contributing factors in
voice modifi cation. Authors have noted that nasal
aerodynamics may have a role in modifying highfrequency
sounds and consonants. The resonance
created within the nasal cavity is characteristic, similar
to a fi nger print and for each person is different (except
in identical twins). Nasal pathologies such as polyps or
rhinitis will directly infl uence the resonance spectrum
and we will”hear“that the person has a cold or something
has changed in his/her voice. 9
MUCOADHESION AS A STRATEGY TO
IMPROVE SYSTEMIC DRUG DELIVERY
VIA THE NASAL ROUTE
Several theories have been put forward to explain the
mechanism of polymer-mucus interactions that lead to
mucoadhesion. To start with, the sequential events that
occur during bioadhesion include an intimate contact
between the bioadhesive polymer and the biological
tissue due to proper wetting of the bioadhesive surface
and swelling of the bioadhesive. Following this is the
penetration of the bioadhesive into the tissue crevices,
interpenetration between the mucoadhesive polymer
chains and those of the mucus. Subsequently low
chemical bonds can become operative. 10, 11 Hydration of
the polymer plays a very important role in bioadhesion.
There is a critical degree of hydration required for
optimum bioadhesion. If there is incomplete hydration,
the active adhesion sites are not completely liberated
and available for interaction. An alternative approach to
the use of chemical enhancers to improve nasal drug
absorption is to increase the duration of formulation
residence within the nasal cavity. This is achieved by
the use of bioadhesive polymers. Apart from these
synthetic and natural polymers, there is now a new class
of promising compounds, the lectins, often referred
to as second-generation mucoadhesive materials.
These are non-immunogenic proteins or glycoproteins
capable of specifi c recognition and reversible binding
to carbohydrate moieties of complex glycoconjugates
without altering the covalent nature of any of the
recognized glycosyl ligands. The use of mucoadhesives
Twarita Deshpande et al.: Nasal Drug Delivery-A Review
can solve a number of problems encountered in
controlled drug delivery. It localizes the formulation at
a particular region in the body, thereby improving the
bioavailability of drugs with low bioavailability. The
increased contact time and localization of the drug
due to the strong interaction between the polymer
and mucus is essential for the modifi cation of tissue
permeability. Furthermore, enzymatic activity can be
locally inhibited to improve the bioavailability of drugs
that are subject to enzymatic degradation. This has
been demonstrated for some mucoadhesive polymers
such as Carbopol 934P and polycarbophil that inhibit
the proteolytic enzyme trypsin, which can thus increase
the stability of co-administered Peptides (Leuben
et al. 1994). Some studies have also demonstrated that
mucoadhesive polymers can also directly interact with the
epithelial tight junctions. 12 The mucoadhesive polymers
reported in the literature are summarized in Table 1.
The theories and mechanisms of bioadhesion are given
Table 2. 13
ADVANTAGES OF MUCOADHESIVE DRUG
DELIVERY SYSTEMS
Mucoadhesive dosage forms have three different advantages
when compared to conventional dosage forms
1. These dosage forms are readily localized in the region
applied to improve and enhance the bioavailability
of drugs. Greater bioavailability of piribedit,
testosterone and its esters, vasopressin, dopamine,
insulin was observed from mucoadhesive dosage
forms when compared to conventional dosage
forms.
Table 1: Mucoadhesive Polymers
Polymer Bioadhesive property
Carboxymethyl cellulose
3
Carbopol 934
3
Polycarbophil
3
Tragacanth
3
Poly(acrylic acid/divinyl benzene)
3
Sodium alginate
3
Hydroxy ethyl cellulose
3
Gum Karaya
3
Gelatin
3
Guar gum
2
Thermally modifi ed starch
2
Pectin
1
Polyvinyl Pyrrolidone
1
Acacia
1
Polyethylene glycol
1
Psyllium
1
Amberlite-200 resin
1
Hydroxy propyl cellulose
1
Chitosan
1
Hydroxy ethyl methacrylate
1
NOTE: 3- Excellent, 2- Fair, 1- Poor.
26 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

2. These dosage forms facilitate intimate contact of the
formulation with the underlying absorption surface.
This allows modifi cation of tissue permeability for
absorption surface. This allows modifi cation of tissue
permeability for absorption of macromolecules, such
as peptides and proteins. Inclusion of penetration
enhancers such as sodium glycholate, sodium
taurocholate and L-lysophosphotidyl choline (LPC)
and protease inhibitors in mucoadhesive dosage
forms resulted in better absorption of peptides and
proteins.
3. Mucoadhesive dosage forms also prolong residence
time of the dosage form at the site of application and
absorption to permit once or twice a day dosing. 14
The absorption enhancement mechanisms can be
grouped into two classes:
NASAL ABSORPTION ENHANCERS
Physicochemical effects: Some enhancers can alter the
physicochemical properties of a drug in the formulation.
This can happen by altering the drug solubility, drug
partition coeffi cient, or by weak ionic interactions with
the drug.
Membrane effects
Many enhancers show their effects by affecting the nasal
mucosa surface. 15 Nasal absorption enhancers involve
two main classes. The most important group involve
microspheres, liposome’s and gels that have been utilized
as drug carriers in the past few years. The second group
Twarita Deshpande et al.: Nasal Drug Delivery-A Review
Table 2: Theories and Mechanisms of Bioadhesion
Theory Mechanism of bioadhesion Comments
Electronic theory Attractive electrostatic forces between
Electrons transfer occurs between the two
glycoprotein mucin network and the
forming a double layer of electric charge at
bioadhesive material.
the surface
Wetting theory Ability of bioadhesive polymer to spread
Spreading coeffi cient of polymers must be
and develop intimate contact with the
positive. Contact angle between polymer and
mucous membrane.
cells must be near to zero.
Adsorption theory Surface force resulting in chemical
Strong primary force: covalent bonds. Weak
bonding.
secondary forces: hydrogen bonds and van
der Waal’s forces
Diffusion theory Physical entanglement of mucin strands
For maximum diffusion and best adhesive
and fl exible polymer chains.
strength, solubility parameters of the
bioadhesive polymer and the mucus
glycoproteins must be similar
Mechanical theory Adhesion arises from an interlocking of
Rough surfaces provide an increased surface
liquid adhesive into irregularities on
area available for interaction along with an
the rough surface.
enhanced viscoelastic and plastic dissipation
of energy during joint failure, which are more
important in the adhesion process than a
mechanical effect.
Fracture theory Analyses the maximum tensile stress
Does not require physical entanglement of
developed during attachment of the
bioadhesive polymer chains and mucous
transmucosal DDS from the mucosal
strands, hence it is appropriate to study the
surface
bioadhesion of hard polymers which lack
fl exible chains
will be discussed under the heading other penetration
enhancers.
Dextran microspheres
Illum et al. introduced well-characterized bioadhesive
microspheres for prolonging the residence time in
the nasal cavity. The slowest clearance was detected
for DEAE-dextran, where 60% of the delivered
dose was still present at the deposition site after 3 h. 16
However, these microspheres were not successful in
promoting insulin absorption in rats. The insulin was
too strongly bound to the DEAE groups to be released
by a solution with an ionic strength corresponding to
physiological conditions. Structural changes due to the
lyophilization process were observed in spheres with
insulin incorporated, which probably further decreased
the release rate. 17
Degradable starch microspheres (DSM)
DSM is the most frequently used microsphere system
for nasal drug delivery and has been shown to improve
the absorption of insulin, gentamicin, human growth
hormone, metoclopramide and desmopressin. Insulin
administered in DSM to rats resulted in a rapid dosedependent
decrease in blood glucose. DSM as a
delivery system for insulin (2 IU.kg_1 ) has also been
tested in sheep. The absolute bioavailability was 4.5%
and the time to reach maximum effect, i.e., a 50%
decrease in plasma glucose, was 60 min. 18 Studies in
rabbits have demonstrated that DSM does not induce
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 27

serious hystopathological changes to the nasal mucosa.
Moreover, the DSM was well tolerated by 15 healthy
volunteers and did not cause signifi cant changes in
mucociliary transport. The effect of starch microspheres
on the absorption enhancing effi ciency of various
enhancer systems with insulin after application in the
nasal cavity of the sheep was investigated. The DSM
was shown to synergistically increase the effect of the
absorption enhancers on the transport of the insulin
across the nasal membrane. 19
Liposomes
Various routes have delivered liposomes. Alpar et al.
studied the potential adjuvant effect of liposomes on
tetanus toxoid, when delivered via the nasal, oral and i.m.
routes compared to delivery in simple solution in relation
to the development of a non-parenteral immunization
procedure, which stimulates a strong systemic immunity.
They found that tetanus toxoid entrapped in DSPC
liposomes is stable and is taken up intact in the gut. The
permeability of liposome entrapping insulin through
the nasal mucosa of rabbit has been studied and
compared with the permeability of insulin solution with
or without pre-treatment by sodium glycocholate (GC).
A comparison of the insulin solution and liposome
suspension showed that the liposome had permeated more
effectively after pre-treatment by GC. The relationship
between the rigidity of the liposomal membrane and
the absorption of insulin after nasal administration of
liposomes modifi ed with an enhancer containing insulin
was investigated in rabbits. The nasal administration
to rabbits showed high fl uidity at 37°C, caused a high
serum glucose reduction, and the reduction effect lasted
for 8 h. The loading and leakage characteristics of the
desmopressin-containing liposomes and the effect
of liposomes on the nasal mucosa permeation were
investigated. The increase of permeability antidiuresis
of desmopressin through the nasal mucosa occured in the
order positively charged liposomes > negatively charged
liposomes > solution. The potential of liposomes as an
intranasal dosage formulation or topical application of
5 (6)-carboxyfl uorescein (CF) was investigated in rats. CF
was rapidly absorbed into the systemic circulation and
no adhesion of CF to the nasal mucosa was observed.
Liposomes suppress drug absorption into the systemic
circulation and concurrently increase drug retention in
the nasal cavity. 20
Gels
Chitin and chitosan have been suggested for use as
vehicles for the sustained release of drugs. Indomethacin
and papaverine hydrochloride were used as model drugs
in gel formulations. It was reported that chitin was able
to control the release of the active agents from gel
Twarita Deshpande et al.: Nasal Drug Delivery-A Review
formulation as compared to the powder formulation.
A similar study done later by Lehr et al. showed that
cationic polymer chitosan was fairly mucoadhesive in
comparison to polycarbophil as a reference substance.
They suggested that a strict distinction should be made
between mucoadhesive of dry polymers on a wet tissue
in air and mucoadhesion of a swollen hydro gel in the
presence of a third liquid phase. 21 Nasal absorption of
nifedipine from gel preparations, PEG 400, aqueous
carbopol gel and carbopol-PEG has been studied in
rats. Nasal administration of nifedipine in PEG resulted
in rapid absorption and high Cmax; however, the
elimination of nifedipine from plasma was very rapid.
The plasma concentration of nifedipine after nasal
administration in aqueous carbopol gel formulation was
very low. The use of PEG 400 in high concentration
in humans should be considered carefully because PEG
400 is known to cause nasal irritation in concentrations
higher than 10%. 22
OTHER PENETRATION ENHANCERS
Cyclodextrin
Several compounds have been investigated for their
nasal absorption enhancement potential using cyclodextrins
as the optimisers. The most studied types are:
cyclodextrins cyclodextrin-cyclodextrin methylcyclodextrin
and Hydroxypropyl-cyclodextrin. Only cyclodextrin
is a compendial substance and is being considered
forGRAS (generally recognized as safe) status. Merkus
et al. reported a study, which investigated the effects of
a dimethyl-cyclodextrin (DM-CD) powder formulation
on intranasal insulin absorption in healthy subjects
and patients with insulin-dependent diabetes mellitus
(IDDM). Mean absolute bioavailabilities of 3.1% and
5.1% were achieved in healthy subjects and diabetics,
respectively. 23
Fusidic acid derivatives
Sodium tauro-24, 25-dihydrofusidate (STDHF) is the
most extensively studied among the derivatives of
fusidic acid. On the basis of its characteristics STDHF
was considered a good candidate for the transnasal
delivery of drugs such as insulin, growth hormone and
octreotide. Lee et al. determined the radioimmunoactive
bioavailability of intranasal salmon calcitonin in
10 healthy human volunteers. The improved nasal
absorption of calcitonin in the presence of STDHF
showed a limited transient irritation of the nasal mucosa
in some subjects. Hedin et al. studied the intranasal
administration of human growth hormone (hGH)
in combination with STDHF at 1% concentration
in patients with hGH defi ciency. They found that in
combination with STDHF, the plasma peak of hGH
28 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

was similar to the endogenous peak. Laursen et al. used a
formulation approach in determining the absorption of
growth hormone in human subjects using didecanoyl-
L-phosphotidylcholine (DDPC) as an enhancer with
different concentrations: 0, 4, 8, and 16%. They
concluded that increasing the relative concentration of
DDPC increases the absorption of nasally administered
hGH. Drejer et al. studied intranasal administration of
insulin with DDPC in healthy human volunteers. They
found that intranasal insulin was absorbed in a dose
dependent manner with slight or no nasal irritation. 24
Phosphatidylcholines (PC)
Phosphatidylcholines are surface-active ampiphilic
compounds produced in biological membranes and
liposomes. Several reports have appeared in the
literature showing that these phospholipids can be used
as enhancers for systemic nasal drug delivery. Newman
et al. investigated the distribution of a nasal insulin
formulation containing DDPC labelled with 99m
Tc-human serum albumin (99m Tc-HAS) in human
volunteers. From the scintigraphic data, the entire dose
from the spray was shown to be deposited in the nasal
cavity with no deposition in the lungs.
The Novo Nordisk study group reported encouraging
results following the nasal administration of an insulin/
DDPC microemulsion formulation in human volunteers.
The study demonstrated good absorption of insulin
whilst preventing or minimizing nasal irritation.
Bile salts and surfactants
Commonly used salts are sodium cholate sodium
deoxy cholate, sodium glycocholate (GC), sodium
taurocholate (TC), sodium taurodeoxycholate (TDC),
and sodiumglycodeoxycholate (GDC). Several studies
indicate that bile salts can be good optimisers in nasal
drug products, there are some reports indicating that
bile salts cause nasal irritation when used above a
concentration of 0.3%. Yokosuka and co-workers
reported a study in which healthy volunteers are dosed
nasally with solution formulation containing insulin
and 1%sodiumglycocholate. Signifi cant decreases in
serum glucose concentrations were observed and
there was a positive correlation between the peak
serum insulin levels and the dose of insulin applied.
Hirata et al. investigated the effi cacy of nasal insulin
formulation containing 1% SGC in healthy volunteers
and diabetic patients. The nasal formulation resulted in
rapid increases in serum insulin levels and decreases in
blood glucose levels in healthy volunteers and diabetics.
Moses and colleagues showed that co administration
of 1% sodium deoxycholate (SDC) enhanced the
intranasal absorption of insulin administered to
human volunteers. High inter-subject variability was
Twarita Deshpande et al.: Nasal Drug Delivery-A Review
observed. Frauman compared the effects of intranasal
and subcutaneous insulin on fasting and post-prandial
blood insulin and glucose concentrations in non-obese
patients with non-insulin-dependent diabetes mellitus
(NIDDM). A nasal solution formulation of insulin
and 1% SGC, administered as a spray, resulted in a
monophasic increase in serum insulin levels. Salzman
et al. investigated the effi cacy of 1% laureth-9 in
enhancing the nasal absorption of insulin in patients
with IDDM and non-diabetic controls. Insulin was
shown to be rapidly absorbed via the nasal route
lowering plasma glucose levels to 50% of basal values
after 45 min in normal subjects compared to 50% in
120 min in diabetics. Paquot et al. investigated the
metabolic and hormonal consequences of an intranasal
insulin formulation administration containing 0.25%
laureth-9 in healthy volunteers. Increase in plasma insulin
levels from 5 to 38 mU.I –1 at 15 min with decreases in
blood glucose concentration from 4.4 to 3.2 mmol.I –1
at 45 min.
DRUG ABSORPTION
The fi rst step in the absorption of drugs from the nasal
cavity is passage through the mucus. Small, uncharged
particles easily pass through this layer. However,
larger or charged particles may fi nd it more diffi cult to
cross. Mucin, the principal protein in the mucus, has
the potential to bind to solutes, hindering diffusion.
Additionally, structural changes in the mucus layer
are possible as a result of environmental changes (i.e.,
pH, temperature, etc) 25 Subsequent to a drug’s passage
through the mucus, there are several mechanisms for
absorption through the mucosa. 26 These include
1. Paracellular Transport Mechanism-It mainly uses
an aqueous mode of transport. Usually the drug
passes through the tight junctions and the open
clefts of the epithelial cells present in the nasal
mucosa. It is a relatively passive mode of transport.
Of Compounds, which are highly hydrophilic in
nature and/or low molecular are most appropriate
for paracellular transport.
2. Transcellular Transport Mechanism-It mainly
encompasses transport via a lipodal route. Small
lipophilc compounds or larger molecules are usually
transported by this route. The transport across nasal
mucosa is mainly a function of lipophilc nature of
the drug. 27
FORMULATION ISSUES
Nasal drug absorption is affected by molecular weight,
particle size, formulation pH, pKa of molecule, and
delivery volume among other formulation characteristics.
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 29

Molecular weight still presents the best correlation to
absorption. 28 The apparent cut-off point for molecular
weight is approximately 1,000, with molecules less than
1,000 having better absorption. Shape is also important.
Linear molecules have lower absorption than cyclicshaped
molecules. Additionally, particles should be larger
than 10 mm, otherwise the drug may be deposited in the
lungs. 29 Hydrophilicity has also been found to decrease
drug bioavailability. 30
Another formulation factor important for absorption
is pH. Both the pH of the nasal cavity and pKa of
a particular drug need to be considered to optimize
systemic absorption. Nasal irritation is minimized
when products are delivered with a pH range of
4.5 to 6.5. Also, volume and concentration are
important to consider. The delivery volume is limited
by the size of the nasal cavity. An upper limit of
25 mg/dose and a volume of 25 to 150mL/nostril has
been suggested. 31
FDA requirements state all nasal drug products sold
in the United States are to be manufactured as sterile
or preserved products. Depending on the drug and/or
formulation characteristics, sterility may be accomplished
via aseptic fi lling processes, terminal sterilization, or both.
Sterility must also be maintained throughout a product’s
shelf life. Unit-dose delivery systems or specialized
multidose delivery systems may be used to ensure
continued product sterility. Alternatively, preservatives
may be added to prevent bacterial growth. The most
commonly used preservative in nasal formulations has
been benzalkonium chloride. 32 However, adverse events,
such as mucosal swelling, irritation, and ciliostastis, are
associated with its use.
Intranasal vaccine administration has also become
a prevalent area of research. One of the major
formulation challenges for nasal vaccination has been
the development of an appropriate adjuvant to stimulate
desirable immune responses. Some examples of
investigational adjuvants include microparticles, genetic
manipulations of Vibrio cholerae and Escherichia
coli toxins, and CpG oligodeoxynucleotides. The
use of adjuvants is especially important for vaccines
composed of recombinant subunits, synthetic peptides,
and plasmid DNA. For instance, when hepatitis B
surface antigen was given alone to mice, little or no
immunogenicity was observed. However, when given
with CpG or cholera toxin, good immunological
responses were achieved. 33
Drugs have been delivered to the nasal cavity in a variety
of formulations, including powders, topical gels, sprays,
drops, and pledgets. As reviewed elsewhere by Behl et al.,
the dosage form should refl ect the intended therapeutic
use, offer easy administration, and provide chemical
stability for the drug. 34
Twarita Deshpande et al.: Nasal Drug Delivery-A Review
With any formulation for intranasal delivery,
interference with normal physiologic processes should
be avoided. The nose is an important defense system
for environmental hazards. Disruption of its normal
physiology or clearance processes may leave a patient
vulnerable to a variety of hosts. Also, formulations
should be designed with patient comfort and acceptance
in mind. Some drugs and excipients have been found
to cause temporary discomfort upon instillation. This
could potentially decrease patient compliance, especially
when chronic administration is indicated. 35–38
NASAL DELIVERY OF VACCINES
Almost all viral, bacterial and parasitic agents causing
common infectious diseases of the intestinal, respiratory
and genital tract enter and infect through the large
surface area made available by mucosal membranes.
The nasal mucosa is an important arm of the mucosal
immune system since it is often the fi rst point of contact
for inhaled antigens and as a consequence, intranasal
immunisation has emerged as possibly the most effective
route for vaccination for both peripheral and mucosal
immunity.
Recently, Partidos has considered the challenges for
intranasal vaccines. He has listed several reasons why the
nose is an attractive route for immunisation.
• Easily accessible.
• Highly vascularized.
• Presence of numerous microvilli covering the nasal
epithelium generates a large absorption
• Surface.
• After intranasal immunisation, both mucosal and
systemic immune responses can be induced.
• Immune response can be induced at distant mucosal
sites owing to the dissemination of effector immune
cells in the common mucosal immune system.
• The nose can be used for the easy immunization of
large population groups.
• Nasal immunisation does not require needles and
syringes, which are potential sources of infection. 39
The utility of intranasal administration for a variety of
clinical applications to include largely the prophylaxis
of disease. Other uses of nasal vaccination have been
reported. For example, Weiner et al. have suggested a
novel mucosal immunlogical approach to Alzheimer’s
disease. Nasal administration of amyloid-beta peptide
decreased cerebral amyloid burden in a mouse model of
the disease. Recently, live attenuated; cold adapted viral
vaccines have been developed as alternatives to inactivated
vaccines. For example, by growing the infl uenza virus
at 25ºC for long periods or using genetic reassortment
methods, it is possible to produce an organism that will
30 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

eplicate effi ciently at 25–28ºC (the temperature of the
nasal passage) but not at 37ºC (the temperature of the
lungs). Clinical studies on live attenuated virus vaccines
have been well reviewed by Wiselka and Keitel and
Piedra. One major goal of intranasal vaccination with
a replicating virus is to induce secretory and systemic
immune responses that more closely resemble those
provided by the natural infection.
BRAIN TARGETING THROUGH NASAL ROUTE
For some time the Blood Brain Barrier has impeded the
development of many potentially interesting CNS drug
candidates due to their poor distribution into the CNS.
The Blood Brain Barrier is a system of layers of cells at
the cerebral capillary endothelium, the choroids plexus
epithelium, and the arachnoid memberanes, which
are connected by tight junctions and which together
separate the brain and the cerebrospinal fl uid from
blood. 40 Owing to the uniqueness of the nose and
the CNS, the intranasal route can deliver therapeutic
agents to the brain bypassing the Blood brain Barrier. 41
Absorption of drug across the olfactory region of the
nose connection provides a unique feature and superior
option to target drugs to brain. When administered
nasally to the rat, some drugs resulted in CSF and
olfactory bulb drug levels considerably higher than
those following intravenous administration. 42 Evidence
of nose to brain transport has been reported by many
scientists. Many previously abandoned potent CNS
drug candidates promise to become successful CNS
therapeutic drugs via intranasal delivery. Recently,
several nasal formulations, such as ergotamine
(Novartis), sumatriptan (GlaxoSmith-Kline), and
zolmitriptan (AstraZeneca) have been marketed to treat
migraine. Scientists have also focused their research
toward intranasal administration for drug delivery to
the brain, especially for the treatment of diseases, such
as, epilepsy, migraine, emesis, depression and erectile
dysfunction. 43,44 Intranasal delivery does not require
any modifi cation of the therapeutic agents and does
not require that drugs be coupled with any carrier.
A wide variety of therapeutic agents, including both
small molecules and macromolecules can be successfully
delivered to the CNS via intranasal method. 45
ADMINISTRATION DEVICE
Drug therapy requires that administration of the dosage
form be accurate and very reproducible, which therefore
places stringent, demands on the device for nasal drug
delivery. The major mechanism of nasal deposition of
particles is by inertial impaction that occurs following a
change in the direction of airfl ow. Other contributory
mechanisms are gravitational sedimentation and
Twarita Deshpande et al.: Nasal Drug Delivery-A Review
Brownian diffusion. Particle deposition by interception
and electrostatic precipitation are of no importance in
nasal deposition (Kublik & Vidgren 1998). Depending
on the type of formulation, a variety of devices have
been used to deliver drugs intranasally. Devices for liquid
formulations include instillation catheter, droppers,
unit-dose containers, squeezed bottle, pump sprays,
airless and preservative-free sprays, compressed air
nebulizers and metered-dose inhalers (MDIs). Devices
for powder dosage forms include insuffl ators, singledose
and multi-dose powder inhalers and pressurized
MDI. Delivery devices are also available for nasal gels
(Kublik & Vidgren 1998). Squeeze bottle delivery
is another option for nasal drug delivery. However
this technique is not able to deliver a measured dose
of drug.
Their angle of insertion into the nostril can infl uence
the part of the nasal cavity that the formulation
comes in contact with initially and as such the overall
deposition pattern. Metered-dose nebulizers and
metered-dose aerosols are superior to other devices in
terms of accuracy and reproducibility (Dondeti 1996).
1985). The particle size of aerosols is very important
with regard to deposition. Particles greater than 10 ml
are deposited within the upper respiratory tract, those
less than 5 ml are inhaled, and those less than 0.5 ml
are exhaled (Sciarra & Cutie 1990; Sanders et al 1997).
A brief description of various intranasal devices is given
as follows Figure 2–8.
1. Caprujet of midazolam–This is a prepackaged
midazolam in 5mg/ml dosing. It is indicated for
treatment of persistent seizure activity. In situations
where the entire volume might not be appropriate,
the syringe can have a indelible mark made by the
clinician or pharmacist at appropriate dosage so the
parent knows how much to give.
2. Direct-Haler powdered Drug Nasal Delivery–This
system enables nasal delivery of fi ne particles for
improved absorption and targeted delivery. Prevents
risk of pulmonary deposition. Prevents risk of
immediate dose swallowing, reduces taste impact
from nasal use.
3. Mucosal Atomization Device(MAD)–Delivers
intranasal medication in a fi ne mist which enhances
absorption and improves bioavailability for fast and
effective drug delivery.
4. Via Nose Electronic Atomizer–This atomizer
creates tiny, Powerful vortices of aerosolized particles,
thus delivering medication more effectively to the
user. This device has a small LCD screen which is
used to specify dose.
5. BDA Accuspray Nasal Spray System–It is a
single use, prefi llable vaccine delivery system which
dispenses a precise dose of fl u vaccine intranasally in
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 31

Figure 2: Nasal Delivery Dosage Forms and Devices.
Figure 3: Caprujet of Midazolam.
the form of a fi ne mist. Recommended for healthy
individuals in the age group of 5–49.
6. Optinose–This device is based on the concept of
a bi-directional nasal drug delivery device. Recently
optinose received positive results from their phase
II clinical studies for chronic rhinosinusitis and
migraine therapies.
Twarita Deshpande et al.: Nasal Drug Delivery-A Review
Regardless of the method chosen to deliver a nasal
medication, several suggestions are worth mentioning:
• Use a highly concentrated form of the medication to
reduce volume and therefore reduce runoff. 1/4 to
1/3 ml per nostril would be preferred. 1/2 to 1 ml per
nostril is tolerable but there will be some loss as the
volume increases. More than one ml per nostril per
dose should likely be split and delivered over several
cycles separated by 10–15 minutes.
• Always use a method that allows the delivery of a
measured dose (syringe or unit dose pump).
• Use a method that fragments the medication into
fi ne particles so the maximal nasal mucosal surface
is covered and minimal volume runs out the nose or
into the throat (atomizer for liquid, powder needs to
be well distributed).
• Utilize both nostrils to double the surface area for
absorption and halve the volume delivered per
nostril.
• Be knowledgeable of the “dead space” within the
delivery device and account for this dead space when
calculating the volume you will deliver to the patient.
Considerations Regarding Device Selection
When selecting a device for nasal administration, it
must be considered, that the administration volume
is comparable low. For liquids, a volume of 100μl is
32 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

Figure 4: Direct-Haler Powdered Drug Nasal Delivery.
Figure 5: Mucosal Atomization Device (MAD).
Twarita Deshpande et al.: Nasal Drug Delivery-A Review
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 33

Figure 6: Via Nase Electronic Atomizer.
Figure 7: BDA Accuspray Nasal Spray System
Twarita Deshpande et al.: Nasal Drug Delivery-A Review
34 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

Figure 8: OptiNose.
optimum per nostrils in a adults, but should be reduced
for children to avoid nasal dripping. Single dose devices
will give best protection for vaccines, but require
highly sophisticated fi lling technology. Multi-dose
spray pumps are option for liquid vaccines, if an in use
microbial contamination of the bottle content can be
prevented. 46
ADVANTAGES OF NASAL DELIVERY
Intranasal delivery is a needle-free, patient-friendly
administration route. Because needles are not involved,
this method of drug delivery is virtually painless. For
patients who fear injections, intranasal administration
offers a more acceptable alternative. Additionally,
the simplicity of nasal delivery would allow for selfadministration
in a home setting. In general, for patients,
the intranasal dosage form provides comfortable, non
threatening, less invasive therapy. This may be especially
important in younger patient populations.
Another major benefi t of intranasal administration, in
contrast to injectables, is that it does not contribute to
biohazardous waste. When the drug has been delivered
intranasally, the administration device may be disposed
of in the normal garbage. There is no need for special
waste containers. Again, this delivery method does not
Twarita Deshpande et al.: Nasal Drug Delivery-A Review
require needles. Hence risk of accidental sticks is not a
concern.
From a pharmacokinetic standpoint, absorption is
rapid, which should provide a faster onset of action
compared to oral and intramuscular administration.
Hepatic fi rst-pass metabolism is also avoided, allowing
increased, reliable bioavailability. In this regard, good
drug candidates for intranasal delivery are those that
undergo extensive fi rst-pass metabolism, display erratic
absorption, or require quick therapeutic onset.
Lastly, patent life of a particular product may be
extended via development of an alternative dosage form,
providing companies the opportunity to maintain their
market share. So from a drug development perspective,
intranasal delivery should stimulate favorable profi t
outcomes. 47
GROWING MARKET FOR NASAL DRUG DELIVERY
Pharmaceutical companies have looked increasingly
towards drug delivery companies for help in lifecycle
management of drugs on the market and with promising
yet hard-to-deliver drugs. The drug delivery market
is currently valued at US$50 billion (or 12.5% of the
global pharmaceutical market) and is expected to reach
US$100 billion by 2005.1 Nasal delivery commands the
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 35

fourth position in market share, with about US$3 billion
in sales, following oral controlled release, pulmonary and
parenteral delivery routes. However, the potential for
growth in this sector is extensive, pending the successful
delivery of proteins and peptides as an alternative to
parenteral delivery. Currently, many nasal drug products
on the market are indicated for the treatment of
local disease such as allergic rhinitis. However, this is
likely to change soon. There are a number of nasally
delivered, systemically acting drugs on the market
in different therapeutic categories, with a growing
number of products in the pipeline. There are many
reasons for this change, including improved patient
compliance (elimination of needles), avoidance of fi rstpass
metabolism and rapid onset of action. Migraine is
a key area where a nasal system (Imitrex ® nasal spray,
GlaxoSmithKline) has provided rapid relief, avoidance
of taking an oral formulation while nauseated, and
pain-free administration circumventing the need for an
injection. Other therapeutic areas where nasal delivery
could provide an alternative to current dosage forms
are crisis situations (seizure and heart attack), erectile
dysfunction, pain management, motion sickness and
psychotropic drugs. Although the market share for nasal
delivery may never take the number one spot enjoyed by
oral controlled release, it remains a drug delivery route
with an enormous potential for growth.
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RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 37

Design and optimization of levofl oxacin
gastroretentive tablets
D. Nagendrakumar 2 , S.B. Shirsand* 1 , M.S. Para 2 , A.D. Chauhan 2
1 Department of Pharmaceutical Technology, H.K.E. Society’s M T R Institute of Pharmaceutical Sciences,
Sedam Road, Gulbarga-585105, India
2Department of Pharmaceutics, S.V.E. Trust’s College of Pharmacy, Kallur Road, Humnabad-585330, India
ABSTRACT
In the present study, gastroretentive fl oating tablets of levofl oxacin hemihydrate were designed with objective
of retention of tablet in acidic pH to improve bioavailability with reduction in dosing frequency. Hydroxypropyl
methyl cellulose of different viscosity grades (K4M and K100LV) was used as polymer and sodium bicarbonate
as gas generating agent to reduce fl oating lag time. Tablets were prepared by direct compression method. The
prepared formulations were evaluated for hardness, friability, weight variation, drug content, swelling index,
in-vitro drug release, short-term stability, fl oating lag time and in-vitro buoyancy. A 32 factorial design was applied
to systematically optimize the drug release profi le.
The proportions of release retardant material HPMC K100LV(X ), sodium bicarbonate (X ) were selected as
1 2
independent variables and t (Y ), and t (Y ) were selected as dependent variables. The promising formulation
50% 1 70% 2
containing levofl oxacin hemihydrate (100 mg), HPMC K100LV (100 mg) and sodium bicarbonate (80 mg) has t50% (5.95 h), t (8.52 h) fl oating lag time was only 10.33 sec and released more than 90% drug in 12 h. This study
70% ,
proved that gastroretentive drug delivery system of levofl oxacin hemihydrate was designed using HPMC K100LV,
which provided excellent gastroretentive property, thus improved the bioavailability of drug.
Key words: Levofl oxacin hemihydrate, Gastroretentive fl oating drug delivery systems, Hydroxypropyl methyl
cellulose, 3² factorial design
INTRODUCTION
The real challenge in the development of an
oral controlled-release drug delivery system
is not just to sustain the drug release, but
also to prolong the presence of the dosage
form within the gastrointestinal tract (GIT)
until the drug is completely released at the
desired period of time. 1 Indeed, gastric
drug retention has received signifi cant
interest in the past few decades. Most of
the conventional oral delivery systems have
shown some limitations related to fast
gastric emptying time. 2
Garg and Gupta3 classifi ed the gastroretentive
dosage forms into four main classes: (i)
fl oating systems4 (ii) expandable systems5 (iii) bioadhesive systems6 and (iv) high
density systems. 7 Floating systems are of
two types: effervescent systems, depending
on the generation of carbon dioxide gas
upon contact with gastric fl uids, and noneffervescent
systems. The non-effervescent
systems can be further divided into four
sub-types, including hydrodynamically balanced
systems, 8 microporous compartment
systems, 9 alginate beads 10 and hollow microspheres/microballons.
11 In addition, superporous
hydrogels 12 and magnetic systems 13
were also described.
As suggested by Singh and Kim, 14 fl oating
drug delivery is of particular interest for the
drugs which: (a) act locally in the stomach;
(b) are primarily absorbed in the stomach;
(c) are poorly soluble at an alkaline pH;
Research Ar cle
Received Date : 11-07-2011
Revised Date : 11-02-2012
Accepted Date : 15-02-2012
DOI: 10.5530/rjps.2012.1.5
Address for
correspondence
S B Shirsand
Department of Pharmaceutical
Technology
H.K.E. Society’s M T R Institute
of Pharmaceutical Sciences
Sedam Road
Gulbarga-585105, India
E-mail: shirsand@rediffmail.
com
www.rjps.in
38 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

D.Nagendrakumar et al.: Design and Optimization of Levofl oxacin Gastroretentive Tablets
(d) have a narrow window of absorption and (e) are
unstable in the intestinal or colonic environment.
Levofl oxacin hemihydrate is a broad spectrum
fl uoroquinolone antibiotic. It is approved for use in
the treatment of Helicobacter pylori infection. H. pylori
was recognized as a major gastric pathogen responsible
for chronic active gastritis, duodenal ulcers and gastric
adeno-carcinoma. 15 The bioavailability of levofl oxacin
hemihydrate is above 99% with a plasma half-life of
6–8 h. It is freely soluble in pH 0.6 to 5.8 range. Above
pH 5.8, the solubility increases rapidly to its maximum
at pH 6.7 and above which the solubility decreases and
reaches a minimum value at a pH of approximately
6.9. 16 Thus solubility of the drug is reduced in intestinal
alkaline pH. Hence, it was selected in the present
investigation as a suitable candidate for the design of
gastric fl oating drug delivery system (GFDDS) with an
improved retention time and bioavailability.
In a full factorial design, two factors were studied at all the
possible combinations, by considering it is most effi cient
in estimating the infl uence of individual variables and
their interactions, using minimum experimentation. In the
present investigation, amount of HPMC K100LV (X 1 )
and amount of sodium bicarbonate (X 2 ) were selected
as independent variables in the 3 2 factorial design. The
time required for drug release of 50% (t 50%, Y 1 ) and 70%
(t 70%, Y 2 ) were selected as dependent variables. A statistical
model incorporating interactive and polynomial
terms was utilized to evaluate the response.
MATERIALS AND METHODS
Levofl oxacin hemihydrate was gifted by Zhejiang Starry
Pharmaceutical Ltd., Mumbai (India), hydroxypropyl
methylcellulose K100LV was gifted by Colorcon
Asia Pvt. Limited, Goa (India). Sodium bicarbonate,
magnesium stearate, talc were purchased from (SD fi ne
chem., Mumbai). All the materials were of analytical or
pharmacopoeial grade and used as received.
PREPARATION OF LEVOFLOXACIN HEMIHYDRATE
FLOATING TABLETS
Preparation
Direct compression method has been employed
to prepare gastroretentive tablets of levofl oxacin
hemihydrate using HPMC K100LV as polymer and
sodium bicarbonate as gas generating agent.
Procedure
All the ingredients including drug, polymer and
excipients were weighed accurately according to the each
tablet formulation (Table 1). The polymer and sodium
bicarbonate were thoroughly mixed on a butter paper
with the help of a stainless steel spatula. The mixture was
mixed with drug and then excipients were added in the
order of ascending weights, the mixture was blended for
10 min in an infl ated polyethylene pouch. The prepared
blend of each formulation was compressed on 10-station
rotary tablet punching machine (Clit, Ahmedabad) to
obtain fl at-faced tablet of 8 mm diameter.
EVALUATION OF GASTRORETENTIVE
FLOATING TABLETS
The prepared formulations were evaluated for hardness,
friability, weight variation, drug content, swelling index,
in-vitro drug release, short-term stability, fl oating lag time
and in- vitro buoyancy.
Hardness test
The crushing strength (kg/cm²) of tablets was
determined by using digital hardness tester 17 (Electro
Lab E01). The mean of three determinations were taken.
Friability test
It was determined by weighing 10 tablets (W ) after
Original
dusting, placing them in the friabilator (Electro lab EF-2)
and rotating the plastic cylinder vertically at 25 rpm for
4 min. 18 After dusting, the total remaining weight of the
tablets (W ) was recorded and the percent friability
Final
was calculated according to Equation 1.
Weight Final − WeightOriginal
Percent friability =
× 100 (1)
Weight
Uniformity of weight
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 39
Original
The weight (mg) of each of 20 individual tablets was
determined using an electronic balance (Shimadzu
BL-220H). The weight data from the tablets were
analyzed for sample mean and percent deviation. 18
Uniformity of drug content
Two tablets were powdered in a glass mortar and the
powder equivalent to 1 mg of drug was placed in a
500 ml conical fl ask. The drug was extracted with
500 ml of pH 1.2 solution with vigorous shaking
on a mechanical gyratory shaker (100 rpm) for 1 h.
Absorbance was measured at 293 nm in Shimadzu
UV-1800 spectrophotometer against blank.
In-vitro fl oating study
Floating time was determined by using USP XXIII tablet
dissolution apparatus (Electro lab TDT-08L) at 50 rpm
using 900 ml of 0.1N HCl at 37 ± 0.5°C temperature.
The fl oating duration was the time during which the
tablet remains buoyant and fl oating lag time was the
time between tablet introduction and its buoyancy.

D.Nagendrakumar et al.: Design and Optimization of Levofl oxacin Gastroretentive Tablets
Table 1: Composition of Factorial Design and Check point Formulations of Levofl oxacin Hemihydrate
Ingredients*
Formulation code
(mg/tablet)
F1 F2 F3 F4 F5 F6 F7 F8 F9 C1 C2 Levofl oxacin hemihydrate 100 100 100 100 100 100 100 100 100 100 100
HPMC K100LV 100 100 100 150 150 150 200 200 200 125 175
Sodium bicarbonate 40 60 80 40 60 80 40 60 80 50 70
Magnesium stearate 4.8 5.2 5.6 5.8 6.2 6.6 6.8 7.2 7.6 5.5 6.9
Talc 2.4 2.6 2.8 2.9 3.1 3.3 3.4 3.6 3.8 2.8 3.5
*Weight expressed as mg per tablet; HPMC-hydroxypropyl methylcellulose; LV-low viscosity; C 1 , C 2 check point formulations.
Duration of fl oating and fl oating lag time was measured
by visual observation.
Swelling index19 The tablet was weighed (W ) and placed in a glass
Wet
beaker, containing 200 mL of 0.1 N HCl, maintained in
a water bath at 37 ± 0.5ºC. At regular time intervals, the
tablet were removed and the excess surface liquid was
carefully removed by a fi lter paper. The swollen tablet
was then reweighed (W ). The swelling index (SI) was
Dry
calculated using the Equation 2.
W − W
Swelling Index =
W
In-vitro dissolution study
wet Dry
Dry
×100 (2)
In-vitro dissolution study of levofl oxacin hemihydrate
fl oating tablets was carried out in USP XXIII tablet
dissolution test apparatus (Electro lab TDT-08L),
employing a paddle stirrer at 50 rpm using 900 ml of
pH 1.2 solution as dissolution medium at 37 ± 0.5ºC.
One tablet was used in each test. At predetermined
time intervals, 5 ml of the samples were withdrawn by
means of a syringe fi tted with pre-fi lter. The volume
withdrawn at each interval was replaced with same
quantity of fresh dissolution medium. The samples were
analyzed for drug release by measuring the absorbance
at 293 nm using Shimadzu UV-1800 spectrophotometer
after suitable dilutions. All the studies were conducted
in triplicate.
Stability study
Short-term stability study was performed at a temperature
of 45° ± 1°C and 75% relative humidity over a period of
thee weeks for the promising GRDF tablet formulation
F . 15 tablets were packed in amber colored, screw-
3
capped bottles and kept in hot air oven maintained at
45° ± 1°C and 75% relative humidity. Samples were taken
at weekly interval for drug content estimation. At the end
of three weeks, dissolution test and in-vitro fl oating study
were performed to determine the drug release profi les,
in-vitro fl oating lag time and fl oating time.
RESULTS AND DISCUSSION
The hardness of the gastroretentive fl oating tablets
of levofl oxacin hemihydrate was found to be in the
range of 6.45 to 6.74 kg/cm² for the factorial design
formulations. The friability of all tablets was from 0.53
to 0.89 % for the factorial design formulations. The
percentage deviation from the mean weights of all the
batches was found to be within the prescribed limits as
per IP. The low values of standard deviation indicated
uniform drug content (Table 2). The swelling index of
the tablets was increased with an increase in the polymer
content and the content of gas generating agent i.e.
sodium bicarbonate (Table 2).
In-vitro fl oating study was performed by placing tablets
in USP XXIII tablet dissolution apparatus containing
900 ml of pH 1.2 maintained at a temperature of
37 ± 0.5ºC. The fl oating lag time and fl oating time were
noted visually. The results were presented in Table 2.
For factorial formulations, lag time was in the range of
10.33 to 64.57 sec. With formulations containing the
same amount of polymer of the same grade, fl oating
lag time was decreased with increase in concentration of
sodium bicarbonate. For formulation F 3 , it was lowest
(10.33 sec) as the drug-polymer (HPMC K100LV) ratio
is 1:1 and sodium bicarbonate is in highest proportion
among the formulations and the tablet remained intact
for 12 h, while for formulation F 7 , lag time was highest
(64.57 sec) as drug-polymer ratio is 1:2 and sodium
bicarbonate is in lowest proportion (40 mg). All the
designed formulations displayed a fl oating time of more
than 24 h.
In-vitro drug release study was performed using USP
XXIII tablet dissolution apparatus From the above data,
it was evident that as the proportion of polymer in the
formulation increased, cumulative percent drug release in
12 h decreased and as the proportion of the gas generating
agent increased, the drug release was increased (Table 3).
Factorial design
Based on the preliminary trial, the levels of independent
variables (X and X ) were fi xed at 100, 150, 200 mg for
1 2
HPMC K100LV (X ) and at 40, 60, 80 mg for sodium
1
bicarbonate (X ) in designing the formulations of 3²
2
40 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

D.Nagendrakumar et al.: Design and Optimization of Levofl oxacin Gastroretentive Tablets
Table 2: Evaluation Results of Factorial Design Formulations of Levofl oxacin Hemihydrate GRDDS
Formulation Code
Mean Hardness
(kg/cm 2 ) Friability (%)
Average
Weight (mg)
Mean Drug Content
Percent* ± SD
Swelling
Index* ± SD
Floating Lag
Time (sec)
Floating
time (h)
F 1 6.61 0.53 248.26 98.60 ± 1.51 4.25 ± 1.35 46.21 >24
F 2 6.55 0.67 268.15 99.64 ± 1.74 13.26 ± 1.87 12.61 >24
F 3 6.45 0.71 289.73 101.48 ± 0.97 49.82 ± 1.79 10.33 >24
F 4 6.69 0.74 300.02 100.05 ± 0.42 07.38 ± 0.06 57.28 >24
F 5 6.62 0.79 319.69 101.87 ± 1.68 23.49 ± 0.48 18.48 >24
F 6 6.51 0.84 342.95 101.62 ± 1.57 53.18 ± 1.42 14.69 >24
F 7 6.74 0.85 352.27 99.74 ± 0.38 10.75 ± 1.29 64.57 >24
F 8 6.67 0.87 369.86 101.85 ± 1.35 28.37 ± 1.15 24.29 >24
F 9 6.59 0.89 393.48 99.39 ± 1.06 56.24 ± 1.45 18.54 >24
*Average of thee determinations, values shown in parenthesis are standard deviations. Formulation F 3 was selected as the best and used for further studies; GRDDSgastroretentive
drug delivery system.
Table 3: In- vitro Cumulative Percent Release of Levofl oxacin Hemihydrate for Factorial Design Batches
Cumulative Percent Drug Release Mean* ± SD
Time (h) F1 F2 F3 F4 F5 F6 F7 F8 F9 01 8.84 ± 0.29 10.37 ± 0.96 13.16 ± 0.65 8.31 ± 0.72 9.51 ± 0.49 09.88 ± 0.34 10.58 ± 0.10 7.51 ± 0.45 6.98 ± 0.17
02 16.96 ± 0.00 18.89 ± 0.90 24.15 ± 0.17 13.24 ± 0.49 13.54 ± 0.89 15.57 ± 0.45 17.40 ± 1.09 14.90 ± 1.44 15.27 ± 1.17
03 24.51 ± 0.50 26.48 ± 0.96 32.33 ± 0.40 20.76 ± 0.79 21.72 ± 1.00 26.13 ± 0.50 24.42 ± 1.17 21.22 ± 1.34 24.29 ± 2.43
04 32.66 ± 2.05 34.06 ± 0.64 40.92 ± 0.72 29.44 ± 1.09 28.88 ± 1.09 36.87 ± 0.47 29.81 ± 2.05 31.91 ± 1.54 34.18 ± 0.61
05 41.65 ± 1.54 42.44 ± 1.15 45.18 ± 0.42 38.36 ± 1.00 35.47 ± 1.12 43.05 ± 0.41 35.70 ± 3.71 39.10 ± 1.20 42.05 ± 0.44
06 48.51 ± 1.41 49.50 ± 0.55 50.05 ± 0.20 46.65 ± 0.80 46.15 ± 0.60 50.13 ± 0.40 44.22 ± 2.75 49.09 ± 1.20 47.15 ± 0.80
07 57.35 ± 1.79 58.25 ± 1.59 63.14 ± 0.56 53.00 ± 1.19 57.67 ± 1.80 54.54 ± 0.54 49.97 ± 1.84 52.58 ± 1.20 56.38 ± 0.61
08 63.41 ± 0.47 65.00 ± 0.74 67.05 ± 1.13 62.39 ± 2.00 60.00 ± 0.37 60.39 ± 0.85 57.16 ± 2.34 56.06 ± 1.20 66.18 ± 0.86
09 71.06 ± 2.42 72.13 ± 0.15 72.18 ± 0.85 66.68 ± 1.34 69.38 ± 1.21 73.51 ± 0.90 64.02 ± 2.05 63.05 ± 1.20 70.05 ± 1.65
10 77.26 ± 2.80 85.61 ± 0.15 88.03 ± 1.02 73.47 ± 2.42 77.36 ± 1.35 79.30 ± 1.95 72.24 ± 2.57 78.54 ± 1.20 72.05 ± 1.14
11 82.58 ± 3.05 91.8 ± 0.37 97.52 ± 0.26 76.78 ± 0.77 80.14 ± 1.43 82.13 ± 0.54 77.26 ± 2.71 81.13 ± 1.32 75.18 ± 1.77
12 87.23 ± 2.67 96.49 ± 0.51 98.91 ± 0.20 83.05 ± 2.50 85.32 ± 1.88 88.91 ± 2.96 81.72 ± 2.22 83.58 ± 1.31 79.13 ± 1.81
*Average of thee determinations, values shown in parenthesis are standard deviations.
Table 4: Dissolution Parameters for 3² Full Factorial Design Batches
Batch Code
Variable Level in
Coded Form
* #
X X 1
2
t 50% (h) t 70% (h)
Cumulative Percent
Drug Release in 12 h
F1 –1 –1 6.20 8.91 87.23
F2 –1 0 6.13 8.71 96.49
F3 –1 1 5.95 8.52 98.91
F4 0 –1 6.68 9.36 83.05
F5 0 0 6.36 9.18 85.32
F6 0 1 6.01 8.74 88.91
F7 1 –1 7.16 9.72 81.72
F8 1 0 6.27 9.47 83.58
F9 1 1 6.32 8.93 79.13
C1 -0.5 +0.5 6.21 8.94 90.98
C2 +0.5 +0.5 6.56 9.23 85.31
C 1 , C 2 check point batches; t 50% , t 70% analyzed by matrix model fi tting using PCP Disso V3 Software. * For HPMCK100LV cps (X 1 ); transformed levels in mg are: –1=100;
‘0’=150, +1=200, –0.5=125, +0.5=175; # For NaHCO 3 (X 2 ), transformed levels in mg are: –1=40; ‘0’=60, +1=80, –0.5=50, +0.5=70; All the batches contained 100 mg of
levofl oxacin hemihydrate,1% talc and 2% magnesium stearate.
full factorial design. The dissolution parameters i.e.,
t 50% , t 70% values were selected as dependent variables.
The formulation codes of the nine batches of factorial
formulations along with dissolution parameter values
(t 50% , t 70% ) and cumulative percent drug released in 12 h
were shown in (Table 4). From the data, it was evident
that formulation F 3 showed highly satisfactory values
for dissolution parameters (t 50% 5.95 h, and t 70% 8.52 h)
and released approximately 98.91% drug in 12 h.
Hence, formulation F 3 was considered as the optimized
promising levofl oxacin hemihydrate gastric fl oating drug
delivery system with improved bioavailability.
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 41

Development of polynomial equations
D.Nagendrakumar et al.: Design and Optimization of Levofl oxacin Gastroretentive Tablets
From the data of dissolution parameters shown in
Table 4 for factorial formulations F 1 to F 9 , polynomial
equations for two dependent variables (t 50% , and t 70% )
have been derived using PCP Disso 2000 V3 software.
The general polynomial equation (Equation 3) for 3² full
factorial design was
2 2 Y = b +b X +b X +b X X +b X +b22X (3)
0 1 1 2 2 12 1 2 11 1 2
Where, Y is dependent variable, b arithmetic mean
0
response of nine batches, and b estimated coeffi cient
1
for factor X . The main effects (X and X ) represented
1 1 2
the average result of changing one factor at a time
from its low to high value. The interaction term
(X X ) showed how the response changes when two
1 2
factors were simultaneously changed. The polynomial
2 2 term (X and X2 ) were included to investigate non-
1
linearity.
The equation derived for t was Y = 6.28 + 0.216X 50% 1 1
– 0.283 X . The negative sign for coeffi cient of X 2 2
indicates that, as the concentration of gas generating
agent (sodium bicarbonate) increases, t value
50%
decreases.
The equation derived for t was Y = 9.02+ 0.31 X –
70% 2 1
0.30 X . 2
The validity of the equations was verifi ed by designing
two check point formulations (C and C ) and studying
1 2
the drug release profi les. The dissolution parameters
predicted from the equations derived and those
observed from experimental results were summarized in
.
Figure 2: Contour plot showing effect of factorial variables on t50% Figure 1: Response surface plot showing effect of factorial vari-
.
ables on t50% Table 5. The closeness of predicted and observed values
for t 50% and t 70% indicated validity of derived equations
for the dependent variables. The computer generated
response surfaces and contour plots for the dependent
variables were shown in Fig. 1–4.
Stability study
Short-term stability study was performed on the
promising formulation F by storing the samples at
3
45 ± 1ºC for 3 weeks. The samples were tested for any
changes in physical appearance and drug content at
weekly intervals. Statistical analysis was performed on
the drug content data and drug release parameters by
using Student t-test. The t value for the drug content
was found to be 3.85. For t and t the t- values
50% 70%,
were found to be 3.46 and 3.00, respectively (Table 6).
These results indicated that there were no signifi cant
changes in drug content and dissolution profi le of
the formulation F during storage at 45ºC for 3 weeks
3
shown no signifi cant effect on physical characteristics
and drug content.
CONCLUSIONS
It was concluded that the gastroretentive fl oating
systems of levofl oxacin hemihydrate with shorter
lag time was prepared by direct compression method
using HPMC K100LV and sodium bicarbonate as
gas generating agent. As the amount of polymer in
the tablet formulation increased, the drug release rate
decreased and as the concentration of gas generating
agent (sodium bicarbonate) increased, the drug release
increased and at the same time fl oating lag time decreased.
42 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

D.Nagendrakumar et al.: Design and Optimization of Levofl oxacin Gastroretentive Tablets
.
Figure 3: Response surface plot showing effect of factorial variables on t70% .
Figure 4: Contour plot showing effect of factorial variables on t70% Table 5: Dissolution Parameters Predicted and Observed
Values for Check point batches
Predicted
Values (h)
Observed
Values (h)
Formulations t50% t70% t50% t70% C1 6.31 9.01 6.20 8.90
C2 6.20 9.02 6.50 9.20
Table.6: Statistical Analysis of Dissolution Parameters
(t 50% , t 70% ) of Stability study for Batch F 3
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 43
Trial
t 50% Values
1st Day
(A)
21st
Day
(B)
A-B
t 70% Values
1st
Day
(A)
21st
Day
(B)
A-B
I 6.01 5.98 0.03 8.69 8.65 0.04
II 5.94 5.93 0.01 8.74 8.73 0.01
III 5.89 5.87 0.02 8.72 8.69 0.03
Mean 5.95 5.92 0.02 8.71 8.69 0.026
SD ± ± 0.060 ± 0.055 ± 0.01 ± 0.025 ± 0.04 ± 0.015
t = 3.46 ;( p

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44 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

Study of the binding properties of hydroxypropyl
guar and its utilization in the formulation and
evaluation of metoprolol tartarate tablets
Swamy N.G.N.*, Dharmarajan T.S., Paranjothi K.L.K
*Department of Pharmaceutics, Government College of Pharmacy, Bangalore – 560 027
ABSTRACT
Derivatization of guar to hydroxypropyl guar enhances the interaction coeffi cient and checks the hydration
rate of the polymer molecule. Hydroxypropyl guar is investigated for its binding property along with sodium
carboxymethylcellulose (1% w/w) as the parallel binding agent. Tablets of metoprolol tartarate were prepared
by wet granulation. Pre-compression parameters such as rate of fl ow, angle of repose, Carr’s index, Hausner’s
ratio, residual moisture content and assay determinations were carried out. Hydroxypropyl guar bound granules
revealed lower Carr’s index value and Hausner’s ratio in comparison to sodium carboxymethylcellulose bound
granules thereby indicating better free fl owing properties. In respect to post compression parameters, tablets
made with hydroxypropyl guar displayed superior hardness, lesser deviation of tablet weight about the mean, a
higher value of (Crushing strength: Friability)/Disintegration time, a faster drug dissolution in comparison to sodium
carboxymethylcellulose bound tablets. While guar with its high intrinsic viscosity restricts its use as a retardant
component in matrix tablets, hydroxypropyl guar with its enhanced interaction coeffi cient and controlled hydration
rate has yielded a promising adjuvant for binding metoprolol tartarate tablets with improved in vitro performance.
Keywords: Carr’s index, crushing strength, disintegration time, friability, hausner’s ratio, hydroxypropyl guar,
interaction coeffi cient, rate of hydration, residual moisture content.
INTRODUCTION
Guar gum1 consists of the endosperm part
of guar plant namely Cyamopsis tetragonolobus
Linn. (Family: Leguminosae). When used in
extemporaneous preparations, guar imparts
colour, which is not desirable. Guar has a
very high intrinsic viscosity but very poor
interaction coeffi cient. 2 Derivatization of
guar to hydroxypropyl guar improves its
interaction coeffi cient. 3 By virtue of its
high intrinsic viscocity, guar gum has been
extensively used as a retarding component
in the formulation of matrix tablets. 4–18
Hydroxypropyl guar with improved
interaction coeffi cient has been investigated
as a gelling agent, 19 as a bodying agent20 in
suppositories, fi lm forming agent21 and
as a suspending agent. 22 Hydroxypropyl
guar with a controlled rate of hydration
is investigated for its solution binding
properties 23 in the formulation of oral
tablets. Sodium carboxymethylcellulose is
tried as the parallel binding agent. 24–28
Metoprolol tartarate 29 (MT), is a selective
β-adrenoreceptor antagonist useful in
the management of hypertension and
ischaemic heart diseases. MT 30 is readily
absorbed after oral administration and
rapidly distributed into body tissues.
The elimination half-life is about 3–7 h.
The drug undergoes extensive fi rstpass
metabolism; only 50% of the orally
administered drug reaches the systemic
circulation. Adult oral dose is 50 mg twice
daily to be optimized subsequently and
not to exceed 400 mg per day. Tablets
containing 50 mg of MT per tablet were
formulated using hydroxypropyl guar
Research Ar cle
Received Date : 26-12-2011
Revised Date : 01-03-2012
Accepted Date : 05-03-2012
DOI: 10.5530/rjps.2012.1.6
Address for
correspondence
Dr. N.G.N. Swamy
Professor
Department of Pharmaceutics
Government College of
Pharmacy,
No. 2, P. Kalinga Rao Road,
Bangalore – 560 027
Mob. No: + 91 9945451772
E-mail: ngnswami@yahoo.co.in
www.rjps.in
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 45

Swamy NGN et al.: Study of the binding properties of hydroxypropyl guar and its utilization in the formulation and evaluation of metoprolol tartarate tablets
(HPG) and sodium carboxymethylcellulose (Na CMC)
as binders.
MATERIALS AND METHODS
MT I.P. was obtained as a gift sample from ASTRA-
IDL ltd. Na CMC (high viscosity grade), Lobachemie
make was used. All other excipients used were of Pharmacopoeial
grade. HPG was obtained by derivatization
of guar, 3 MT tablets weighing around 150 mg each and
containing 50 mg of the drug were made by wet granulation
by employing 1% W/V aqueous dispersions
of HPG and Na CMC respectively as solution binders.
1% W/V HPG dispersion revealed a viscosity of
6400 cps; Na CMC displayed a viscosity of 448 cps.
The aqueous polymer dispersions were stabilized with
0.18% W/V methyl paraben and 0.02% W/V propyl
paraben.
The formulation details of the tablets are shown in
Table 1.
PART-I: PREPARATION OF GRANULES
The quantity of MT as specifi ed in Table 1 was mixed
with lactose IP in the ascending order of weights; the
granulating agent was added little by little so as to get
a damp mass with a pliable consistency. The batter was
allowed to stand for 15 min and passed through sieve
20; the granules so obtained were dried in an oven at 60°
for 1 h duration. The agglomerates were regranulated by
using sieve 20 superimposed on sieve 40. While granules
retained on sieve 20 were used for compression, the
fi nes retained on sieve 40 were used for improvement
of fl ow properties of granules.
PART-II: STUDY OF GRANULE PROPERTIES
(a) Determination of compressibility factor
The untapped and the tapped bulk densities were
obtained by the 10 point method. The 10 cc tared
measuring jar was fi lled with granules up to 1 cc and
the weight was recorded; likewise, the weight of 2 cc,
3 cc ... up to 10 cc was fi lled with granules and the weight
was recorded without tapping. Similarly the procedure
was repeated with the granules fi lled up to 1 cc, tapped
so as to compact the granules, excess granules was added
to record a volume of 1 cc after tapping. The weight
of 2 cc, 3 cc ... up to 10 cc with tapping each time after
the addition of excess granules to adjust the volume
was recorded. A graph of weight values (gm) vs corresponding
volume (cc) was constructed. The bulk density
was arrived from the slope of the linear relationship.
Compressibility factor ‘C’ was calculated using the formula,
c
b a
=
b
− (1)
Table 1: The formulation details of Metoprolol tartarate
tablets
Tablet wt Tablet wt
Ingredient
in gm in gm
Metoprolol tartarate 30 30
Lactose I.P 60 60
Hydroxypropyl guar 1% w/v aqueous
dispersion
Q.S –
Sodium CMC 1% w/v aqueous
dispersion
– Q.S
Talc 2% 2%
Where, b=Tapped bulk density and a = Untapped bulk
density. The graphical representations are shown in
fi gure 1and 2. Compressibility factor multiplied by 100
gives Carr’s index values.
(b) Determination of the optimum amount of
Lactose fi nes to be incorporated to the 20/40
mesh granules to improve the fl ow properties
Since 1% w/v HPG bound granules revealed the lowest
Carr’s index value, 31 1% w/v HPG bound lactose
granules were used for the evaluation.
(i) Determination of angle of repose32 Carried out by employing fi xed bed method using the
formula,
tan θ = ;
h
(2)
r
Where, θ = angle of repose, h = height of the heap
and r = radius of the heap. The exact value of θ was
arrived at by referring to the table on natural tangents.
The advantage of this method being, the dimension of
the base is fi xed, whereas, the height alters as the angle
of repose changes.
(ii) Determination of the rate of fl ow33 Carried out using a glass funnel having an orifi ce and
base diameters of 0.8 cm and 9 cm respectively and
securely clamped to a retort stand. A 50 gm sample was
introduced into the funnel and the powder was allowed
to fall freely under the gravitational force. The fl ow rate
was calculated using the expression:
Flow rate = amount of powder (gm) / time of fl ow (s). (3)
The above studies were conducted on 20/40 # lactose
granules blended with 5% w/w, 10% w/w and 15%
w/w, lactose fi nes. The average value of three such
determinations is recorded in fi gures 3 and 4.
(c) Determination of optimum concentration of
the glidant (Talc) to be added to dummy lactose
granules containing 10% w/w of Lactose fi nes
The θ values and the fl ow rate values were determined
for the above mentioned granules blended with 1%, 2%,
3%, 4% and 5% w/w talc powder. The infl uence of the
46 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

Swamy NGN et al.: Study of the binding properties of hydroxypropyl guar and its utilization in the formulation and evaluation of metoprolol tartarate tablets
Figure 1: Untapped and tapped BD recording for 20/40#MT granules made with 1% w/v of HPG.
Figure 2: Untapped and tapped BD recording for 20/40#MT granules made with 1% w/v of NaCMC.
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 47

Swamy NGN et al.: Study of the binding properties of hydroxypropyl guar and its utilization in the formulation and evaluation of metoprolol tartarate tablets
Figure 3: Influence of percentage lactose fines on the angle of repose for 20/40# lactose granules.
Figure 4: Influence of percentage lactose fines on the rate of flow for 20/40# lactose granules.
talc % w/w on the θ and the fl ow rates is depicted in
fi gure 5 and 6.
From the studies carried out at “b” and “c” above, it
could be concluded that the optimum fi nes concentration
was 10% w/w and optimum glidant concentration was
2% w/w of talc powder.
(d) Determination of Carr’s index for 20/40 mesh
MT granules containing 10% w/w fi nes and
blended with 2% w/w of Talc powder
The graphical representation for granules made with
1% w/v HPG as binder are shown in fi gure 7 and
for granules made with 1% w/v NaCMC is shown
in fi gure 8. Carr’s Index is calculated by using the
formula
CI = (Dt – Db/ Dt) × 100 (4)
(e) Hausner’s ratio
It is an index of ease for powder fl ow. It is calculated by
the following formula,
Hausner s ratio Dt
' = (5)
Db
48 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

Swamy NGN et al.: Study of the binding properties of hydroxypropyl guar and its utilization in the formulation and evaluation of metoprolol tartarate tablets
Figure 5: Influence of % w/w talc added on the angle of repose of 20/40 mesh lactose granules blended with 10% w/w of lactose fines.
Figure 6: Influence of % w/w talc added on the flow rate of 20/40 mesh lactose granules blended with 10% w/w of lactose fines.
Table-2: Infl uence of additives on the angle of repose and the fl ow rate for dummy 20/40 # Lactose granules
Granules composition
20/40 mesh lactose
granules bound with
1% w/v HPG,
20/40 mesh lactose
granules bound with
1%w/v NaCMC,
Angle of
repose for
plain granules
Angle of repose
with the added
additives
Fall in angle
of repose
Flow rate in gm/s
for granules with
additives
Flow rate in
gm/s for plain
granules
Enhancement
in fl ow rate
(gm/s)
40°7’ 38°42’ 1°25’ 3.508 3.385 0.123
39°11’ 37°18’ 1°53’ 3.533 3.496 0.037
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 49

Swamy NGN et al.: Study of the binding properties of hydroxypropyl guar and its utilization in the formulation and evaluation of metoprolol tartarate tablets
Weight (gm)
Figure 7: Untapped and tapped BD recording for 20/40# MT granules made with 1% w/v HPG blended with 10% w/w fines and 2% w/w talc.
Where Dt is the tapped bulk density and Db is the
untapped bulk density.
(f) Determination of angle of repose θ
Angle of repose determination and fl ow time
determination were carried out on 20/40 mesh dummy
granules blended with 10% w/w lactose and 2% W/W
talc powder.
The infl uence of additives on the fl ow properties of
20/40 mesh dummy granules is depicted in Table 2
(g) Determination of residual moisture content
Residual moisture content was determined by drying the
granules at 60° for 15 min. 34
(h) Assay of granules
(i) Carried out to ascertain uniform distribution of drug
in tablets over the entire range of sample (ii) To enable
us to adjust the die volume/ the total weight of the tablet
so as to get tablets having the required drug content. MT
has a λmax at 275 nm in 95% v/v ethanol. It obeys Beer’s
Lamberts law in the concentration range of 10–100
μg/ml. A calibration curve was constructed; the linear
relationship had a slope value of 0.0046 and an intercept
value of 0.0034 which were used in the calculation
of the drug content in the assay of sample. The precompression
parameters are contained in Table 3.
PART-III: COMPRESSION OF GRANULES
INTO TABLETS
The compression was carried out using a 10 station
rotary tablet machine. The capacity of the die cavity was
so adjusted as to accommodate a weight of granules
containing 50 mg of MT. 7mm punches were used and
a compression pressure of 6 Kg was applied on the
rollers for all tablets. The tablets were preserved in tight
screw capped bottles until further evaluation tests were
carried out.
PART-IV: EVALUATION OF TABLETS
The compressed tablets were subjected to the following
evaluation tests.
(i) Hardness test 35
Carried out using Monsonto hardness tester; a mean of
three readings was recorded.
(ii) Determination of disintegration time36 Disintegration time was recorded as per I.P procedure;
6 tablets were subjected to the study.
(iii) Weight variation 36
The test was carried out on 20 tablets. The tablets were
weighed collectively and the average weight was arrived
50 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

Swamy NGN et al.: Study of the binding properties of hydroxypropyl guar and its utilization in the formulation and evaluation of metoprolol tartarate tablets
Table 3: Pre-compression parameters
Granule composition
20/40 mesh MT granules bound with
1% w/v HPG, blended with 10% w/w fi nes,
2% w/w talc.
20/40 mesh MTgranules bound with
1%w/v NaCMC, blended with 10% w/w
fi nes, 2% w/w talc.
RMC* Residual moisture content.
Weight (gm)
Untapped bulk
density (Db) gm/cc
at. The weights of 20 tablets were recorded individually
and the deviation from the mean was calculated. Not
more than 2 tablets to deviate beyond ±7.5% and not
even one to deviate beyond ±15%.
(iv) Friability Test 37
The test was carried out on 20 tablets weighed collectively.
The tablets were loaded on to a Roche friabilator and
Tapped bulk
density (Dt) gm/cc
Carr’s
Index
0.5638 0.5761 2.13
Hausner’s
Ratio
1.0218
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 51
RMC*
Assay Value
(mg)
1.34 47.51
0.5346 0.5728 6.66 1.0714 1.24 48.71
Volume (cc)
Figure 8: Untapped and tapped BD recording for 20/40# MT granules made with 1%w/v NaCMC blended with 10% w/w fines and 2%w/w talc .
Table 4: Post compression parameters
Hardness
in Kg/cm2 Disintegration Friability Assay
time in minutes %
(mg) (CS:FR)/DT* t in min
80%
Tablets made with 1% w/w
HPG as binder
4.5 8.20 0.24 47.86 2.3 20
Tablets made with 1% w/v
of NaCMC as Binder
3.0 7.50 0.18 47.45 2.1 35.45
(CS:FR)/DT*; CS = Crushing strength, FR = Friability and DT = Disintegration time.
rotated at 25 rpm for 4 min; tablets weighed again and
% friability was calculated. It is desirable that the weight
loss is less than 1.0%.
(v) Assay
As mentioned under granules; 5 tablets36 are randomly
powdered; a quantity equivalent to 100 mg of MT was
taken and dissolved in 95% of ethanol; subsequent

Swamy NGN et al.: Study of the binding properties of hydroxypropyl guar and its utilization in the formulation and evaluation of metoprolol tartarate tablets
Figure 9: Dissolution profile of 1% HPG and 1% Na CMC bound Metoprolol Tartarate tablets.
dilutions were made with ethanol to have MT
concentration in the range of 10–100 μg/ml; absorbance
was read at 275 nm; concentration of MT was calculated
by making use of slope and intercept values obtained
from the calibration curve. Post compression parameters
are compiled in Table 4.
(vi) Dissolution profi le
Dissolution testing was carried out in 0.1N Hydrochloric
acid. Amount of medium used was 1000 ml; rpm 50.
Volume withdrawn was 10 ml, samples withdrawn at
the end of 15, 30, 45, 60, 75 and 90 min and analyzed
spectrophometrically at 275 nm. The dissolution profi les
of HPG bound and Na CMC bound tablets are depicted
in fi gure 9.
RESULT AND DISCUSSION
In this study, 1% w/v aqueous dispersion of HPG
has been tried as a binder for MT; 1%w/v aqueous
dispersion of Na CMC is used as the parallel binder.
20/40 mesh lactose granules were made and mixed
with optimum amounts of 10% w/w lactose fi nes
and 2% w/w of talc powder. 20/40 mesh granules as
mentioned afore said were mixed with 10% w/w fi nes
and 2%w/w talc powder and subjected to untapped and
tapped density determination; from this the percentage
compressibility factor i.e. Carr’s index and Hausner’s
ratio were calculated. Granules made with HPG as
binder revealed a Carr’s index value of 2.13 in contrast
to a value of 6.66 for granules made with 1% w/v Na
CMC dispersion. Granules having Carr’s index value in
the range of 5 to 12 reveal excellent fl ow characteristics.
Granules made with HPG revealed a Hausner’s ratio of
1.022 in contrast to a value of 1.071 in case of Na CMC
granules. Lower Hausner’s ratio (1.25). It is a well verifi ed fact
that the more compressible a bed of particles is, the less
fl owable the powder or granules will be. A material having
a compressibility value of less than 16 is considered to
be a free fl owing material; this is in close agreement with
the data compiled in table 3. Repose angle also needs
to be considered along with compressibility; Repose
angle 34 for the experimental samples are in the range of
37–38° which is marginally higher than the optimum
value i.e. 30°. When the angle of repose exceeds 50°,
the fl ow is rarely acceptable for manufacturing process.
Table 5: Flow properties and corresponding angle of
repose
Flow property Angle of repose (Degrees)
Excellent 25–30
Good 31–35
Fair 36–40
Passable 41–45
Poor 46–55
Very poor 56–65
Very, Very Poor >66
52 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

Swamy NGN et al.: Study of the binding properties of hydroxypropyl guar and its utilization in the formulation and evaluation of metoprolol tartarate tablets
The infl uence of angle of repose 38 on fl ow properties
of granules is depicted in Table 5.
The fl ow rate was determined by passing 50 gm of
granules through a glass funnel with orifi ce and base
diameters of 0.8 cm and 9 cm respectively. By and large,
lower the compressibility factor and smaller the repose
angle, the faster is the predicted fl ow rate of granules.
HPG granules have revealed a signifi cant enhancement
in the fl ow rate after the inclusion of additives i.e.
0.123 gm/s in contrast to an enhancement value of
0.037 gm/s incase of granules made with Na CMC.
For trouble free compression, a moisture content of
around 1.5% is desirable. HPG bound granules revealed
a moisture content of 1.34% in contrast to a value of
1.24 in case of granules made with Na CMC as binder.
Moisture content of less than 1% can cause capping and
lamination whereas above 2%, some sticking problem
may develop.
In respect to post compression parameters, from the
weight variation data, it is observed that in case of HPG
bound tablets, the % deviation from the mean was found
to range between (-) 5.86 to (+) 6.73 in contrast to a
value of (-) 7.39 to (+) 8.94 for Na CMC bound tablets.
This observation correlates well with the lower ‘C’
values obtained for HPG bound granules. Tablets made
with HPG as binder revealed a hardness of 4.5 kg/cm 2
in contrast to a value of 3 Kg/cm 2 for tablets made
with Na CMC as binder. The friability value for HPG
bound tablets was 0.24% in contrast to a value of 0.18%
in case of Na CMC bound tablets. Disintegration time
for HPG bound tablets was 8.3 min which was slightly
higher than the Disintegration time value of 7.8 min for
Na CMC bound tablets. Crushing strength – friability
ratio (CS:FR) which is the quotient of the crushing
strength (CS) value divided by the friability (FR) value, is
an index used as a measure of the mechanical strength
of tablets. But the (CS:FR)/DT ratio would be a better
index for measuring tablet quality. 39 It is so because, in
addition to measuring tablet strength (crushing), and
tablet weakness (friability), it does evaluate all negative
effects of these parameters on disintegration time. 40
HPG bound tablets revealed a (CS:FR)/DT ratio of
2.26 in contrast to a value of 2.1 in case of Na CMC
bound tablets. A higher value of (CS:FR)/DT is
indicative of a better balancing act between the cohesive
binding and disintegrating behavior of the tablets. From
the dissolution profi le, a t 80% value of 20 min has been
displayed in case of HPG bound tablets in contrast to a
value of 35.45 min in Case of Na CMC bound tablets.
CONCLUSION
The hydroxypropyl guar with it’s enhanced interaction
coeffi cient and controlled rate of hydration has proved
to be a superior solution binder in achieving suffi ciently
rapid release of the drug from the tablet formulation in
comparison to the conventionally used tablet solution
binder namely sodium carboxymethylcellulose.
ACKNOWLEDGEMENT
The authors wish to thank Shri P. Guruswamy, The
then plant manager, Caryll Pharma, Sarakki main road
Bengaluru, for providing tablet compression facility;
Shri K.R.P Shenoy, Controller, QA and Technical
services, ASTRA-IDL for gifting Metoprolol Tartarate
and Shri Chandrashekhar, Managing Director, Ce-Chem
Pharmaceuticals Pvt Ltd. Peenya, Bengaluru for the gift
Sample of Lactose I.P.
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54 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

Phytochemical investigation of root extract
of the plant Carissa spinarum
Karunakar Hegde 1 , D. Satyanarayana 2 , Arun B. Joshi 3
1 Department of Pharmacology, Srinivas College of Pharmacy, Valachil, Post- Parangepete, Mangalore- 574 143,
Karnataka, India
2Department of Pharmachemistry, N G S M Institute of Pharmaceutical Sciences, Mangalore- 574 160, Karnataka, India
3Department of Pharmacognosy, Goa College of Pharmacy, Panaji- 403 001, Goa, India
ABSTRACT
From the petroleum ether extract of the roots of Carissa spinarum Linn. (Apocynaceae), six compounds namely
stigmasterol, ursolic acid, lupeol, campesterol, 17-hydroxy-11-oxo-nor-β-amyrone and urs-12-ene-3β, 22β-diol-
17-carboxylic acid have been isolated by column chromatography. Their structures were characterized by m.p.,
IR, 1HNMR, 13CNMR and mass spectral data. However, the compounds stigmasterol, campesterol, 17-hydroxy-
11-oxo-nor-β-amyrone and urs-12-ene-3β, 22β-diol-17-carboxylic acid were reported for the fi rst time from the
root of this plant.
Keywords: Carissa spinarum, Root extract, Sterol, Triterpenoids
INTRODUCTION
Carissa spinarum Linn. (Carissa opaca Stapf
ex Haines) is a evergreen shrub with short
stem and strong thorns in pairs, belonging
to family Apocynaceae. The plant is widely
distributed throughout the dry, sandy and
rocky soils of India, Ceylon, Myanmar and
Thailand. 1 In traditional system of medicine
the plant is used as purgative, for the
treatment of rheumatism, cleaning worm
infested wounds of animals and in snake
bite. 1,2 Earlier studies have shown that the
extract of the plant possesses cardiotonic, 3
anticonvulsant, 4 hepatoprotective, 5 antiarthritic,
6 antibacterial, 7 potent antioxidant8 and CNS depressant activitiy. 9 Various
cardiac glycosides, 10 caffeic acid, 11 ursolic
acid, naringin12 germacrane sesquiterpene
and lignans8 were reported from this plant.
Since the chemical investigation of the
roots of Carissa spinarum has not been dealt
in detail, in the present study an effort was
made to establish and report the chemical
investigations, of the petroleum ether
extract of the roots of this plant.
MATERIALS AND METHODS
Chemicals and Instruments
All the chemicals and solvents were
of analytical grade and procured from
Ranbaxy Fine Chemicals Ltd., Mumbai,
India. The melting points were determined
in a Toshniwal melting point apparatus
and were uncorrected. The IR spectra of
the compounds were recorded using KBr
pellet method on Rx-1 Perkin-Elmer FTIR.
1 13 HNMR and CNMR spectra were run on
Bruker Avance II 400 spectrophotometer
using CDCl as a solvent. Mass spectra
3
(FAB-MS) were obtained on a JEOL SX
102/DA-6000 mass spectrometer.
Plant Material
The roots of C. spinarum were collected from
Sirsi, Uttara Kannada District, Karnataka,
Research Ar cle
Received Date : 28-11-2011
Revised Date : 03-02-2012
Accepted Date : 08-02-2012
DOI: 10.5530/rjps.2012.1.7
Address for
correspondence
Karunakar Hegde
Department of Pharmacology
H.K.E. Society’s M T R Institute
Srinivas College of Pharmacy
Valachil, Post- Parangepete
Mangalore- 574 143
Karnataka, India
E-mail: khegde_sh2003@
yahoo.co.in
Phone: +91-824-2274722
Fax: +91-824-2274725
www.rjps.in
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 55

Karunakar Hegde et al.: Phytochemical Investigation of Root Extract of the Plant Carissa spinarum
India during May 2007. It was authenticated by
Dr. Gopalakrishna Bhat, Department of Botany, Poorna
Prajna College, Udupi, Karnataka, India. A voucher
specimen no. 105b is deposited in the herbarium of our
institute.
Extraction and Isolation
The shade dried roots of the plant (1000 g) were soaked
in 1.5 L of 95% ethyl alcohol and extracted in the
cold for 4 days with occasional shaking. After 4 days
the ethanol layer was decanted off. The process was
repeated for 4 times. The solvent from the total extract
was fi ltered, the concentrate was evaporated to dryness
under reduced pressure and low temperature (40oC) on a rotary evaporator to give the ethanolic extract
(13% w/w yield), which was stored at 4oC until use.
The ethanol extract (125 g) was suspended in distilled
water and extracted with petroleum ether (60–80oC, 8 ×
500 ml). All the fractions were then washed with distilled
water (30 ml), dried over anhydrous sodium sulphate
and freed of solvent by distillation to give petroleum
ether (60–80ºC) soluble fraction (32 g). Petroleum ether
fraction (25 g) was saponifi ed by refl uxing for 6 h in
500 ml 5% methanolic KOH and allowed to stand at
room temperature for 20 h. The unsaponifi able portion
was extracted with diethyl ether. All the ethereal fractions
Figure 1: Structure of isolated compounds.
were combined and washed with distilled water. The
solvent was evaporated and dried over anhydrous sodium
sulphate to afford a yellowish residue (9 g). 13
The residue (8 g) was dissolved in chloroform (10 ml)
and adsorbed on to silica gel (60–120 mesh, 20 g). After
evaporation of the solvent, it was subjected to column
chromatography over silica gel (150 g) prepared in
n-hexane. The elutions were carried out with n-hexane:
ethyl acetate graded mixture (95:5) afforded compound
I (65 mg) and n-hexane: ethyl acetate graded mixture
(90:10) resulted into compound II (93 mg) and III
(125 mg). Further, the elutions carried out with n-hexane:
ethyl acetate graded mixtures (85:15) resulted into
compound IV (95 mg), V (90 mg) and VI (80 mg)
respectively. The individual compounds were purifi ed
by preparative TLC on silica gel G and further purifi ed
by recrystallization with n-hexane.
RESULTS
Compound I (stigmasterol)
Pearl white crystals (Figure 1); R 0.63 in n-hexane:
f
ethyl acetate (95:05); m.p. 167–170oC; IR v (KBr):
max
3422, 2951, 2851, 1669, 1619, 1465, 1072 cm –1 ; 1HNMR (CDCl , 400 MHz): δ 0.79, 0.78, 0.82, 0.91, 0.94, 1.08
3
(m, 18H), δ 1.13 to δ 2.53 (m, 18H, 9x CH and 8H),
2
56 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

Karunakar Hegde et al.: Phytochemical Investigation of Root Extract of the Plant Carissa spinarum
3.54 (1H, dd, J= 9.1, 6.0 Hz, H-α3), 5.33 ( s, 1H, H-3),
5.11 and 5.15 (2H, br); 13 CNMR (CDCl 3, 400 MHz): δ
33.79 (C-1), 78.98 (C-3), δ 145.83 (C-5), δ 121.81 (C-6), δ
32.50 (C-7), δ 35.35 (C-8), δ 45.99 (C-9), δ 30.02 (C-14),
δ 56.93 (C-17), δ 19.91 (C-19), δ 138.39 (C-22), δ129.35
(C-23), δ 30.35 (C-24), δ 18.89 (C-28); FAB-MS m/z
(rel.int): 412 [M + ] (C 29 H 48 O) (100), 397 (20), 369 (10), 351
(70), 329 (65), 300 (40), 299 (23), 273 (31), 255 (28).
Compound II (ursolic acid)
Pale yellow powder (Figure 1); R 0.51 in n-hexane: ethyl
f
acetate (90:10); m.p. 271–274oC; IR v (KBr): 3455, 2924,
max
2853, 1638, 1460, 1382, 1035 cm –1 ; 1HNMR (CDCl , 400 3
MHz): δ 1.00 to 1.91 (m, 22H), 3.20 (1H, dd, J= 10, 5.7
Hz, H-3), 2.17 (1H, d, J= 4.52 Hz, H-18), 2.28 (m, 1H,
OH), 5.34 (s, 1H, H-12); 13CNMR (CDCl 400 MHz):
3,
δ 37.30 (C-1), 78.49 (C-3), 39.98 (C-4), 41.18 (C-5),
51.87 (C-9), 125.25 (C-12), 137.48 (C-13), 55.39 (C-17),
38.95 (C-18), 32.54 (C-19), 18.45 (C-25), 16.95 (C-26),
23.69 (C-27), 177.48 (C-28), 20.12 (C-29); FAB-MS m/z
(rel.int): 456 [M + ] (C H O ) (71), 428 (50), 289 (49),
30 48 3
277 (35), 248 (100), 149 (27), 85 (11), 83 (44).
Compound III (lupeol)
Pearl white crystals (Figure 1); R 0.56 in n-hexane: ethyl
f
acetate (90:10); m.p. 213–215oC; IR v (KBr): 3435,
max
2928, 2850, 1648, 1455, 1373, 1038, 880 cm –1 ; 1HNMR (CDCl , 400 MHz): δ 0.76 to 1.02 (m, 18H, Me-28,
3
Me-23, Me-24, Me-25, Me-26, Me-27), 1.66 (3H, d,
J= 0.7 Hz, Me-30), 3.21 (1H, dd, J= 9.5, 6.2 Hz, H-α3),
4.57 ( s, 1H, H-3); 13CNMR (CDCl 400 MHz): δ 38.55
3,
(C-1), 78.49 (C-3), 38.98 (C-4), 54.34 (C-5), 18.44 (C-6),
39.68 (C-8), 49.87 (C-9), 42.28 (C-14), 42.98 (C-17),
48.24 (C-18), 47.42 (C-19), 150.48 (C-20), 28.25 (C-21),
16.51 (C-25), 15.96 (C-26), 109.29 (C-29); FAB-MS m/z
(rel.int): 426 [M + ] (C H O) (50), 409 (10), 395 (13), 318
30 50
(9), 218 (74), 203 (65), 187 (46), 175 (30), 161 (39), 147
(64), 125 (71), 121 (70), 93 (100).
Compound IV (campesterol)
Pearl white crystals (Figure 1); R 0.79 in n-hexane:
f
ethyl acetate (85:15); m.p. 157–159oC; IR v (KBr):
max
3425, 2935, 2843, 1642, 1474, 1032, 601 cm –1 ; 1HNMR (CDCl , 400 MHz): δ 0.76, to 1.08 (m, 18H, Me-18,
3
Me-21, Me-26, Me-27, Me-24’, Me-19), δ 1.12 to δ 1.26
(m, 16H, 8xCH ), δ 1.39 to δ 1.86 (m, 9H), 3.22 (m, 1H,
2
H-3a), 5.36 (1H, t, J= 3.4 Hz, H-12); 13CNMR (CDCl3, 400 MHz): δ 32.96 (C-1), δ 31.92 (C-2), δ 72.98 (C-3),
δ 150.96 (C-5), δ 124.43 (C-6), δ 50.46 (C-9), δ 40.85
(C-12), δ 43.01 (C-13), δ 29.98 (C-14), δ 56.32 (C-17),
δ 12.66 (C-18), δ 19.80 (C-19), δ 19.39 (C -21), δ 33.86
C-23), δ 31.18 (C-26), δ 21.09 (C-24’); FAB-MS m/z
(rel.int): 400 [M + ] (C H O) (70), 394 (21), 351 (35), 300
28 48
(12), 271 (65), 255 (54), 213 (28), 159 (23), 145 (9), 133
(15), 105 (34), 83 (16), 55 (100 %).
Compound V (17-hydroxy-11-oxo-nor-β-amyrone)
Semisolid compound (Figure 1); R f 0.59 in n-hexane:
ethyl acetate (85:15); m.p. 85–87 o C; IR v max (KBr): 3445,
2931, 1789, 1649, 1454, 1376, 1082, 882 cm –1 ; 1 HNMR
(CDCl 3 , 400 MHz): δ 0.79 (s, 3H, 1xCH 3 , H-27), δ 0.87
(s, 6H, 2xCH 3 , H-25 & H-26), δ 0.91 (s, 6H, 2xCH 3 , H-23
& H-24), δ 0.97 (s, 6H, 2xCH 3 , H-28 & H-29), δ 1.13 to
1.61 (m, 18H, 9xCH 2 ), δ 1.68 to δ 2.31 (s, 3H, 3xCH), δ
3.61 (s, 1H, OH), δ 5.18; 13 CNMR (CDCl 3, 400 MHz): δ
38.94 (C-1), δ 206.82 (C-3), δ 55.34 (C-5), δ 40.02 (C-8),
δ 47.72 (C-9), δ 198.36 (C-11), δ 124.45 (C-12), δ 139.61
(C-13), δ 75.68 (C-17), δ 59.11 (C-18), δ 39.88 (C-19),
δ 39.39 (C-20), δ 41.87 (C-22), δ 28.10 (C-28), δ 15.67
(C-29); FAB-MS m/z (rel.int): 440 [M+H] + (C 29 H 44 O 3 )
(52), 425 (19), 409 (100), 391 (78), 353 (26), 325 (45).
Compound VI (urs-12-ene-3β, 22β-diol-
17-carboxylic acid)
White crystalline powder (Figure 1); R 0.54 in n-hexane:
f
ethyl acetate (85:15); m.p. 211–214oC; IR v (KBr):
max
3455, 2948, 2850, 1642, 1462, 1374, 1055 cm –1 ; 1HNMR (CDCl , 400 MHz): δ 1.00 to 1.91 (m, 23H), δ 2.36 (1H,
3
d, J=7.3 Hz, H-18), δ 3.38 (dd, 1H, OH), δ 3.61 (dd,
1H, OH), δ 5.28 (s, 1H); 13CNMR (CDCl 400 MHz): δ
3,
38.30 (C-1), δ 78.98 (C-3), δ 54.34 (C-5), δ 33.02 (C-7),
δ 39.38 (C-8), δ 46.72 (C-9), δ 123.25 (C-12), δ 139.35
(C-13), δ 41.37 (C-14), δ 48.35 (C-17), δ 38.89 (C-19),
δ 70.68 (C-22), δ 14.99 (C-23), δ 179.33 (C-28), δ 16.61
(C-30); FAB-MS m/z (rel.int): 472 [M] + (C H O ) (100),
30 48 4
456 (45), 428 (12), 289 (30), 277 (35), 241 (70), 234 (25),
149 (9), 85 (42), 83 (20).
DISCUSSION
Compound I was obtained as pearl white crystals
and gave characteristic color reaction for sterol. The
FAB-MS spectrum showed a molecular ion peak at m/z
412 corresponding to molecular formula C H O. The
29 48
IR spectrum exhibited strong absorptions at 3422 cm –1
(hydroxyl group), 2951 and 1669 cm –1 . The 1HNMR spectrum exhibited six tertiary methyl groups at δ 0.79,
0.78, 0.82, 0.91, 0.94, and 1.08, one vinylic proton at δ 5.33
and two olefi nic protons at δ 5.11 and 5.15 respectively.
In 13CNMR spectrum the most down fi eld signals at δ
145.83 was accommodated for sp2 (olefi nic) carbon at
C-5 and the next downfi eld signal at δ 138.39 ppm and
δ 129.35 ppm to C-22 and C-23. The downfi eld signal at
δ 121.81 is to C-6. The oxygenated carbon at C-3 gave
a downfi eld signal at δ 78.98 ppm. The next downfi eld
signal at δ 56.93 ppm was accommodated for C-17.
Other carbon atoms of the steroidal skeleton except
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 57

Karunakar Hegde et al.: Phytochemical Investigation of Root Extract of the Plant Carissa spinarum
that in the side chain appeared in the range δ 45.99 to
δ 30.02 ppm. The angular methyl groups and the side
chain methyl carbons gave signals in the region δ 19.91
to δ 18.89 ppm. 13 CNMR spectral data matched exactly
with that of Stigmasterol 14 . Its identity as stigmasterol
was confi rmed by m.p., IR, 1 HNMR, 13 CNMR and
mass spectral data and by co-chromatography with an
authentic sample.
Compound II was obtained as pale yellow powder and
it gave characteristic color reaction for triterpenoids..
The FAB-MS showed a molecular ion peak at m/z 456
corresponding to a molecular formula C 30 H 48 O 3 . It was
unambiguously identifi ed as ursolic acid on the basis
of its m.p., IR, 1 HNMR, 13 CNMR and mass spectral
data 15 .
Compound III was obtained as pearl white crystals and
it gave characteristic color reaction for triterpenoids.
The FAB-MS spectrum showed a molecular ion peak at
m/z 426 corresponding to molecular formula C 30 H 50 O.
The IR spectrum exhibited strong absorptions at
3435 cm –1 (hydroxyl group) and 2928, 1648, 880 cm –1
(exomethylene group). The 1 HNMR spectrum exhibited
six tertiary methyl groups at δ 0.76, 0.78, 0.82, 0.91,
0.94 and 1.02, a methane group at δ 1.66, a secondary
carbinol group at δ 3.21 and an exomethylene group at
δ 4.56 and 4.68 implies a typical pentacyclic triterpenoid
of the lupeol. Based on the m.p., IR, 1 HNMR, 13 CNMR
and mass spectral data the compound was identifi ed as
lupeol. 16,17
Compound IV obtained as pearl white crystals and it
gave characteristic color reaction for sterol. The peak at
1642.47 cm –1 in IR spectra indicated C=C streching, while
the peaks at 1474.27 cm –1 and 1032.65 cm –1 indicated the
C-H deformation in gem dimethyl and C-O streching of
secondary alcohol respectively. The peak at 3425.25 cm –1
indicated the presence of OH group. The proton NMR
signal at δ 2.17 indicated OH at C–3 position. The signal
at δ 5.36 gave singlet which indicated one vinylic proton
at C-6 position, while the signal at δ 5.12 gave doublet
which indicated two allylic protons at C–7 position. The
13 CNMR spectrum revealed 28 signals which were duly
assigned as CH 3 , CH 2 , CH and -C- group represent in the
compound. The FAB-MS spectrum showed a molecular
ion peak at m/z 400 corresponding to molecular formula
C 28 H 48 O. The mass fragmentation was typical to that of
campesterol. Based on the m.p., IR, 1 HNMR, 13 CNMR
and mass spectral data the compound was identifi ed as
campesterol.
Compound V obtained as semisolid mass and gave
characteristic color reaction for triterpenoids. The peak at
1789.68 cm –1 in IR spectra indicated C=O streching. The
peak at 882.12 cm –1 indicated =C-H out plane bending.
The peak at 1649.64 cm –1 indicated C=C streching and
peak at 1376.69 cm –1 indicated the C-H deformation
in gem dimethyl. The peak at 3445.42 cm –1 indicated
the presence of OH group. The proton NMR signals
at δ 5.18 indicates vinylic protons and another singlet
at δ 3.61 indicated OH at C-17 position. The 13 CNMR
spectrum revealed 29 signals which were duly assigned
as CH 3 , CH 2 , CH, -C- and -C=C- group represent in the
compound. The FAB-MS spectrum showed a molecular
ion peak at m/z 400 corresponding to molecular formula
C 29 H 44 O 3 . The mass fragmentation was typical to that
of β-amyrone skeleton. From the above evidences the
compound was identifi ed as 17-Hydroxy-11-oxo-norβ-amyrone
18 .
Compound VI obtained as white crystals and gave a
characteristic color reaction for triterpenoids. The FAB-
MASS spectrum displayed the characteristic retro-Diels-
Alder fragment peak at m/z 234 indicating a C-12 /
C-13 double bond which suggested an ursane structure 19
substituted by two hydroxyl groups (-OH), one located
at A/B rings and one on D/E rings. These results
are in good agreement with the IR spectrum, which
showed absorption bands at 3455.84 cm –1 assigned to
the hydroxyl groups and 1642.89 cm –1 corresponds to
carbonyl group of the carboxylic acid. The 1 HNMR
spectrum displayed seven methyl signals. In addition,
the presence of the olefi nic triplet at δ 5.28 and two
hydroxyl bearing methine protons exhibiting two double
doublets at δ 3.38 and δ 3.61. On the basis of the
1 HNMR spectrum of the compound, a β-confi guration
of the two hydroxyl groups was assigned according to
the fact that their respective geminal protons appeared
as dd thus confi rming axial orientation. 20 Further the
13 CNMR spectrum revealed 30 signals which were duly
assigned as CH 3 , CH 2 , CH and -C- group represent in the
compound. Therefore the structure of compound VI is
identifi ed as Urs-12-ene-3β, 22β-diol-17-carboxylic acid,
whose mass value obtained as 472 by FAB-MASS which
corresponds to the C 30 H 48 O 4 ; the molecular formula of
the compound.
CONCLUSION
From the spectral data, the compounds I-VI was analyzed
as stigmasterol, ursolic acid, lupeol, campesterol,
17-hydroxy-11-oxo-nor-β-amyrone and urs-12-ene-3β,
22β-diol-17-carboxylic acid respectively. However, the
compounds stigmasterol, campesterol, 17-hydroxy-11oxo-nor-β-amyrone
and urs-12-ene-3β, 22β-diol-17carboxylic
acid were reported for the fi rst time from the
root of this plant.
REFERENCES
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58 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

Karunakar Hegde et al.: Phytochemical Investigation of Root Extract of the Plant Carissa spinarum
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of Carissa spinarum Linn. The Indian J Pharm 1971;33:76–7.
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RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 59

Synthesis and antimicrobial activity of
4-hydroxy-1-methyl/phenyl-3- (substituted
anilinoacetyl) quinolin-2(1H)-one
Girish Bolakatti 1 , Manjunatha S. Katagi* 1 , S.N. Mamledesai 2 , Sujatha. M.L. 3,
Prakash Dabadi 1 , Narayana Miskin. 1
1 Bapuji Pharmacy College, Davangere 577 004, Karnataka, India
2 P.E.S’s College of Pharmacy education and Research, Ponda 403 401, Goa, India
3 Bapuji Institute of Engineering and Technology, Davangere 577 004, Karnataka, India
ABSTRACT
A series of new 2-quinolone derivatives were synthesized, purifi ed and characterized on the basis of IR, 1H NMR, 13C NMR and Mass spectral studies. Same compounds were evaluated for antimicrobial activity against
Staphylococcus aureus, Bacillus substilis, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and
Asparagillus niger. Among eighteen synthesized novel compounds, in which fi ve compounds (3a, 3b, 3d, 4a, 4c)
shows promising antibacterial activity as compared to Ciprofl oxacin (100 μg/ml), however most of them (3a, 3b,
3d, 3e, 4a, 4b, 4d, 4e) showed potent antifungal activity as compared to Fluconazole (100 μg/ml).
Key words: Mass spectral, 2-quinolone, antibacterial and antifungal
INTRODUCTION
Although considerable advances have been
achieved over recent decades in the research
and development of new structural prototypes
as effective antimicrobials, current antimicrobial
chemotherapy still suffers from
two major limitations. The fi rst is the lack
of selectivity of conventional antimicrobial
agents, which in turn brings about unwanted
side effects. The second is acquisitions by
the microorganism of multi drug resistance.
The design of new agents, active against
resistant organism is of critical importance.
In the fi eld of quinolone antimicrobial
agents, the new generation of quinolone
has achieved signifi cant improvements in
terms of potency, spectrum and pharmacokinetic
properties. But these agents faced
a rapid increase of resistance from grampositive
organisms. Therefore, enhancing
the potency of quinolone especially against
gram-positive organism has become most
urgent. 1
The resistance to antimicrobial drugs is
wide spread, the development of new
antimicrobial agents and understanding
their mechanisms of action are becoming
vital nowadays. Nitrogen containing heterocyclic
compounds is an indispensable
structural unit for both the chemist and
the biochemist. Among the antimicrobial
agents discovered in recent years the various
2-quinolone as potent and selective antimicrobial
agent has stimulated remarkable
interest in the synthesis of 2-quinolones
bearing heterocycles. 2 Numerous biological
activities of 2-quinolone have been
described; antimicrobial, 3–5 antioxidant and
anti-infl ammatory, 6 antitumor, 7 Fornesyl
transferase inhibitor 8 , antiangiogenic 9 and
anti-tuberculosis. 10–13
EXPERIMENTAL PROCEDURE
All the chemicals and solvents were
supplied by Merck, S.D Fine-Chem.
Research Ar cle
Received Date : 19-11-2011
Revised Date : 02-03-2012
Accepted Date : 05-03-2012
DOI: 10.5530/rjps.2012.1.8
Address for
correspondence
Manjunatha S. Katagi
Bapuji Pharmacy College
S.S. Layout
Davangere-577 004
Karnataka, India
Phone: 08192-221459
Mobile: +919886499160
Fax: 08192-222561
Email: manju_mpharm@
rediffmail.com
www.rjps.in
60 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

Girish Bolakatti et al.: Synthesis and antimicrobial activity of 4-Hydroxy-1-methyl/phenyl-3- (Substituted anilinoacetyl) quinolin-2(1H)-one
Limited, Mumbai. All the solvents were distilled before
use and chemicals were purifi ed by either distillation or
recrystallisation before use. The melting points were
taken on the Veego (VMP–MP) melting point apparatus
and are uncorrected. The IR spectra of the compounds
were recorded using KBr on Jasco FTIR spectrometer
(model-4100). The 1 H NMR and CHN elemental
analysis of the synthesized compounds was recorded on
Bruker avance II 400 NMR spectrometer (with TMS as
internal references) and Perkin Elmer 2400 respectively
at Sophisticated Analytical and Instrumentation Facility
(SAIF), Punjab University (Chandigarh). Mass spectra
were recorded on Shimadzu LC MS-2010A at Quest
Research and Training Institute (Pvt) Ltd, Bangalore.
4-Hydroxy-6-methyl/phenyl-2H-pyrano [3,2-c] quinoline-
2,5(6H)-dione, were synthesized according to literature
procedure and subjected to hydrolysis to yield 1a and
1b, 14 further 1a and 1b subjected to bromination to yield
3-Bromoacetyl-4-hydroxy-1-methyl/phenyl quinolin-
2(1H)-one 2a and 2b. The synthesis of title compounds
3a-h and 4a-h were accomplished by condensing with
different substituted aniline. The compounds thus
obtained were characterized by IR, 1 HNMR and mass
spectral data. The physicochemical properties of titled
compounds were given in Table 1 and Table 2.
General procedure for synthesis of
3-(Bromoacetyl)-4-hydroxy-1-methyl/
phenylquinolin-2(1H )-one 2a, 2b
A solution of 3-Acetyl-4-hydroxy-1-methyl/phenyl
quinolin-2(1H )-one (0.065 mol) in glacial acetic acid
(50 ml) was heated (80–100°)( follow the instruction
provided on page no 5 of instruction to authors) with
constant stirring, then bromine (0.065 mol) in glacial
acetic acid (20 ml) was added drop wise over a period
of 1 h and heated the solution until slight change in the
color of the solution was seen. The reaction mixture was
then cooled to room temperature. The product obtained
as yellow crystals and recrystallized from ethanol.
3-(Bromoacetyl)-4-hydroxy-1-methylquinolin-
2(1H )-one 2a
This was prepared and purifi ed as per the above
mentioned procedure: yield 10.26 g (73%), mp 174–176
°C, IR (KBr, v, cm –1 ): 756.14 cm –1 and 1190.18 (CH 2 -Br),
1615.47 cm –1 (– C = O amide), 3046.24cm –1 (aromatic
–C–H stre). 1 H NMR (400MHz, CDCl 3 ) δ (ppm): δ
3.78 (s, 3H, N–CH 3 ), 4.98 (s, 2H, CH 2 –Br), 7.72–8.35
(m, 4H, Ar–H), 15.82 (s, 1H, OH).
3-(Bromoacetyl)-4-hydroxy-1-phenylquinolin-
2(1H )-one 2b
This was prepared and purifi ed as per the above
mentioned procedure: yield 12.24 g (67.43%), mp
192–194°, IR (KBr, v, cm –1 ): 786.11 cm –1 and 1210.06
(CH 2 -Br), 1620.31 cm –1 (– C = O amide), 3058.11cm –1
(aromatic –C – H stre). 1 H NMR (400MHz, CDCl 3 ) δ
(ppm): δ 5.23 (s, 2H, CH 2 -Br), 7.65–8.85 (m, 9H, Ar–H),
15.91 (s, 1H, OH).
General procedure for synthesis of 4-Hydroxy-
1-methyl/phenyl-3-(Substituted anilinoacetyl)
quinolin-2(1H )-one 3a-h and 4a-h
A solution of 3-(Bromoacetyl)-4-hydroxy-1-methyl/
phenylquinolin-2(1H)-one 2a, 2b (0.004 mol) in acetone
Table 1: Characterization data of 4-Hydroxy-1-methyl -3-(Substituted anilinoacetyl)-quinolin-2(1H)-one 3a-h
Analysis( % )Found (Calcd) in % Yield
Comp R Mol formula M.P. °C * Rf C H N
3a -2,5-Dichloro C H Cl N O 18 14 2 2 3 210–212 0.56 57.31 (57.16) 3.74 (3.61) 7.43 (7.37)
3b -2,4-Dichloro C H Cl N O 18 14 2 2 3 206–208 0.53 57.31 (57.19) 3.74 (3.64) 7.43 (7.39)
3c -p-Nitro C H N O 18 15 3 5 198–200 0.47 61.19 (61.08) 4.28 (4.21) 11.89 (11.79)
3d -p-Fluro C H FN O 18 15 2 3 204–206 0.41 66.25 (66.18) 4.63 (4.51) 8.58 (8.49)
3e p-Chloro C H ClN O 18 15 2 3 211–213 0.42 63.07 (63.01) 4.41 (4.35) 8.17 (8.11)
3f -3-Chloro-4-fl uro C H ClFN O 18 14 2 3 220–222 0.77 59.93 (59.85) 3.91 (3.86) 7.76 (7.71)
3g -2,4-Dimethyl C H N O 20 20 2 3 225–227 0.51 71.41 (71.37) 5.99 (5.91) 8.33 (8.24)
3h -3,4-Dimethyl C H N O 20 20 2 3 222–224 0.53 71.41 (71.35) 5.99 (5.92) 8.33 (8.25)
*TLC Solvent system: Chloroform: Methanol: Strong ammonia-(10:5:3) .
OH O
N
CH 3
3a-h
O
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 61
NH
R

Girish Bolakatti et al.: Synthesis and antimicrobial activity of 4-Hydroxy-1-methyl/phenyl-3- (Substituted anilinoacetyl) quinolin-2(1H)-one
Table 2: Characterization data of 4-Hydroxy-1-phenyl -3-(Substituted anilinoacetyl) quinolin-2(1H)-one 4a-h
(50 ml) was cooled at 0º, primary amine (0.004 mol)
dissolved in acetone (25 ml) was added with stirring
at 0–5° followed by drop wise addition of sodium
hydroxide (0.004 mol) in water (25 ml). Contents were
stirred for 3 hour and poured in to ice water acidifi ed
with dil. HCl, fi ltered, washed, dried and purifi ed by
crystallization from a ethanol + water (9:1).
3-{2-[(2,5-dichlorophenyl)amino]acetyl}-4hydroxy-1-methylquinolin-2(1H)-one
3a
This was prepared and purified as per the above
mentioned procedure: yield 0.75 g (49.73%); IR
(KBr, v, cm –1 ): 1617.61 cm –1 (– C = O amide), 3156.20
cm –1 (– CH stre), 3266.33 cm –1 (– NH), 3363.09 cm –1
(– OH); 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm):
2.85 (s, 3H, N- CH 3 ), 3.87 (s, 1H, NH), 4.81 (s, 2H,
COCH 2 ), 6.45–7.5 (m, 7H, Ar–H), 15.24 (s, 1H,
OH); 13 C NMR (400MHz, DMSO-d 6 ) δ (ppm): 31.5
(N-CH 3 ), 59.8 (CH 2 ), 108.01 115.12, 115.65, 119.32,
121.23, 121.93, 124.99, 126.11, 128.84, 132.36,
134.24, 135.85, 145.40 (Aromatic carbons), 164.53
(exocyclic ketone), 166.32 (Hydroxyl carbon), 196.85
(Carbonyl carbon); LCMS: C 18 H 14 Cl 2 N 2 O 3 (M + ) m/z
377.29; calcd. 377.22.
Analysis( % )Found (Calcd) in % Yield
Comp R Mol formula M.P. 0C * Rf C H N
4a -2,5-Dichloro C H Cl N O 23 16 2 2 3 240–242 0.58 62.88 (62.81) 3.67 (3.60) 6.38 (6.32)
4b -2,4-Dichloro C H Cl N O 23 16 2 2 3 236–238 0.55 62.88 (62.83) 3.67 (3.62) 6.38 (6.34)
4c -p-Nitro C 23 H 17 N 3 O 5 245–247 0.45 66.50 (66.41) 4.12 (4.01) 10.12 (10.01)
4d -p-Fluro C 23 H 17 FN 2 O 3 251–253 0.43 71.13 (71.02) 4.41 (4.32) 7.21 (7.16)
4e p-Chloro C 23 H 17 ClN 2 O 3 211–213 0.45 68.23 (68.17) 4.23 (4.14) 6.92 (6.87)
4f -3-Chloro-4-fl uro C 23 H 16 ClFN 2 O 3 208–210 0.73 65.33 (65.23) 3.81 (3.74) 6.63 (6.52)
4g -2,4-Dimethyl C H N O 25 22 2 3 246–248 0.47 75.36 (75.23) 5.57 (5.48) 7.03 (6.97)
4h -3,4-Dimethyl C H N O 25 22 2 3 251–253 0.49 75.36 (75.28) 5.57 (5.45) 7.03 (6.98)
* TLC Solvent system: Chloroform: Methanol: Strong ammonia-(10:5:3).
OH O
N
3-{2-[(2,4-dichlorophenyl)amino]acetyl}-4hydroxy-1-methylquinolin-2(1H)-one
3b
This was prepared and purifi ed as per the above
mentioned procedure: yield 0.71 g (47.08%); IR (KBr,
v, cm –1 ): 1620.54 cm –1 (– C = O amide), 3158.51 cm –1
(– CH stre), 3271.31 cm –1 (– NH), 3359.05 cm –1 (– OH);
1 H NMR (400MHz, DMSO-d6 ) δ (ppm): 2.82 (s, 3H,
N–CH 3 ), 3.85 (s, 1H, NH), 4.75 (s, 2H, COCH 2 ), 6.35–
7.58 (m, 7H, Ar–H), 15.04 (s, 1H, OH).
3-[2-(4-Nitrophenylamino)acetyl]-4-hydroxy-
1-methylquinolin-2(1H)-one 3c
This was prepared and purifi ed as per the above
mentioned procedure: yield 0.86 g (60.90%); IR (KBr,
v, cm –1 ): 1623.11 cm –1 (– C = O amide), 3186.22 cm –1
(– CH stre), 3252.38 cm –1 (– NH), 3345.23 cm –1 (– OH);
1 H NMR (400MHz, DMSO-d6 ) δ (ppm): 2.91 (s, 3H, N-
CH 3 ), 4.06 (s, 1H, NH), 4.96 (s, 2H, COCH 2 ), 6.86–7.92
(m, 8H, Ar–H), 14.52 (s, 1H, OH); 13 C NMR (400MHz,
DMSO-d 6 ) δ (ppm): 32.5 (N-CH 3 ), 60.2 (CH 2 ), 107.25,
114.16, 115.21, 121.76, 121.99, 124.65, 126.11, 128.84,
135.05, 136.56, 153.65 (Aromatic carbons), 164.36
(exocyclic ketone), 166.32 (Hydroxyl carbon), 196.12
62 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012
O
NH
4a-h
R

Girish Bolakatti et al.: Synthesis and antimicrobial activity of 4-Hydroxy-1-methyl/phenyl-3- (Substituted anilinoacetyl) quinolin-2(1H)-one
(Carbonyl carbon) ; LCMS: C 18 H 15 N 3 O 5 (M + ) m/z
353.33; calcd. 353.32.
3-[2-(4-Fluorophenylamino)acetyl]-4-hydroxy-
1-methylquinolin-2(1H)-one 3d.
This was prepared and purifi ed as per the above
mentioned procedure: yield 0.56 g (42.94%); IR (KBr,
v, cm –1 ): 1613.16 cm –1 (– C = O amide), 3180.01 cm –1
(– CH stre), 3245.02 cm –1 (– NH), 3305.03 cm –1 (– OH);
1 H NMR (400MHz, DMSO-d6 ) δ (ppm): 2.75 (s, 3H, N-
CH 3 ), 3.89 (s, 1H, NH), 4.53 (s, 2H, COCH 2 ), 6.45–7.53
(m, 8H, Ar–H), 14.96 (s, 1H, OH).
3-[2-(4-Chlorophenylamino)acetyl]-4-hydroxy-
1-methylquinolin-2(1H)-one 3e
This was prepared and purifi ed as per the above
mentioned procedure: yield 0.68 g (49.63%); IR (KBr,
v, cm –1 ): 1613.51 cm –1 (– C = O amide), 3123.26 cm –1
(– CH stre), 3243.36 cm –1 (– NH), 3345.12 cm –1 (– OH);
1 H NMR (400MHz, DMSO-d6 ) δ (ppm): 2.63 (s, 3H, N-
CH 3 ), 3.91 (s, 1H, NH), 4.56 (s, 2H, COCH 2 ), 6.49–7.62
(m, 8H, Ar–H), 14.99 (s, 1H, OH).
3-[2-(3-Chloro-4-fl uorophenylamino)acetyl]-
4-hydroxy-1-methylquinolin-2(1H)-one 3f
This was prepared and purifi ed as per the above
mentioned procedure: yield 0.78 g (54.09%); IR (KBr,
v, cm –1 ): 1613.05 cm –1 (– C = O amide), 3123.12 cm –1
(– CH stre), 3243.56 cm –1 (– NH), 3355.10 cm –1 (– OH);
1 H NMR (400MHz, DMSO-d6 ) δ (ppm): 2.83 (s, 3H, N-
CH 3 ), 3.99 (s, 1H, NH), 4.63 (s, 2H, COCH 2 ), 6.49–7.51
(m, 7H, Ar–H), 14.82 (s, 1H, OH).
3-[2-(2,4-dimethylphenylamino)acetyl]-4-hydroxy-
1-methylquinolin-2(1H)-one 3g
This was prepared and purifi ed as per the above
mentioned procedure: yield 0.81 g (60.26%); IR (KBr, v,
cm –1 ): 1632.05 cm –1 (– C = O amide), 3178.28 cm –1 (– CH
stre), 3288.88 cm –1 (– NH), 3355.51 cm –1 (– OH); 1 H NMR
(400MHz, DMSO-d 6 ) δ (ppm): 2.32 (s, 6H, CH 3 ), 2.42 (s,
3H, N- CH 3 ), 3.75 (s, 1H, NH), 4.85 (s, 2H, COCH 2 ),
6.38–7.53 (m, 7H, Ar–H), 15.23 (s, 1H, OH); 13 C NMR
(400MHz, DMSO-d 6 ) δ (ppm): 21.9, 24.6 (2 CH 3 ), 31.5
(N–CH 3 ), 60.2 (CH 2 ), 107.25, 113.16, 115.21, 121.5,
121.76, 124.99, 126.76, 126.96, 128.84, 131.75, 135.05,
143.65 (Aromatic carbons), 164.10 (exocyclic ketone),
166.82 (Hydroxyl carbon), 196.65 (Carbonyl carbon) ;
LCMS: C 20 H 20 N 2 O 3 (M + ) m/z 336.32; calcd. 336.38.
3-[2-(3,4-dimethylphenylamino)acetyl]-4-hydroxy-
1-methylquinolin-2(1H)-one 3h
This was prepared and purifi ed as per the above
mentioned procedure: yield 0.96 g (71.42%); IR (KBr,
v, cm –1 ): 1632.00 cm –1 (– C = O amide), 3178.28 cm –1
(– CH stre), 3288.88 cm –1 (– NH), 3355.51 cm –1 (– OH);
1 H NMR (400MHz, DMSO-d6 ) δ (ppm): 2.15 (s, 6H,
CH 3 ), 2.42 (s, 3H, N- CH 3 ), 3.75 (s, 1H, NH), 4.85 (s, 2H,
COCH 2 ), 6.38–7.53 (m, 7H, Ar–H), 15.23 (s, 1H, OH).
3-{2-[(2,5-dichlorophenyl)amino]acetyl}-4hydroxy-1-phenylquinolin-2(1H)-one
4a
This was prepared and purifi ed as per the above
mentioned procedure: yield 0.95 g (54.10%); IR (KBr, v,
cm –1 ): 1625.61 cm –1 (– C = O amide), 3166.00 cm –1 (– CH
stre), 3260.83 cm –1 (– NH), 3392.08 cm –1 (– OH); 1 H
NMR (400MHz, DMSO-d 6 ) δ (ppm): 3.89 (s, 1H, NH),
4.76 (s, 2H, COCH 2 ), 6.60–7.64 (m, 12H, Ar–H), 15.20
(s, 1H, OH); 13 C NMR (400MHz, DMSO-d 6 ) δ (ppm):
59.91 (CH 2 ), 107.5, 113.01, 115.22, 118.21, 118.32,
118.93, 119.21, 119.31, 120.23, 127.84, 128.36, 129.7,
131.1, 133.25, 137.2, 140.7, 145.4 (Aromatic carbons),
159.53 (exocyclic ketone), 166.30 (Hydroxyl carbon),
196.85 (Carbonyl carbon) ; LCMS: C 23 H 16 Cl 2 N 2 O 3 (M + )
m/z 439.23; calcd. 439.29.
3-{2-[(2,4-dichlorophenyl)amino]acetyl}-
4-hydroxy-1-phenylquinolin-2(1H)-one 4b
This was prepared and purifi ed as per the above
mentioned procedure: yield 0.92 g (52.39%); IR (KBr,
v, cm –1 ): 1620.54 cm –1 (– C = O amide), 3158.51 cm –1
(– CH stre), 3271.31 cm –1 (– NH), 3359.05 cm –1 (– OH);
1 H NMR (400MHz, DMSO-d6 ) δ (ppm): 3.93 (s, 1H,
NH), 4.81 (s, 2H, COCH 2 ), 6.63–7.69 (m, 12H, Ar–H),
15.05 (s, 1H, OH).
3-[2-(4-Nitrophenylamino)acetyl]-4-hydroxy-
1-phenylquinolin-2(1H)-one 4c
This was prepared and purifi ed as per the above
mentioned procedure: yield 1.06 g (63.85%); IR (KBr,
v, cm –1 ): 1628.65 cm –1 (– C = O amide), 3145.06 cm –1
(– CH stre), 3245.38 cm –1 (– NH), 3354.23 cm –1 (– OH);
1 H NMR (400MHz, DMSO-d6 ) δ (ppm): 4.06 (s, 1H,
NH), 4.82 (s, 2H, COCH 2 ), 6.86–7.92 (m, 13H, Ar–H),
14.82 (s, 1H, OH).
3-[2-(4-Fluorophenylamino)acetyl]-4-hydroxy-
1-phenylquinolin-2(1H)-one 4d
This was prepared and purifi ed as per the above
mentioned procedure: yield 0.49 g (31.57%); IR (KBr,
v, cm –1 ): 1619.13 cm –1 (– C = O amide), 3188.05 cm –1
(– CH stre), 3251.02 cm –1 (– NH), 3389.03 cm –1 (– OH);
1 H NMR (400MHz, DMSO-d6 ) δ (ppm): 4.15 (s, 1H,
NH), 4.91 (s, 2H, COCH 2 ), 7.12–8.13 (m, 13H, Ar–H),
14.92 (s, 1H, OH).
3-[2-(4-Chlorophenylamino)acetyl]-4-hydroxy-
1-phenylquinolin-2(1H)-one 4e.
This was prepared and purifi ed as per the above
mentioned procedure: yield 0.56 g (34.61%); IR
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 63

Girish Bolakatti et al.: Synthesis and antimicrobial activity of 4-Hydroxy-1-methyl/phenyl-3- (Substituted anilinoacetyl) quinolin-2(1H)-one
(KBr, v, cm –1 ): 1619.51 cm –1 (– C = O amide), 3187.26
cm –1 (– CH stre), 3256.31 cm –1 (– NH), 3385.18 cm –1
(– OH); 1 H NMR (400MHz, DMSO-d 6 ) δ (ppm): 4.11
(s, 1H, NH), 4.67 (s, 2H, COCH 2 ), 7.05–8.11 (m, 13H,
Ar–H), 14.91 (s, 1H, OH).
3-[2-(3-Chloro-4-fl uorophenylamino)acetyl]-
4-hydroxy-1-phenylquinolin-2(1H)-one 4f
This was prepared and purifi ed as per the above
mentioned procedure: yield 0.88 g (52.07%); IR (KBr,
v, cm –1 ): 1610.05 cm –1 (– C = O amide), 3142.02 cm –1
(– CH stre), 3251.16 cm –1 (– NH), 3420.15 cm –1 (– OH);
1 H NMR (400MHz, DMSO-d6 ) δ (ppm): 3.85 (s, 1H,
NH), 4.68 (s, 2H, COCH 2 ), 6.84–8.11 (m, 12H, Ar–H),
13.92 (s, 1H, OH).
3-[2-(2,4-dimethylphenylamino)acetyl]-
4-hydroxy-1-phenylquinolin-2(1H)-one 4g
This was prepared and purifi ed as per the above
mentioned procedure: yield 0.83 g (52.13%); IR (KBr,
v, cm –1 ): 1653.12 cm –1 (– C = O amide), 3251.06 cm –1
(– CH stre), 3296.80 cm –1 (– NH), 3395.22 cm –1 (– OH).
3-[2-(3,4-dimethylphenylamino)acetyl]-
4-hydroxy-1-phenylquinolin-2(1H)-one 4h
This was prepared and purifi ed as per the above
mentioned procedure: yield 0.96 g (60.30%); IR (KBr,
v, cm –1 ): 1612.00 cm –1 (– C = O amide), 3178.33 cm –1
(– CH stre), 3269.28 cm –1 (– NH), 3358.01 cm –1 (– OH);
1 H NMR (400MHz, DMSO-d6 ) δ (ppm): 2.13 (s, 6H,
CH 3 ), 3.78 (s, 1H, NH), 4.96 (s, 2H, COCH 2 ), 6.98–8.35
(m, 12H, Ar–H), 14.01 (s, 1H, OH).
ANTIMICROBIAL SCREENING15–18 All the synthesized compounds 3a-h and 4a-h have
been screened in vitro for their antibacterial activity
against gram-negative bacteria Escherichia coli (ATCC
10536), Pseudomonas aeruginosa (ATCC 10145) and grampositive
bacteria Staphylococcus aureus (ATCC11632),
Bacillus substilis (ATCC 60511), while antifungal activity
against Candida albicans (ATCC 2501) and Asparagillus
niger (ATCC 1781) at 100 μg/ml concentration by cupplate
agar diffusion method using dimethylsulfoxide
as solvent. After 24 and 48h of incubation at 37° ±1,
the antibacterial and antifungal activity respectively
was determined by measuring the zones of inhibition
in mm. Standard antibacterial ciprofl oxacin and
fungicide fl uconazole were used under similar condition
for comparison. Control test with solvent were
performed for every assay but showed no inhibition
of microbial growth. The observed zone of inhibition
for antibacterial and antifungal activity is presented in
Table 3
Table 3: In vitro antimicrobial activities of the 2-quinolone
derivatives at a concentration of 100 µg/ml (zone of
inhibition in mm)
Zone of inhibition
Compounds Sa Bs Ec Pa Ca An
3a 19 22 29 28 28 33
3b 18 23 28 26 30 34
3c 10 12 13 NA 16 18
3d 20 23 25 27 31 34
3e 09 07 10 08 27 33
3f 07 08 NA 10 15 16
3g 08 10 13 15 17 12
3h 11 13 NA 17 13 19
4a 19 24 31 29 28 31
4b 11 12 11 13 27 30
4c 21 25 32 30 11 16
4d 12 16 19 21 29 28
4e 09 07 10 11 32 34
4f 12 11 13 NA 13 15
4g 09 07 10 11 NA NA
4h 08 NA NA NA 10 NA
Ciprofl oxacin 23 26 34 31 ND ND
Fluconazole ND ND ND ND 33 36
Sa = Staphylococcus aureus, Bs = Bacillus substilis, Ec = Escherichia coli,
Pa = Pseudomonas aeruginosa, Ca = Candida albicans, An = Asparagillus niger, NA = No
activity, ND = Not determined.
RESULTS AND DISCUSSION
4-Hydroxy-6-methyl/phenyl-2H-pyrano[3,2-c]
quinoline-2,5(6H)-dione, were synthesized according to
literature procedure14 and subjected to hydrolysis to yield
1a and 1b, further 1a and 1b subjected to bromination
to yield 3-Bromoacetyl-4-hydroxy-1-methyl/phenyl
quinolin-2(1H)-one 2a and 2b. The synthesis of title
compounds 3a-h and 4a-h were accomplished by
condensing with different substituted aniline.
The structures of newly synthesized compounds were
confi rmed by their spectral data. The characteristic
IR absorption peaks 1617.61 cm –1 was due to amide
carbonyl, 3156.20 cm –1 mainly because aromatic –C – H
stretching, 3266.33 cm –1 may be due to free amine group
of acetamido and 3363.09 cm –1 of hydroxyl group
indicates the completion of the reaction. Their structure
was further supported by their 1H NMR spectral data
that exhibited three proton of N-methyl signal found
at δ value 2.85, where as aromatic proton signal shows
the δ value 6.45–7.5. Subsequent purifi cation yielded
fi nal compounds in moderate to higher yields. Physical
data of the synthesized compounds are listed in Table
1 and Table 2. Some of these compounds have shown
antibacterial and potent antifungal agents.
64 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

CONCLUSION
Girish Bolakatti et al.: Synthesis and antimicrobial activity of 4-Hydroxy-1-methyl/phenyl-3- (Substituted anilinoacetyl) quinolin-2(1H)-one
4-Hydroxy-1-methyl/phenyl-3-(Substituted
anilinoacetyl)quinolin-2(1H)-one compounds were
synthesized and evaluated for antimicrobial activity
against Staphylococcus aureus, Bacillus substilis Escherichia coli,
Pseudomonas aeruginosa, Candida albicans and Asparagillus
niger. Among synthesized novel compounds 3a, 3b, 3d,
4a, 4c shows promising antibacterial activity as compared
to Ciprofl oxacin (100 μg/ml), however most of them
3a, 3b, 3d, 3e, 4a, 4b, 4d, 4e showed potent antifungal
activity as compared to Fluconazole (100 μg/ml).
Suitable molecular modifi cation of these compounds
may generate potent antimicrobial agents in future.
REFERENCE
1. Alireza F, Saeed E, Masood M, Mohammad HM, Abbas S. Synthesis
and antibacterial activity of N-[2-(5-bromothiophen-2-yl)-2-oxoethyl] and
N-[2-(5-bromothiophen-2-yl)-2-oximinoethyl] derivatives of piperazinyl
quinolones. Bioorg Med Chem Lett 2005; 15: 4536–9.
2. Thomas K. The pyrano route to 4-hydroxy-2-quinolone and 4-hydroxy-
2-pyridones. IL farmaco 1999; 54:309–15.
3. Jayashree BS, Seeja T, Yogendra N. Design and synthesis of
2-quinolones as antioxidants and antimicrobials: a rational approach.
Med Chem Res 2010;19:193–209.
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 65

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4. El-Dine SA, El-Khawass SM. Synthesis of 4-[(2-alkylaryl or arylalkyl)
amino-1,3,4-thiadiazol-5-yl]-2-synthesized quinolines and compounds
reasonable antifungal and antimicrobial activity. Pharmazie 1979;
34:537–8.
5. Joseph P, Charles B, Gladys D, Trevor V. Refomatsky reactions
with N-arylpyrrolidine-2-thiones: Synthesis of tricyclic analogues of
quinolone antibacterial agents. Tetrahedron 2001;57:9635–48.
6. Anastasia D, Dionysia B, Kyriakos CP, Maria K, Giorgos A, Georgia
M. Design and synthesis of novel quinolinone-3-aminoamides and
their α-lipoic acid adducts as antioxidant and anti-infl ammatory agents.
J Med Chem 2007;50:2450–8.
7. Joseph S, Francis D. Aryl-2-quinolone derivatives having in vivo and
in vitro antitumour activity. J Med Chem 2002;45:2543.
8. Qun Li, Keith WW, Weibo W, et al. Design, synthesis, and activity of
achiral analogs of 2-quinolones and indoles as non-thiol farnesyltransferase
inhibitors. Bioorg Med Chem Lett 2005;15:2033–9.
9. Gisela CM, Mariela B, Ana MB, Silvia EA. Evaluation of antiparasitic,
antituberculosis and antiangiogenic activities of 3-aminoquinolin-2-one
derivatives. J Chil Chem Soc 2006; 51:859–63.
10. Anquetin G, Greiner J, Mahmoudi N, et al. Design, synthesis and activity
against Toxoplasma gondii, Plasmodium spp., and Mycobacterium
tuberculosis of new 6-fl uoroquinolones. Eur J Med Chem 2006;41:
1478–93.
11. Alexandra A, Veziris N, Cambau E, Truffot-Pernot C, Jarlier V, Fisher LM.
Novel Gyrase Mutations in Quinolone-Resistant and Hyper susceptible
Clinical Isolates of Mycobacterium tuberculosis: Functional Analysis of
Mutant Enzymes. Antimicrob Agents Chemother 2006;50:04–112.
12. Cheng AF, Yew WW, Chan EW, Chin ML, Hui MM, Chan RC. Multiplex
PCR amplimer conformation analysis for rapid detection of gyrA
mutations in fl uoroquinolone-resistant Mycobacterium tuberculosis
clinical isolates. Antimicrob Agents Chemother 2004; 48:596.
13. Ginsburg AS, Grosset JH, Bishai WR. Fluoroquinolones, tuberculosis,
and resistance. Lancet Infect Dis 2003;3:432–42.
14. Roschger P, Fiala W, Sradlabauer W. Nucleophilic substitution and
ringclosure reactions of 4-chloro-3-nitro-2-quinolones. J Heterocycl
Chem 1992;29:225–31.
15. Fairbrother RW, Martyn G. The Disc Technique for Determining
Sensitivity to the Antibiotics. J Clin Pathol 1951;4:374–77.
16. Gould JC, Bowie JH. The determination of bacterial sensitivity to
antibiotics. Edinb med J 1952;59:178–99.
17. Handan A, Oznur A, Seher B, Gulten O, Melten U, Dilek S. Synthesis
of mannich bases of some 2,5-disubstituted-4-thiazolidinones and
evaluation of their antimicrobial activities. Turk J Chem 2005;29:425–35.
18. Wolfson J, Hooper DC. Fluoroquinolone Antimicrobial agents. Clini
Microbiol Rev 1989;2(4): 378–24.
66 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

Evaluation of gastroprotective ability of
Amorphophallus paeoniifolius corms against
indomethacin induced gastric ulcers
H.N. Nataraj 1 , R.L.N. Murthy 1 , Ramachandra Setty 2
1 Dept. of Pharmacognosy, T.V.M. College of Pharmacy, Bellary
2 Dept. of Pharmacology, Govt. College of Pharmacy, Bangalore
ABSTRACT:
Peptic ulcer is regarded as a multifactorial gastrointestinal disorder in its pathophysiology including free radical
generations. Thereby free radical scavengers can play an important role in such diseases. The methanolic extract
of Corms of Amorphophallus paeoniifolius exhibited remarkable anti-oxidant activity in various In-vitro models.
Further, preliminary phytochemical screening revealed the presence of polyphenolic compounds; fl avonoids and
tannins which are known to possess anti-ulcer activity. In light of these fi nding, it was under taken to investigate the
gastroprotective activity of methanolic extract gainst NSAID–Indomethacin (30 mg/kg p.o.) induced gastotoxicity
in Wistar albino rats wherein the animals were orally administered with two different doses of test extract
(250 and 500 mg/kg b.w.) or with reference drug Lansoprozole (8 mg/kg p.o.). Animals were analyzed for Ulcer
score, and in vitro estimation of GSH and LPO. Extract showed signifi cant (p

H.N Nataraj et al.: Evaluation of gastroprotective ability of Amorphophallus paeoniifolius corms against indomethacin induced gastric ulcers
antisecretory ctivity can prove effectively in PUD
(Peptic Ulcer Disease). Acid neutralization is being recognized
as effective treatment for many centuries ago,
but with the understanding of pathogeneses of PUD
the treatment has become more effective. A number
of antiulcer drugs by various mechanisms like gastric
anti-secretory drugs, H 2 -receptors antagonists, antimuscarine
drugs, proton pomp inhibitors and mucosal protective
agents are in most common usage as a remedy
for peptic ulcer.
In ancient system of medicine, herbal preparations are
being used for treating duodenal ulcers. Presently, a large
section of the world’s population relies on traditional
and medicinal herbs due to their less cost and easy
access. 3 In the Indian Pharmaceutical Industry, antacids
and antiulcer share 6.2 million rupees and occupy 4.3%
of the market share. 4 Some of the phytoconstituents
known to possess antiulcer activity include fl avonoids,
saponins, tannins, gum and mucilages.
Amophophallus paeoniifolius (Dennst.) Nicolson (Family:
Araceae) is a perennial herb with underground tuber
commonly known as ‘Suran’ in Hindi. The plant is widely
distributed in India, Bangladesh, and Africa. 5–6 These
tuberous roots of the plant have been used traditionally
for the treatment of piles, abdominal pain, tumours,
enlargement of spleen, asthma and rheumatism. 5,6 The
tuberous roots have been reported to possess tonic,
stomachic and appetizer properties. 6,7
It was revealed that the corms are reported in the
management of Haemorroids, 8 Obesity; 9 evaluated
for Antiprotease, 10 Analgesic, 11 Immunomodulatory, 12
Anthelmintic, 13 Hepatoprotective 14 and Cytotoxic
activity. 15
Previously we have reported that the methanolic extract
of corms Amophophallus paeoniifolius (MECAP) found to
be containing appreciable quantities of phenolics and
fl avonoids as upon their quantitative estimations, 16 based
on which the possible free radical scavenging potential
by various models of MECAP was anticipated. 17
The present investigation was aimed to evaluate the
gastroprotective potential of Amophophallus paeoniifolius
corms against indomethacin-induced gastric ulcer in
albino rats in pursuit of newer gastro protectant.
MATERIALS AND METHODS
Plant material and preparation of extract
The corms of Amorphophallas paeoniifolius were collected
from cultivated lands from Hassan district of Karnataka
and authenticated by Dr. Kotresh Botany department,
Karnataka University, Dharwad. The voucher specimens
of these plants and tubers were preserved in the
herbarium of the pharmacognosy department of this
institution.
The air dried powdered plant material-corm was
extracted with methanol in soxhelt extraction apparatus
as methanol is one of best solvents for phenolics and
it gave more quantitative colour reaction for phenolics
than hydroalcoholic extract. The methanolic extract was
fi ltered and evaporated to dryness in vacuum (at 35ºC &
0.8Mpa) in a Buchi evaporator, R-114. The dry extract
(MECAP-Methanolic Extract Corms of Amorphophallas
paeoniifolius) was kept in vacuum desiccators until use
and the preliminary phytochemical analysis revealed the
presence of mainly polyphenols, tannins, fl avonoids,
coumarins and triterpenoids.
Animals
Healthy adult Wistar albino rats weighing 200–250 g
were used for the present investigation. They were
housed in clean polyacrylic cages with not more than
four animals per cage and maintained under standard
laboratory conditions (temperature 25 ± 2°C, relative
humidity 55–65%, with dark/light cycle 12/12 h).
They were allowed free access to standard pellet diet
and water ad libitum. The animals were acclimated to
laboratory condition for one week prior to experiment.
All the procedures were performed in accordance with
the institutional Animal Ethics Committee (IAEC)
constituted as per the direction of the Committee
for the Purpose of Control and Supervision of
Experiments of Animals (CPCSEA), under Ministry
of Animal Welfare Division, Govt. of India,
New Delhi. The experiment protocol was approved
by IAEC S.C.S. College of Pharmacy, Harapanahalli,
Karnataka (SCSCP/753/2009-10/Sl No.15).
EXPERIMENTAL
Acute Oral toxicity Studies and selection of dose
Fixed dose method of Organization of Economic
Co-operation and Development (OECD) Guideline
No.423 (Annex 2d) given by CPCSEA18 was adopted for
toxicity studies and it was concluded that the extract was
unclassifi ed w.r.t. Globally Hormonized Classifi cation
system and their LD was infi nite i.e. more than
50
5000 mg/kg. Hence, the doses 500 mg/kg and 250 mg/
kg (1/10th & 1/20th of 5000 mg/kg) were selected for
pharmacological screening of the MECAP.
Antiulcer Activity Studies
Antiulcer activity was evaluated in indomethacin
induced gastric ulcer model according to the method
described by Datta GK et al. 19 In this method, animals
were divided into four groups (each group contains
six rats) and animals were fasted for 36 h prior to the
experiment, but allowed free access to water.
68 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

H.N Nataraj et al.: Evaluation of gastroprotective ability of Amorphophallus paeoniifolius corms against indomethacin induced gastric ulcers
Grouping and Treatment of Animals
The different groups were treated as follows;
Group-I : + ve control water, 10 ml/kg,
P.O. + Indomethacin, 30 mg/kg, P.O.
Group-II : Standard Lansoprazole, 8 mg/kg,
P.O. + Indomethacin, 30 mg/kg, P.O.
Group-III : Methanolic extract 250 mg/kg,
P.O. + Indomethacin, 30 mg/kg, P.O.
Group-IV : Methanolic extract 500 mg/kg
P.O.+ Indomethacin, 30 mg/kg, P.O.
Groups of 6 animals each were pretreated with water
or standard drug or methanolic extract of corm (250,
500 mg/kg P.O.) 30 minutes before the administration
of indomethacin at dose 30 mg/kg orally.
Determination of gastric ulcer index
Four hours later the albino rats were sacrifi ced by an
overdose of anesthetic ether. Subsequently, stomachs
were removed and cut open along the greater curvature
and pinned on a soft board. The number of ulcers in the
glandular portion per stomach were noted and severity
of gastric mucosal lesions scored microscopically with
the help of hand lens (10x) and scoring was done as
fallows. 20
0 = Normal stomach 0.5 = Red coloration
1 = Spot ulcers 1.5 = Haemorrhagic streaks
2 = Ulcer > 3 mm < 5mm 3 = Ulcers > 5mm
Mean ulcer score for each animal was expressed
as ulcer index. The percentage protection was
calculated by using the formula; Percentage protection
= 1–U t /U c × 100 ; Where, U t = Ulcer index of treated
group; U c = Ulcer index of control group (Table 1).
GSH estimation in Indomethacin induced
gastric ulceration
Tissue samples of stomach were homogenized in ice
cold Trichloroacetic acid (1 gm tissue plus 10 ml 10%
TCA) in an ultra turrax tissue homogenizer. Glutathione
measurements were performed using the modifi cation
of the Ellamn procedure. 21 Briefl y, after centrifugation
at 3000 rpm for 10 minutes, 0.5 ml supernatant was
added to 2 ml of 0.3 M disodium hydrogen phosphate
solution. A 0.2 ml solution of dithiobisnitrobenzoate
(0.4 mg/ml in 1% sodium citrate) was added and the
absorbance at 412 nm was measured immediately
after mixing. Percentage increase in OD was directly
proportional to the increase in the levels of Glutathione.
Hence, Percentage increase in OD was calculated by the
formula: Percentage increase = { (Test OD - Control
OD) / Control OD } x 100 (Figure 1).
In vivo lipid peroxidation estimation in gastric
ulceration
The degree of lipid peroxide formation was assayed by
monitoring thiobarbituric reactive substance formation. 22
Combined 1.0 ml of tissue samples of stomach
biological sample (0.1–2.0 mg of membrane protein or
0.10.2μ mol of lipid phosphate) with 2.0 ml of TCA-
TBA-HCl (15% w/v trichloroacetic acid; 0.375% w/v
thiobarbituric acid; 0.25N hydrochloric acid) and mixed
thoroughly. The solution was heated for 1 hr in a boiling
water bath. After cooling, the fl occulent precipitate was
removed by centrifugation at 1000 rpm for 10 mins. The
absorbance of the sample was determined at 535 nm
against a blank that contains all the reagents minus the
lipid. The malondialdehyde concentration of the sample
was calculated by using an extinction coeffi cient of
1.56 × 105 M –1 cm –1 [Percentage inhibition = { (Control
– Test) / Control OD } × 100] (Figure 2).
Statistical analysis
All the results were expressed as mean ± SEM, n = 6.
Statistical analysis was performed with one way analysis
of variance (ANOVA) followed by Tukey-Kramer
Multiple Comparisons Test by using Graph Pad Instat
3.06 Software P value less than < 0.05 was considered
to be statistically signifi cant. * P < 0.05, **

H.N Nataraj et al.: Evaluation of gastroprotective ability of Amorphophallus paeoniifolius corms against indomethacin induced gastric ulcers
Figure 1: Effect of MECAP on tissue GSH levels.
Figure 2: Effect of MECAP on tissue GSH levels.
of ethyl acetate were spotted in the form of bands on
precoated and preactivated aluminium silica gel 60GF 254
HPTLC plates (10 cm × 10 cm width, 0.2 mm thickness-
E-Merk) by means of Linomat IV automatized spray
on band applicator equipped with a 100 μL syringe. The
linear ascending development was carried out in Camag
HPTLC twin trough chamber saturated with mobile
phase consisting of chloroform : ethyl acetate : formic
acid (5:4:1). Subsequent to the development the plates
were dried in a current of air with the help of an air dryer.
Densitometric scanning was performed on Camag TLC
scanner III in absorbance mode at 254 nm (Figure 3).
For references, HPTLC fi nger print analysis of standards
markers viz., Gallic acid and Quercetin were developed
70 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

H.N Nataraj et al.: Evaluation of gastroprotective ability of Amorphophallus paeoniifolius corms against indomethacin induced gastric ulcers
Figure 3: HPTLC Finger print profile after densitometric scan of Sample (EtOAc fraction of MeOH extract) under uv 254 nm (Chloroform:
Ethyl acetate: Formic acid - 5:4:1).
with same solvent system. Densitometric scanning was
performed and R f values were recorded (Table 2).
RESULTS
Effect of MECAP on tissue GSH & LPO levels,
Gastric ulceration
In pylorus ligation induced gastric ulcer there was
marked reduction in the tissue GSH levels in control
group. Treatment with MECAP increased GSH levels
by both the doses 250 and 500 mg/kg to 47.01% &
73.09% respectively and inhibition of lipid peroxidation
generation up to 29.27% and 36.81% respectively in a
dose dependant manner. Besides, MECAP at both
concentrations showed signifi cant (p

H.N Nataraj et al.: Evaluation of gastroprotective ability of Amorphophallus paeoniifolius corms against indomethacin induced gastric ulcers
role in gastric ulceration induced by several types of
stress. ROS also decrease the levels of endogenous
antioxidants, such as GSH, α-tocophenol and ascorbate
and make more prone to oxidative damage. 26 Most of
the world’s population relies on traditional remedies
for its treatment. It is recommended that natural drugs
containing antiulcer, antioxidant and antisecretory
activity can exhibit effectiveness in peptic ulceration.
The well known Phytoconstituents possessing antiulcer
activity includes polyphenols, tannins, fl avonoids, gums
and mucilages, saponins etc.
In the present study indomethacin induced gastric ulcer
model experiment, ulcer index parameter was used
for the evaluation of gastroprotective activity. Oral
administration of indomethacin (30 mg/kg) has induced
ulcers as indicated by higher ulcer index values. Further
it also reduced the gastric tissue glutathione levels
and enhanced the lipid peroxidation. Indomethacin
induced gastric damage particularly due to inhibition of
cycloxygenase pathway of arachidonic acid metabolism
which has a consequence diversion of pathway resulting
in over production of leukotrienes 27,28 and these
leukotrienes have been attributed an important role
in gastrointestinal ulceration. 29 Apart from this, these
agents break the mucosal barrier, provoke an increase in
the gastric mucosal permeability to H + and Na + ions and
drop in the transmucosal potential differences inducing
the formation of erosions and ulcer and in addition
indomethacin markedly decrease gastric mucosal PGE 2
levels causing ulceration. 30
Whereas, the Methanolic extract of corms of
Amorhophallus paeoniifolius (MECAP) signifi cantly reduced
the ulcer score in dose dependent manner compared to
control group. Along with this, it also lowered destructive
lipid peroxidation (LPO) levels and restored back the
protective GSH levels.
Preliminary phytochemical screening of MECAP indicated
the presence of the phytoantioxidants viz., Tannins,
Flavonoids, Sterols, Polyphenolics and Coumarins
which are well known powerful natural antioxidants due
to their electron donating property which either scavenge
the principal propagation free radicals or halt the
radical chain. The HPTLC analysis revealed the presence
of nine phenolics including Gallic acid and Quercetin
in the tested extract.
Besides, various mechanisms have been thought to be
involved in the ulcer production; hence it is not possible
to propose a single mechanism for anti-ulcer effect to a
particular drug or especially for an extract. It has been
demonstrated that the antioxidant compounds could
be active in producing antiulcerogenic effect as these
substances are capable of settling on the membrane
and counteracting lipid peroxidation. 31 Along with
this the fl avonoids present in the test extract not only
exhibit antioxidant activity 32 but also have effects in
gastrointestinal tract, including mucosal protection in
rat colitis. 33 In addition to this, it is being reported that
fl avonoids are able to protect the gastric mucosa against
a variety of ulcerogenic agents via several mechanisms
of action, mainly free radical scavenging and antioxidant
properties, increased mucus production. 34 Thus the
antiulcerogenic effect observed with oral administration
of MECAP could be related to the presence of fl avonoids
and other phytoantioxidants detected in test extract.
However, further studies are required to establish the
exact mode of action and the active principles involved
in its gastroprotection.
CONCLUSION
In conclusion, from the results of the present study, it is
clear that Amorphophallus paeoniifolius corms methanolic
extract has shown signifi cant gastroprotective
activity in indomethacin induced gastric ulceration
animal model. The MECAP at both doses exhibited
signifi cant gastoprotection in dose dependent manner
in comparison to standard Lansoprazole. In addition to
tannins, sterols, fl avonoids, coumarins and nine phenolic
compounds including quercetin and gallic acid (resolved
in HPTLC analysis) present in extract and some other
compounds might have contributed to the protection
offered. Further, evaluation of the said compounds for
gastroprotection is under study to ascertain the claim.
ACKNOWLWDGMENTS
The authors are grateful to principal and management,
T.V.M. College of Pharmacy, Bellary and authorities
of SCS College Pharmacy, Harapanahalli for providing
necessary facilities. We wish to extend our thanks to
Dr. Kotresh also for authentication of plant.
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of Ethanolic and Aqueous extracts of Amorphophallus campanulatus’ ,
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RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 73

Immunomodulatory activity of methanolic
extracts of Pongamia glabra Vent. seeds and
bark in cyclophosphamide induced mice
Sanjeev Heroor 1 , Arunkumar Beknal 1 , Nitin Mahurkar 2
1 Dept. of Pharmacognosy and Phytochemistry, HKES’s MTR institute of Pharmaceutical Sciences, Gulbarga, Karnataka, India
2 Dept. of Pharmacology, HKES’s MTR institute of Pharmaceutical Sciences, Gulbarga, Karnataka, India
ABSTRACT
Immune activation is an effective as well as a protective and novel approach against emerging infectious
diseases. Traditionally Pongamia glabra Vent claimed to cure infectious diseases needs scientifi c validation as
immunomodulatory agent. Methanolic extracts of seeds and barks at the doses of 250 mg/kg and 500 mg/kg
(per oral) of Pongamia glabra Vent. were studied for the assessment of immunomodulatory activity on
cyclophosphamide induced immunosuppression in mice. The activity was assessed by determining the RBC,
Hb%, platelet, total WBC and differential counts. Methanolic extracts of seeds and barks of Pongamia glabra
Vent. showed dose dependent highly signifi cant counteracting effect (p

Sanjeev Heroor et al.: Immunomodulatory activity of methanolic extracts of Pongamia glabra Vent. seeds and bark in cyclophosphamide induced mice
Karanja, a tree found all over India bearing imparipinnate
leaves and pinkish white colored fl owers. 8 The seeds of
the plant reported to contain fi xed oil and traces of
essential oil. Bark contains a bitter alkaloid, resin and
mucilage. 9 The ancient folklore claims the uses of the
seeds to treat rheumatic joints and anti-paracytic. Bark is
astringent and powdered seeds are used as febrifuge and
tonic. 10 The present study was aimed at screening of seeds
and bark methanolic extracts of Pongamia glabra Vent.
for immunomodulatory activity in cyclophosphamide
induced immunosuppressed albino mice.
MATERIALS AND METHODS
Plant material
Pongamia glabra Vent. seeds and bark were collected from
local areas of North Karnataka and a voucher specimen
has been deposited at the departmental herbarium. The
plant was authenticated by Dr. Srinathrao, Prof. and
HOD, Dept. of Botany, Gulbarga University, Gulbarga
and Ref. No. GUG/BOT/Herbarium/2008–09/09. The
mentioned parts of the plant were dried and pulverized
to particle size (#) 40 and then were fi rst defatted with
petroleum ether (40–60°C) and extracted with methanol
by continuous hot percolation method using Soxhlet
apparatus at 40°C for 48 h to obtain methanolic extracts
of seeds and bark of the plant respectively. The fi ltrates
of the extracts were concentrated to dryness at 40°C
under reduced pressure in a rota fl ash evaporator. The
yields of the methanolic extracts of seeds and barks were
43.16 gm (30.59%w/w) and 37.76 gm (22.19%w/w)
respectively.
Preliminary Phytochemical Studies
The methanolic extracts of seeds and bark were
subjected for preliminary qualitative chemical tests
and the presence of major phytoconstituents were
confi rmed by Thin Layer Chromatography (TLC)
studies11,12 (Table 1).
Thin Layer Chromatography studies
• TLC profi le for fl avonoids
Solvent system: ethyl acetate: formic acid: glacial
acetic acid: water
(10:1.1:1.1:2.6).
Detection: UV 365 (blue fl uorescent spots).
• TLC profi le for alkaloids
Solvent system: toluene: ethyl acetate: diethylamine
(7:2:1)
Detection: wagner’s reagent (brown colored spots).
• TLC profi le for steroids
Solvent system: petroleum ether: acetone (7:3).
Detection: anisaldehyde: sulphuric acid reagent (pink
to red colored spots)
• TLC profi le for saponins
Solvent system: chloroform: glacial acetic acid:
methanol: water (6.4:3.2:1.2:0.8).
Detection: anisaldehyde: sulphuric acid reagent (pink
colored spots).
Animals
Swiss albino mice of either sex, weighing 25–30 g
housed in standard conditions of temperature, humidity
and light were used. They were fed with standard rodent
diet and water ad libitum. The study was approved by
Institutional Animal Ethical Committee, Ref. No.
HKECOP/IAEC/45/2011–12.
Acute Toxicity Studies
Acute toxicity studies were conducted as per OECD
guideline by 425 method. 13 The animals did not show
any mortality at the dose of 5000 mg/kg and hence its
1/10th dose i.e. 500 mg/kg and 1/20th dose i.e. 250 mg/
kg were used as the therapeutic doses for the methanolic
extracts of the study.
Test Samples
Weighed quantities of test extracts were suspended
in 1% sodium carboxy methyl cellulose to prepare a
suitable dosage form. 14 The control animals were given
an equivalent volume of sodium CMC vehicle.
Drugs
Cyclophosphamide was used as a standard immunosuppressant,
Cycloxan ® (Biochem–pharmaceutical
indus tries Ltd., Mumbai) containing 200mg – cyclosphosphamide
, was procured from the market and dilutions
were made using sterile water for injection as mentioned
on the label of the marketed product.
Cyclophosphamide Induced Myelosuppression15 Animals were divided into six groups of six animals
each. Group I served as control group and received
Table 1: Thin Layer Chromatography (TLC) results of Pongamia glabra Vent. seeds and bark methanolic extracts
Flavonoids Alkaloids Steroids Saponins
S.No. Extracts No. of spots Rf value No. of spots Rf value No. of Spots Rf Value No.of spots Rf value
1 Seed Ext. 2 0.59, 0.63 2 0.89, 0.80 2 0.61, 0.76 2 0.58, 0.65
2 Bark Ext. 2 0.62, 0.71 2 0.82, 0.53 4 0.64,0.72, 0.78, 0.88 2 0.72, 0.87
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 75

Sanjeev Heroor et al.: Immunomodulatory activity of methanolic extracts of Pongamia glabra Vent. seeds and bark in cyclophosphamide induced mice
the vehicle (1% sodium CMC) for 13 days. Group
II (Cyclophosphamide group) received the vehicle
(1% sodium CMC) for a period of 13 days and on 11th,
12th and 13th days was injected with cyclophosphamide
(30 mg/kg intraperitonial). Groups III and IV were
administered methanolic extracts of seeds of the plant
at the doses of 250 mg/kg and 500 mg/kg (per oral)
daily for 13 days respectively. Similarly Groups V and
VI were administered methanolic extracts of bark of
the plant at the doses of 250mg/kg and 500mg/kg (per
oral) daily for 13 days respectively. The groups III, IV, V
and VI were injected with cyclophosphamide (30 mg/kg
intraperitonial) on the 11th, 12th and 13th days, 1 hour
after the administration of the Respective oral treatment.
Blood samples were collected on 14th day of the
experiment by retro orbital puncture and hematological
parameters were studied for RBC, Hb%, platelets, total
WBC counts and differential leucocytes counts (DLC).
Statistical Analysis
Data were expressed as mean ± SEM and differences
between the groups were statistically determined by
analysis of variance (ANOVA) followed by Dunnet’s
test. p-values

Sanjeev Heroor et al.: Immunomodulatory activity of methanolic extracts of Pongamia glabra Vent. seeds and bark in cyclophosphamide induced mice
Immune activation is an effective as well as protective
approach against emerging infectious diseases. 17
Immunomodulatory activity of methanolic extract of
seeds and bark of Pongamia glabra Vent. was explored
by evaluating their effects on cyclophosphamide
induced myelosuppression in mice at 2 dose levels of
250 mg/kg and 500 mg/kg (per oral) Results of the
study revealed the dose dependent counteracting effect
of the extracts to the cyclophosphamide induced bone
marrow activity suppression i.e. myelosuppression, as
indicated by increase in RBC, total WBC platelet counts,
Hb% and DLC in the extract treated groups (Group III,
IV, V and VI), when compared to cyclophosphamide
treated group (Group II). However the counteracting
activity to reduction in blood cell counts of the seeds
extract was more than that of the bark extract at the
mentioned doses, but both extracts showed similar level
of signifi cance of activity. The potentiated activity of
seed extract may be due to presence of excess number
of fl avonoids and steroids in the seed extract. The
results indicate modulation of bone marrow activity,
viz., – suppression when used cyclophosphamide alone
and stimulation to counteract the cyclophosphamide
induced myelosuppression in pretreated methanolic
extract groups of seeds and barks of Pongamia
glabra Vent.
CONCLUSION
From the phytochemical investigation, it was found
that the major chemical constituents of the methanolic
extracts of seeds and barks were steroids, saponins,
tannins, fl avonoids, alkaloids, proteins and carbohydrates.
Saponins are either triterpenoid or steroidal glycosides
proven as important phytoconstituent with different
pharmacological activities such as antiallergic,
cytotoxic, antitumour, antiviral, immunomodulating,
antihepatotoxic, and antifungal activities. Recently three
diosgenyl saponins isolated from Paris polyphylla were
reported for immunostimulating activity. 18 Tannins are
also known to possess immunostimulating activites. The
well known ayurvedic formulation, Triphala contains
Terminalia chebula, Terminalia belerica and Emblica offi cinalis,
which are rich in tannins reported for immunostimulating
activity. 19 Hence the collective presence of steroids,
saponins, tannins and fl avonoids in the methanolic
extracts would be attributed for immunostimulating
activity. However this is a preliminary research work
and the precise mechanism(s) of immunomodulatory
action infl uenced by potent bio-active constituents of
methanolic extracts of seeds and bark of Pongamia glabra
Vent. against cyclophosphamide induced immunesuppression
needs to be investigated.
ACKNOWLEDGMENT
Authors are thankful to authorities of HKE Society and
MTR Institute of Pharmaceutical sciences, Gulbarga,
Karnataka India, for providing necessary facilities to
carry out the study.
REFERENCES
1. Charak Samhita (Trans.). Shree Gulab Kunvera Ayurvedic Society,
Jamnagar, India. 1949; 249–50.
2. Patwardhan B, Kalbag D, Patki P. S, Nagasampagi B.A. Search of
Immunomodulatory agents: a review. Indian Drugs 1990;28(2):56–63.
3. Wagner H. Immunomodulatory agents. Proceedings of the Alfred
Benzon Symposium. 1983:20;559.
4. Makare N, Bodhankar S, Rangari V. Immunomodulatory activity of
alcoholic extracts of Mangifera indica Linn in mice. J. Ethnopharmacol.
2001;78:133–7.
5. Baumann F, Preiss R. Cyclophosphamide and related anticancer drugs,
B. chrmoatogr. B. Biomed Sci. Appl.2001;764:173–92.
6. Goodman J. W. In : Stites D. P., Terr. A.I., Parslow T. G. (Eds.), The
Immune response in Basic and Clinical Immunology. 8th Edn. NJ:
Prentice-Hall, Engle wood Cliffs; 1994; 40–7.
7. Angulo I., Jimenez M.B., Garcia Bustos J.F, Gargallo D. Candida albicans
infection enhances immunosuppression induced by cyclophosphamide
by selective priming of suppressive myeloid progenitors. cell immunol
2002:218;46–58.
8. Kirtikar K. R, Basu B.D. Indian Medicinal Plants, Oriental Enterprises.
Dehradun, 2nd Ed. 1984;I:830–32.
9. Nadkarni K. M. Indian Materia Medica, Popular Prakashan.1996;
I: 1001–04.
10. Hartwell J. L. Plants used against cancer. A Survey, Lloydia.1967–1971;
30–34.
11. Khandelwal K.R. Practical Pharmocognosy Techniques and
Experiments. 10th Ed. Pune: Nirali Prakashan; 2003; 149–58.
12. Hildebert Wagner, Sabine Bladt. Plant Drug Analysis-A Thin layer
Chomoatography Atlas, Springer-verlag. 2nd Ed. New York: Berlin
Heidelberg; 2001; 1–3, 195–197, 305–206.
13. Committee for the purpose of control and supervision of Experimental
Animals (CPCSEA), OECD Guidelines for the testing of Chemicals,
revised draft guidelines 425: Acute oral toxicity-Acute toxic class method,
revised document. India: Ministry of Social Justice and Empowerment;
2008, No.26.
14. Satpute K. L, Jadhav M. M, Karodi R. S, Patil M.J. Immunomodulatory
activity of fruits of Randia dumetorum Lamk. J. Pharmocog. and
Phytother. 2009;1:1–5.
15. Manjarekar P. N, Jolly C.L, Narayan S. Comparative studies of
immunomodulatory activity of Tinospora cordifolia and Tinopora
sinensis. Fitoterpia 2001;71:254–7.
16. Pelczar M.J, Chan E.C.S, Krieg N. R. Microbiology, 5th Ed. New Delhi:
Tata Mcgraw Hill; 1990; 703–15.
17. Hackett C J. Allergy. Clin. Immunol. 2003;112:686–94.
18. Xiu-feng Z, Yan C, Jiajun H. Ya-Zhou Z, Zhou N. Ya-Lin T, Yang L.
Immunostimulating properties of diosgenyl saponins isolated from Paris
polyphylla. Bioorganic and Med. Chem. Letters 2007;7:255–9.
19. R Srikumar, Parthasarathy N.J, Sheeladevi R. Immunomodulatory
activity of Triphala on Neutrophil Functions. Biol. Pharm. Bull. 2005;
28(8):1398–1403.
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 77

Simultaneous fi rst derivative UV
spectrophotometric estimation of
ramipril and olmesartan
Santosh R. Karajgi, Simpi C.C., Kalyane N.V.
Department of Pharmaceutical Chemistry, BLDEA’s College of Pharmacy, BLDEA University Campus, Solapur Road,
Bijapur, Karnataka, India
ABSTRACT
A simple, precise and economical procedure for the simultaneous estimation of Olmesartan Medoxomil and
Ramipril in tablet formulation has been developed. Olmesartan and Ramipril are antihypertensive agents belonging
to category of angiotensin-converting enzyme inhibitor. The present study involves the simultaneous estimation
done by fi rst derivative UV Spectrophotometric method using Shimadzu 1700 spectrophotometer. Olmesartan
has zero crossing point at 240 nm in methanol and Ramipril has zero crossing point at 246 nm in methanol.
Both these drugs obey Beer’s law in the concentration range employed for the present method. The result
of analysis has been validated statistically by recovery studies. The slope and intercept for Olmesartan were
0.0364 and 0.0078 and for Ramipril were 0.0010 and –0.0001 respectively as determined by the method of
least squares.
INTRODUCTION
Ramipril is a prodrug belonging to the
class of ACE inhibitors used to treat
hypertension and congestive heart failure
where as Olmesartan is an ARB used
to treat high blood pressure. Both ACE
inhibitors and ARBs are widely used in
renal failure patients in the treatment of
hypertension, left ventricular dysfunction,
and diabetic nephropathy. Their effi cacy
in these conditions is well established, and
generally both classes of drugs are well
tolerated, with a low incidence of side
effects. 1 Combination of ACE inhibitors and
ARBs is proved to be a useful combination
therapy for the treatment of ischemic heart
diseases. 2
Review of literature revealed that there
are very few methods reported for the
estimation of Ramipril and Olmesartan
individually and in combined dosage
forms, 3–26 no derivative spectrophotometric
method has been so far reported for
simultaneous estimation of these drugs in
combined dosage forms. Therefore, the
aim and objective of present study were
to develop a fi rst derivative UV-Visible
spectrophotometric analytical method for
the estimation of Ramipril and Olmesartan
in bulk and formulated dosage form in
combination without prior separation and
to establish a simple, sensitive, standard,
reproducible method for the quality control
of Ramipril and Olmesartan.
MATERIALS AND METHODS
Materials
Ramipril and Olmesartan were gift samples
from Ajantha Pharmaceuticals, Aurangabad.
The commercial formulations were
purchased from local market. Methanol
A.R. Grade (Qualigens, Fine chemicals)
was used as the solvent.
Research Ar cle
Received Date : 27-08-2011
Revised Date : 06-02-2012
Accepted Date : 08-02-2012
DOI: 10.5530/rjps.2012.1.11
Address for
correspondence
Santosh R. Karajgi
Department of Pharmaceutical
Chemistry, BLDEA’s College of
Pharmacy, BLDEA
University Campus,
Solapur Road, Bijapur,
Karnataka, India
Email: santosh.karajgi@gmail.
com
Mobile: +919739619395
www.rjps.in
78 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

Equipments
Santosh R. Karajgi et al.: Simultaneous First Derivative UV Spectrophotometric Estimation of Ramipril and Olmesartan
Shimadzu spectronic 1700 double beam UV-visible
spectrophotometer with 1 cm matched quartz cells was
used for all the absorbance measurements.
Method
The solutions of Ramipril and Olmesatan were
prepared separately in methanol at a concentration
range of 10 μg/ml. Both these solutions were scanned
in the wavelength range of 200 nm to 300 nm. Data
were recorded at the wavelength interval of 1 nm in the
derivative mode to obtain fi rst order derivative spectra
at N=2. After examining the overlain fi rst derivative
spectra, wavelengths for analysis were selected where
one drug showed zero crossing and the other drug
showed substantial absorbance. The wavelength selected
for Ramipril analysis was 240 nm where Olmesartan
has zero absorbance and the wavelength selected for
Olmesartan analysis was 246 nm where the absorbance
of Ramipril is zero. The overlain derivative spectra of
the drugs are given in the Figure 1.
Linearity
Standard stock solutions of Ramipril and Olmesartan
were prepared by dissolving 100 mg each of standard
drug samples in 100 ml volumetric fl asks separately
and the volume was made up with methanol to
get a concentration of 5 – 50 μg/ml for each drug.
Figure 1: Zero crossing points of Ramipril and Olmesartan.
The absorbances of derivative spectra were measured
at 240 nm and 246 nm for Ramipril and Olmesartan
respectively. Five replicate analyses were carried out.
Absorbance against concentrations were plotted to
obtain the calibration graph. Both the drugs obeyed
Beer’s law with the above concentration range with
the R 2 value of 0.9994 and 0.9996 for Ramipril and
Olmesartan respectively (Figure 2 and 3).
Analysis of marketed formulation
Twenty tablets were weighed accurately and powdered.
An accurately weighed quantity of powder equivalent
to 20 mg of Ramipril and 20 mg of Olmesartan was
taken in a 50 ml volumetric fl ask and dissolved in
methanol. The volume was made up with methanol so
that the theoretical concentrations of both drugs were
10μg/ml and the concentrations were determined at
240 nm for Ramipril and 246 nm for Olmesartan by
using calibration graph. (Table 1). The overlain spectra
of synthetic mixture, Ramipril bulk drug, Olmesartan
bulk drug and combined tablet formulation of Ramipril
and Olmesartan is given in the Figure 4.
Recovery Studies
To determine the accuracy of the method, recovery
study was performed using the method of standard
addition. To the pre- analyzed marketed formulation
powder of combined Ramipril and Olmesartan, diluted
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 79

Santosh R. Karajgi et al.: Simultaneous First Derivative UV Spectrophotometric Estimation of Ramipril and Olmesartan
Table 1: Analysis of marketed formulation
Label claim (mg) Amount found (mg) Label claim %
Sample Ramipril Olmesartan Ramipril Olmesartan Ramipril Olmesartan
1 10 10 10.08 10.18 100.8 101.8
2 10 10 10.11 10.17 101.1 101.7
3 10 10 9.99 10.11 99.9 101.1
4 10 10 10.04 10.18 100.4 101.8
5 10 10 9.98 9.88 99.8 98.8
Mean 10.04 10.10 100.4 101.0
S.D. 0.0561 0.1285 0.561 1.285
Figure 2: Calibration graph of Ramipril at 240 nm.
Figure 3: Calibration graph of Olmesartan at 246 nm.
Figure 4: First derivative spectra of Ramipril, Olmesartan, Synthetic mixture and Tablet formulation.
80 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

Santosh R. Karajgi et al.: Simultaneous First Derivative UV Spectrophotometric Estimation of Ramipril and Olmesartan
to 10 μg/ml with methanol, weighed quantities of bulk
drugs were added at four levels, i.e. 50 %, 75 %, 100 %
and 125 % for both drugs and the total drug contents
were determined as described for formulation. The
percentage recovery was determined (Table 2).
RESULTS AND DISCUSSION
Simultaneous analysis of Ramipril and Olmesartan by zero
order UV-Visible spectrophotometric method appears to
be quite impossible because of the total overlap of bands.
(Figure 5). But derivative spectrophotometric technique
involves the differentiation of the normal spectrum with
respect to the wavelength. The fi rst derivative spectra of
the Olmesartan shows an absorbance at 246 nm, where
the absorption for Ramipril is zero and Olmesartan can
be specifi cally measured at that wavelength. Ramipril
on the other hand, has absorbance at 240 nm where
absorbance for Olmesartan is zero; hence Ramipril is
specifi cally measured at that wavelength.
Figure 5: Overtain zero order spectra of Ramipril and Olmesartan.
Table 2: Recovery study of marketed formulation
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 81
Drug
Amount present in
formulation (μg/ml)
Amount
added (%)
Mean
Recovery (%)
Ramipril 10.11 50 99.62
75 100.93
100 98.89
125 99.85
Olmesartan 10.04 50 100.43
75 99.93
100 100.18
125 99.75
The percentage RSD values in precision shows that
proposed method provides acceptable variation of
Ramipril and Olmesartan which were found to be less
than 2% shows its capacity to remain unaffected by
small variations in method parameters and provides
an indication of its reliability during normal usage.
The good recovery values indicate the accuracy of the
Table 3: Validation of the proposed method
Sl. No. Parameters
Ramipril
Experimental value
Olmesartan
ICH Limits
1 Beer’s Range 5–50 μg/ml 5–50 μg/ml –
2 Linearity (R2 ) 0.9994 0.9996 0.9990
3 Precision (% RSD) 0.1754 0.0349 2.0000
4 Accuracy (% Recovery) 99.62–100.93 % 99.75–100.43 % 97–103 %
5 Intercept -0.0001 +0.0078 –
6 Slope +0.0010 +0.0364 –

Santosh R. Karajgi et al.: Simultaneous First Derivative UV Spectrophotometric Estimation of Ramipril and Olmesartan
method (Table 3). Method validation was carried out as
per ICH guidelines. 27
CONCLUSION
The described method gives accurate and precise results
for determination of Ramipril and Olmesartan mixtures
in tablets without prior separation and are easily applied
for routine analysis. This method also provides simple
and reproducible quantitative analysis without any
interference from the excipients.
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82 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

Purifi cation and characterization of thermostable
amylase from a strain of thermoactinomyces
thalpophilus KSV 17
K. Sreenivasa Rao 1 , P. Ellaiah 2 , Karnakumar V. Biradar 3
1 Department of Pharmaceutics, RRKS College of Pharmacy, Bidar-585402, India
2 Department of Biotechnology, College of Pharmaceutical Sciences, Visakhapatnam, Andhra University, Andra Pradesh, India
3 Department of Pharmacology, RRKS College of Pharmacy, Bidar-585402, India
ABSTRACT
This research reported the Purifi cation and Characterization of Thermostable Amylase from a strain of
T. Thalpophilus KSV 17. The result showed that the purifi ed enzyme specifi c activity of 145.80 U mg –1 , this was
an increase of 21 fold than the crude enzyme extract. The analysis of SDS- polyacrylamide gel electrophoresis
showed that the molecular weight of the enzyme was 52 kDa. The Optimum pH of the purifi ed enzyme showed
maximum activity at pH 7.0, but the enzyme was stable in the pH range of 5.5–7.0. The optimum temperature
of the purifi ed enzyme was 85°C in absence of 10 mM CaCl while 90°C in presence of 10 mM CaCl K and
2 2., m
V values for the purifi ed enzyme were calculated as 5.2 mg ml max –1 , 0.45 mg ml –1 /minute respectively. The thermal
stability of the purifi ed enzyme at 80°C in absence of CaCl and 85°C in presence of CaCl . The purifi ed enzyme
2. 2
mostly inhibited by diethyl pyrocarbonate and N-bromosuccinimide and at 5 mM conc. Ca2+ , Na + and Mg2+ showed stimulatory effect while Cu2+ , Zn2+ , Hg2+ and Mn2+ shown inhibitory effect. The purifi ed enzyme showed
good stability and compatibility on commercial detergents and on being stored for 60 minutes at 85°C.
Key words: Purifi cation, Characterization, α-Amylase, Sephadex G-200, SDS-PAGE
INTRODUCTION
Microbial enzymes and coenzymes are
widely used in several industries, notably
in detergent, food processing, brewing
and pharmaceuticals. They are also used
for diagnostic, scientifi c and analytical
purposes. Since ancient times they have
been used in the preparation of fermented
foods, especially in oriental countries. 1
The activity of an enzyme is determined
by the enzyme concentration, substrate
concentration and its availability,
concentration of co-factors, allosteric
effectors, the concentration and type of
inhibitors, ionic strength, pH, temperature
and time since start of the reaction. The
way in which each of these parameters
affects enzyme activity can be determined
by studying the enzyme kinetics, many assay
procedures for determining the enzyme’s
activity are available. Some of them are
typically dependent on measurement of
chemical changes Eg: Biuret Method,
Lowry Method, Bradford Method etc.
and others are based on physical changes.
Some of these assays are also designed
to investigate particular properties of the
enzyme preparations and physical changes
Terms, which are frequently encountered,
especially in investigations of enzyme
purifi cation and use are, specifi c activity and
molecular activity per milligram of enzyme.
Where the enzyme preparation is not pure,
specifi c activity is frequently expressed in
units per milligram of protein or in units
per milligram of enzyme preparation. Due
to wide range of application of α-Amylase,
in order to get not only high quality and but
Research Ar cle
Received Date : 26-12-2011
Revised Date : 04-03-2012
Accepted Date : 06-03-2012
DOI: 10.5530/rjps.2012.1.12
Address for
correspondence
K.Sreenivasa Rao
R.RK.S. College of Pharmacy
Naubad, Bidar-585401,
Karnataka, India
Email: bidarkaran@gmail.com
www.rjps.in
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 83

K. Sreenivasa Rao et al.: Purifi cation and Characterization of Thermostable Amylase from a Strain of Thermoactinomyces thalpophilus KSV 17
also high grade α-Amylase we made objectives behind
the sudy.
In this research, α-amylase was purifi ed from a strain of
T. Thalpophilus KSV 17 in three steps of purifi cation
processes, namely precipitation with ammonium sulfate,
precipitation with zink sulfate and Sephadex G-200
gel fi ltration chromatography. The enzyme was then
characterized. The enzyme activity was determined
based on Bernifi eld method, 2 while protein content
was determined by Lowry method. 3
MATERIAL AND METHODS
Chemicals and Microorganism Used
Agarose, acrylamide, bis-acrylamide, sodium dodecyl
sulfate (SDS), TEMED, ammonium per sulfate and
other chemicals for polyacrylamide gel electrophoresis
(SDS-PAGE) were purchased from Sigma Chemical Co.,
U.S.A. A strain of T. thalpophilus mutant KSV 17 was
used for the study. The research phases done were as
follows: Production, Purifi cation and Characterization
of purifi ed enzyme.
Production of α-Amylase
The amylase production was carried out in a 2 liter
fermentor, containing 1.5 L modifi ed production
medium containing Starch 2%, Yeast extract 0.05%, Mg
SO 4 0.01 and salt solution (KH 2 PO 4 0.15%, K 2 HPO 4
0.22% CaCl 2 .2H 2 0 0.0025% and FeSO 4 . 7H 2 0 0.00025%).
A 10% (v/v) level of inoculum was added and the
fermentor was run at 55°C for 48h. Whole fermentation
broth was centrifuged at 10,000 g at 4°C and the clear
supernatant was separated. The supernatant (crude
enzyme) was subjected to recovery and purifi cation
processes.
Purifi cation of α-Amylase
The purifi cation of enzyme was done by following steps,
Ammonium Sulfate Precipitation
Ammonium sulfate was added at different
concentrations ranging from 40 to 80 % saturation.
The precipitates so obtained were suspended in cold
saline solution (2ml) and tested for amylase activity and
total protein content. The salting out concentration of
crude enzyme was established to be 60% on the basis
of enzyme activity. To obtain complete precipitation
of the crude enzyme, the remaining harvest fl uid was
subjected to ammonium sulfate precipitation at 60%
saturation. For this purpose, solid ammonium sulfate
(195g) was added gradually with mechanical stirring
to 500ml of harvest fl uid at 4˚C to a saturation of
60%. The precipitate so formed was separated by
centrifugation (8000g) for 15 min., again suspended in
cold saline solution (100ml) and dialyzed in cold against
1 L of 0.05M Phosphate buffer (pH 7) for 20 h. After
dialysis, the solution was centrifuged and supernatant
obtained was designated as fraction-I. It was used for
zinc sulfate precipitation.
Zinc Sulfate Precipitation
About 100ml of the pooled supernatant (fraction-I)
with activity of 20 U/mg protein was diluted with
1.4 L of 0.05M Phosphate buffer (pH 7) to obtain 2
units of absorbance at 280 nm. To this solution 75 ml
of 0.1M zinc sulfate was added drop wise with stirring
at 0˚C in an ice bath. The precipitate thus formed was
separated by centrifugation (8000g) for 15 min. and
re-suspended in 50ml of 0.4M sodium citrate. This
solution was dialyzed in cold (4ºC) against 240 ml of
0.05M Phosphate buffer, pH 7. The resulting solution
was designated as fraction-II, and subjected to gel
fi ltration chromatography.
Sephadex G-200 Gel Filtration Chromatography
The dialyzed enzyme (fraction-II) was centrifuged at
8000rpm for 15 min. and supernatant was chromatographed
on a column of Sephadex G-200. The sample
(fraction-II) was loaded onto a column of Sephadex
G-200 (1.5cm × 24cm) equilibrated with 0.05M phosphate
buffer (pH 7). The column was eluted at a fl ow
rate of 4.0ml /h with a discontinuous gradient from
0.1 M to 0.8 M phosphte buffrer in the same buffer.
A total of 25 fractions were collected. Fractions (15–18)
with high amylase activity were pooled together, dialyzed,
concentrated by lyophilization and used for further
studies. It was labeled as fraction-III.
Sodium Dodecyl Sulfate Polyacrylamide
Gel Electrophoresis (SDS-PAGE)
After Sephadex G-200 column chromatography, the
fractions (19–23) showing the highest specifi c activity
were pooled, dialyzed, lyophilized and then subjected to
SDS-PAGE. The SDS-PAGE was performed according
to Laemmli (1970) 4 using 12% acrylamide.
Characterization of Purifi ed Enzyme
The Characterization of purifi ed enzyme includes:
Determination of optimum pH, Temperature, effects
of metal ions, effects of inhibitors and chelators and
stability towards temperature, pH, and detergents, kinetic
data, Activity test of α-amylase and protein content.
Determination of Optimum pH of Purifi ed Enzyme
The pH was adjusted using the following buffers: 0.05 M
phosphate (pH 5.0–7.0), Tris-HCl (pH 8.0) and glycine-
NaOH (pH 9.0–12.0). Reaction mixtures were incubated
at 55ºC for 30 min. and the activity of the enzyme
84 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

K. Sreenivasa Rao et al.: Purifi cation and Characterization of Thermostable Amylase from a Strain of Thermoactinomyces thalpophilus KSV 17
was measured. For stability study, the purifi ed enzyme
was diluted in different relevant buffers (pH 6.0–12.0)
in two sets. One set incubated for 2h and another set for
20 h at 55ºC. The relative activity at each exposure was
measured as per assay procedure.
Determination of Optimum Temperature
of Purifi ed Enzyme
The activity of the purifi ed enzyme was determined by
incubating the reaction mixture at different temperatures
ranging from 30 to 95°C for 30 min. in the absence and
presence of 10 mM CaCl 2 . Thermostability of the purifi ed
enzyme was determined in the presence and absence
of CaCl 2 (10mM) by pre-incubating enzyme samples in
phosphate buffer for 30 minutes at temperatures ranging
from 30 to 95ºC.
Determination of Amylase Activity on
Various Metal Ions
Metal ions viz. Ca2+ , Mg2+ , Hg2+ , Fe3+ , Na + , Zn2+ , Mn2+ and Cu2+ (10 mM) were investigated by adding them
into the reaction mixture. The mixture was incubated
for 30 min. at 55°C and the relative amylase activities
were measured.
Determination of Amylase Inhibitors and
Chelators on Purifi ed Enzyme
The effect of various amylase inhibitors (at 5mM), such
as Diethyl pyrocarbonate (DEPC), N-bromo succinimide
(N-BS) cysteine-inhibitors p-chloromercuric benzoate
(pCMB) and β-mercaptoethanol (β-MCE), and a
chelator of divalent cations [Ethylene diamine tetra
acetic acid (EDTA)] were determined by preincubation
with the enzyme solution for 30 min at 55°C before the
addition of substrate. The relative amylase activity was
measured.
Determination of Enzyme Stability in
Presence of Detergents
The compatibility of KSV-17 amylase with local
laundry detergents was investigated in the presence of
10 mM CaCl . The detergents were diluted in distilled
2
water (0.7% w/v), incubated with 0.1ml of enzyme
(500U/ml) for 4 h at 55°C and the residual activity was
determined.
Determination of Enzyme Kinetic Data
of Purifi ed Enzyme
Initial rates of soluble starch hydrolysis was determined
with various concentrations at various substrate
concentrations ( g/100ml: 0.10, 0.20, 0.30, 0.40, 0.50,
0.75, 1.00, 1.25, 1.50, 1.75 and 2.00). The kinetic
constants K m and V max were determined by the method
of Lineweaver-Burk.
Activity Test of α-Amylase and Protein
Content Determination
The α- amylase activity was done using 3, 5-dinitro
salicylic acid as a coupling agent by following mandels
(1976) method. The content of protein was determined
based on method of lowry et al. (1951). 3
RESULTS AND DISCUSSION
Table 1: Summary of purifi cation steps of amylase from T. Thalpophilus KSV 17
Purifi cation step
Total enzyme
activity (U)
Total
protein (mg)
Purifi cation of Amylase of Thermoactinomyces
Thalpophilus KSV 17
The enzyme production was carried in a 2 L fermentor
and the extraction of growth media that contain the
extracellular enzyme with cold centrifugation to get
the raw extract enzyme which further precipitated
by salt of ammonium sulfate at 60% of saturation
grade, and obtained supernatant precipitated with zinc
sulfate, resulting solution was subjected for gel fi ltration
column chromatography using Sephadex G-200, then
subjected for SDS-PAGE was performed according
to Laemmli et al.(1970) 4 using 12% acrylamide fi naly
α- amylase activity was done using 3,5-dinitro salicyclic
acid as a coupling agent according to Mande l (1976)
method. Purifi cation steps were showed in Table1.
Gel fi ltration Chromatography using
Sephadex G-200
The purifi cation of enzyme with Gel fi ltration
chromatography using Sephadex G-200, used phosphate
buffer 0.05M, pH 7 and column eluted with 0.1 M to
0.8 M NaCl as elution buffer. The protein pattern (A ) 280
and activity of α-amylase obtained from gel fi ltration
chromatography using Sephadex G-200. From the
fi gure 1 showed only one peak showed α-amylase activity
observed at 0.4M NaCl concentration that was fraction
(15th–18th), the highest activity was fraction 18th as
showed in fi gure 1.
Specifi c
activity (U/mg)
Purifi cation
fold % Recovery
Crude enzyme 175000 26800 6.52 1.0 100
(NH4)2SO4 precipitation 131000 7890 16.60 2.5 74.85
Sephadex G-200 13560 93 145.80 22.3 7.74
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 85

K. Sreenivasa Rao et al.: Purifi cation and Characterization of Thermostable Amylase from a Strain of Thermoactinomyces thalpophilus KSV 17
Figure 1: Chromatography of α-amylase from Thermoactinomyces thalpophilus KSV 17 at Gel filtration column chromatography using
Sephadex G-200, elution solution of buffer phosphate 0.05M, pH 7.0, column size 1.5 × 24 cm and flow rate 1.0 ml/h.
Homogenity test of Purifi ed Enzyme by SDS-
Polyacrylamide gel Electrophoresis
The result of electrophoresis SDS-polyacrylamide of
purifi ed enzyme can be seen in electrophoregram as
shown in Figure 2. This fi gure showed that the molecular
weight of purifi ed enzyme was 52 kDa.
Characterization of Purifi ed Enzyme
Determination of Optimum pH of Purifi ed Enzyme
The activity (%) of purifi ed enzyme and stability can
be seen in fi gure 3. It showed optimum pH of purifi ed
enzyme is 6.0 and stable in the pH range of 4–7.5 in
case of 2h. preincubation and at 20h. preincubation the
enzyme was stable in the pH range 5.5–7.0 (Figure 3).
Determination of Optimum Temperature
of Purifi ed Enzyme
The activity (%) of purifi ed enzyme at various
temperatures in presence and absence of CaCl 2 can be
seen in fi gure 4. The recorded optimum temp of purifi ed
enzyme was 85ºC in the absence of CaCl 2 , while 90ºC in
the presence of CaCl 2 (Figure 4).
Determination of Inhibitors and Chelators
effect on Purifi ed Enzyme
The inhibitors tested (at 5 mM conc.), diethyl
pyrocarbonate (DEPC) was able to inhibit the almost
completely, N-bromo succinimide (N-BS) exhibited
89% inhibition. Results are similar to Bolton et al.,
(1997) 5 study. This indicated that it is an alpha amylase
and, 51% inhibition was observed with EDTA and slight
stimulation effect by β -mercaptoethanol (fi gure 5).
Determination of Compatibility with
Detergents on Purifi ed Enzyme:
Besides pH, a good detergent amylase is expected to
be stable in presence of commercial detergents. The
amylase from KSV 17 showed excellent stability and
compatibility in the presence of locally available detergents
(Nirma, Wheel, Henko, Surf excel, Ariel and Rin).
Determination of Metal ions Effect on
Purifi ed Enzyme
The data indicates that Ca 2+ , Mg 2+ and Na + have slight
stimulatory effect activation of the enzyme may be
due to activation of metal ions while other ions have
slight inhibitory effect on the enzyme. These results
suggest, the metal ions apparently protected the enzyme
against thermal denaturation and played a vital role in
maintaining the active confi rmation of the enzyme at high
temperatures (Donaghy and McKay (1993) 6 (Figure 6).
Determination of Kinetic data of
Purifi ed Enzyme
The graph of Determination of K and V values
m max
of the purifi ed enzyme can be seen in fi gure 7. From
the graph, the K (Michaelis constant) and V m max
were calculated as 5.2 mg/ml and 0.45 mg/ml/min
respectively. (Figure7). Aguilar et al., (2000) reported
a K of 3.44 mg/ml and V of 0.45 mg/ml/min for
m max
alpha amylase from Lactobacillus. A K of 0.90 g/l was
m
reported for a thertmostable alpha amylase from Bacillus
licheniformis by Ivanova et al., (1993). 7 Krishnan and
Chandra, (1983) 8 reported a K of 1.274 mg/ml and
m
V of 0.738 mg/ml/min for alpha amylase obtained
max
from Bacillus licheniformis CUMC 305.
86 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

K. Sreenivasa Rao et al.: Purifi cation and Characterization of Thermostable Amylase from a Strain of Thermoactinomyces thalpophilus KSV 17
Figure 2: SDS-PAGE of Amylase from T. Thalpophilus KSV 17. Lane A, crude enzyme; Lane B, purified enzyme; Lane C, molecular mass
markers. The molecular weight of standard protein markers used are: BSA (67 kDa), Ovalbumin (45 kDa), Carbonic anhydrase (30 kDa),
Trypsinogen (24 kDa) and α-lactalbumin (14 kDa).
Figure 3: The optimum pH of purified enzyme 6.0, same figure also showed that the purified enzyme was stable in the pH range of 4–7.5
in case of 2h. preincubation group. But in the case of 20 h. preincubation group, the enzyme was stable between pH 5.5 and pH 7.
RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012 87

K. Sreenivasa Rao et al.: Purifi cation and Characterization of Thermostable Amylase from a Strain of Thermoactinomyces thalpophilus KSV 17
Figure 4: For the study temperature ranging from 30 to 90 o C in the absence and presence of 10 mM CaCl 2 . The result indicates purified
enzyme is stable at 80 o C and showed high activity at 85 o C. The optimum temperature recorded was 85 o C for purified enzyme in the
absence of CaCl 2 , while 90 o C in the presence of CaCl 2 .
Figure 5:: The inhibitors like DEPC, N-BS exhibited 89% inhibition., 51% inhibition was observed with EDTA and slight stimulation effect
by α-mercaptoethanol.
CONCLUSION
Based on above results and discussion, the following
conclusions are made: The analysis of SDS- PAGE
indicates the molecular weight of α-Amylase is 52 kDa
depending on the relative mobility. The purifi ed enzyme
specifi c activity was an increase of 21 fold than the crude
enzyme extract. The purifi ed enzyme has optimum
pH 7.0. The enzyme was stable between pH 6 and
88 RGUHS J Pharm Sci | Vol 2 | Issue 1 | Jan–Mar, 2012

K. Sreenivasa Rao et al.: Purifi cation and Characterization of Thermostable Amylase from a Strain of Thermoactinomyces thalpophilus KSV 17
Figure 6: The metal ions Ca 2+ , Mg 2+ and Na + have slight stimulatory effect while other slight inhibitory effect on the enzyme.
Figure 7: Amylase activity done according to Bernfield (1955), using 3,5- dinitro salicylic acid as a coupling agent.
pH 7 even after 20 h. preincubation. The optimum
temperature for purifi ed enzyme activity was 85°C
in absence of CaCl 2 while 90ºC in the presence of
CaCl 2 . Amylase inhibitors like DEPC, N-BS and
pCMB showed exhibitory however Ca 2+ , Mg 2+ and
Na + ions have slight stimulatory effect. This purifi ed
enzyme showed good compatibility and stability with
commercial detergents.
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3. Lowry OH. Rosebrough N J, Farr AL, Randall RJ. Adenyl cyclase
activity in human platelets. J Biol Chem1951;193:265–71.
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Castor. Nature1970;227:680–7.
5. Bolton DJ, Kelly CT, Fogarty WM. Purifi cation and charecterization of
Amylase of Bacillus. Enz Microb Technol 1997;20:340–3.
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charecteristics thermostable Amylase enzyme. J Biotechnol 1993;28:
277–89.
7. Krishnan T, Chandra AK. Effect of oilseed cakes on alpha-amylase
production by Bacillus licheniformis CUMC305. Appl Environ Microbiol
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8. Domingues CM, Peralta RM. Production of amylase by soil fungi and
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