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Protease Inhibitors: A review

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 Vol.50 Issue No.02 February 2013

INDIAN DRUGS 50(02) February 2013 1

2 INDIAN DRUGS 50(02) February 2013
 Vol. 50
No. 02

February 2013

review article
- Protease Inhibitors: A Review
Sapkale P. V., Jadhav S. B. and Sable P. N. ................................................................. 5

original research articles

- Method Development and Validation of Olanzapine in Pure and
Pharmaceutical Dosage form by Rp-Hplc Method
Mastiholimath V.S., Dandagi P.M., Gadad A.P., Murali Krishna N.V.
and Mannur V................................................................................................................ 20

- Quantitative Determination of Deferasirox in Bulk and Pharmaceutical

Formulation by Uv Spectrophotometric Method
Marathe G. M., Pande V. V., Patil P. H., Mutha R. E. and Bari S. B. ........................... 27

- Preclinical evaluation of nootropic activity of Glabridin Rich Extract of

Glycyrrhiza glabra using Passive Avoidance Paradigm in rats
Desai S. K., Pandey C. H. and Mulgaonkar S.M. . ........................................................ 33

- Antihyperglycemic and In Vitro Antioxidant Activities of Punica Granatum Linn.

in Alloxan Induced Diabetic Rats
Patil U. S., Bandawane D. D., Bibave K. H. and Chaudhari P. D. ................................ 39

short notes

- A Novel Method For Isolation of Mangiferin from Mangifera Indica L. Bark

Gholkar M. S and Laddha K. S. .................................................................................... 47

- Anti-Inflammatory Effect of Seeds of Tamarindus Indica in Wistar Rats

Hivrale M. G., Mali A. A. and Bandawane D. D............................................................. 49

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INDIAN DRUGS 50(02) February 2013 3

 Founder Editor
*Dr. A. Patani, D.Sc. (Germany)
Editorial Committee
Editor
Dr. Gopakumar G. Nair, Ph.D.
Associate Editors
Mr. J. L. Sipahimalani, B. Pharm. Hons. (London), MRCS, FRPharmS
Dr. Nagaraj Rao, D.Sc. (Germany)
Dr. George Patani, Ph.D.
Consulting Editor
Dr. S. G. Deshpande, M.Sc. (Tech.), Ph.D.

Editorial Board Editorial Advisory Board

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Prof. Padma Devarajan, M.Pharm., Ph.D. (Tech.) Dr. B. N. Dhawan, M.D.
Dr. Prashant M. Dikshit, Ph.D. Prof. S. S. Handa, Ph.D.
Prof. A. K. Gadad, M.Pharm., Ph.D. Dr. C. I. Jolly, Ph.D.
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Prof. Saranjit Singh, M.Pharm., Ph.D. Dr. G. N. Singh, M.Pharm., Ph.D.
Prof. N. Udupa, M.Pharm., Ph.D. Prof. R.T. Sane, Ph.D.
Dr. K. Valliappan, M.Pharm., Ph.D. Prof. M. N. Saraf, M.Pharm., Ph.D.
Dr. A. A. Natu, Ph.D. Dr. P. D. Sethi, Ph.D.
Dr. N. G. N. Swamy, Ph.D. Dr. Ashok Vaidya, M.D., Ph.D., F.A.I.M.
Dr. J. S. Yadav, Ph.D., FNA

4 INDIAN DRUGS 50(02) February 2013

 review article

Protease Inhibitors : A Review

Sapkale P. V., Jadhav S. B*. and Sable P. N.

(Received 29 August 2012) (Accepted 19 December 2012)

Abstract

HIV protease inhibitors were first invented between 1989 and 1994 by researchers working for the
pharmaceutical companies of Hoffmann- La Roche Inc. (in Nutley, New Jersey), Abbott Laboratories
and Merck & Co., Inc. HIV protease inhibitors are used in the treatment of patients with AIDS and were
considered the first breakthrough in over a decade of AIDS research. Currently, there are five HIV
protease inhibitors approved by FDA for the treatment of HIV infection. These drugs work at the final
stage of viral replication and attempt to prevent HIV from making new copies of itself by interfering with
the HIV protease enzyme. As a result, the new copies of HIV are not able to infect new cells. Occurrence
of protease along with structural properties, classification of inhibitors like Saquinavir, Ritonavir, Indinavir,
Nelfinavir etc and life cycle of virus confirm the role of protease inhibitor. Other parameters like adverse
effect, application, structure activity relationship and dose regime shows need of medication for person
suffering from HIV virus.

Keywords: Viral replication, enzyme, Lifecycle, • Metalloproteases

Structural properties, Adverse reaction. • Glutamic acid proteases

INTRODUCTION The threonine and glutamic-acid proteases were

Protease not described until 1995 and 2004, respectively. The
mechanism used to cleave a peptide bond involves
A protease (also termed peptidase or proteinase) making an amino acid residue that has the cysteine
is any enzyme that conducts proteolysis, that is, begins and threonine (proteases) or a water molecule
protein catabolism by hydrolysis of the peptide bonds (aspartic acid, metallo- and glutamic acid proteases)
that link amino acids together in the polypeptide chain nucleophilic so that it can attack the peptide carboxyl
forming the protein. group. One way to make a nucleophile is by a catalytic
triad, where a histidine residue is used to activate
Classification of Protease serine, cysteine, or threonine as a nucleophile. Within
Proteases are currently classified into six broad each of the broad groups proteases have been
groups: classified, by Rawlings and Barrett, into families of
related proteases. For example, within the serine
• Serine proteases proteases families are labelled Sx where S denotes
• Threonine proteases the serine catalytic type and the ‘x’ denotes the number
• Cysteine proteases of the family, for example S1 (chymotrypsins). An
up to date classification of proteases into families is
• Aspartate proteases
found in the MEROPS database 1, 2.

*For correspondence Occurrence of protease

Modern College of Pharmacy, Sector 21
Yamuna nagar, Nigdi, Pune-411044. Proteases occur naturally in all organisms. These
E-mail: sbjadhav_pharma@yahoo.co.in enzymes are involved in a multitude of physiological

INDIAN DRUGS 50(02) February 2013 5

reactions from simple digestion of food proteins to
highly regulated cascades (e.g., the blood-clotting
cascade, the complement system, apoptosis
pathways, and the invertebrate prophenoloxidase-
activating cascade). Proteases can either break
specific peptide bonds (limited proteolysis), depending
on the amino acid sequence of a protein, or break
down a complete peptide to amino acids (unlimited
proteolysis). The activity can be a destructive change,
abolishing a protein’s function or digesting it to its
principal components; it can be an activation of a
function, or it can be a signal in a signaling pathway.
Bacteria also secrete proteases to hydrolyze (digest)
the peptide bonds in proteins and therefore break
the proteins down into their constituent monomers.
Bacterial and fungal proteases are particularly
important to the global carbon and nitrogen cycles in
the recycling of proteins, and such activity tends to be
regulated by nutritional signals in these organisms.
The net impact of nutritional regulation of protease
activity among the thousands of species present
in soil can be observed at the overall microbial
community level as proteins are broken down in
response to carbon, nitrogen, or sulfur limitation.
A secreted bacterial protease may also act as an
exotoxin, and be an example of a virulence factor in
bacterial pathogenesis. Bacterial exotoxic proteases
destroy extracellular structures. Protease enzymes
are also used extensively in the bread industry as
bread improver3.
Proteases, also known as proteinases or
proteolytic enzymes, are a large group of enzymes.
Proteases belong to the class of enzymes known as
hydrolases, which catalyze the reaction of hydrolysis
of various bonds with the participation of a water
molecule. Proteases are involved in digesting
long protein chains into short fragments, splitting
the peptide bonds that link amino acid residues.
Some of them can detach the terminal amino
acids from the protein chain (exopeptidases, such
as aminopeptidases, carboxypeptidase A) while
others attack internal peptide bonds of a protein
(endopeptidases, such as trypsin, chymotrypsin,
pepsin, papain, elastase)4.
6 INDIAN DRUGS 50(02) February 2013
Proteases are divided into four major groups
according to the character of their catalytic active
site and conditions of action: serine proteinases,
cysteine (thiol) proteinases, aspartic proteinases, and
metallo proteinases. Attachment of a protease to a
certain group depends on the structure of catalytic
site and the amino acid (as one of the constituents)
essential for its activity. Proteases are used throughout
an organism for various metabolic processes. Acid
proteases secreted into the stomach (such as pepsin)
and serine proteases present in duodenum (trypsin
and chymotrypsin) enable us to digest the protein in
food; proteases present in blood serum (thrombin,
plasmin, Hageman factor, etc.) play an important role
in blood-clotting, as well as lysis of the clots, and the
correct action of the immune system. Other proteases
are present in leukocytes (elastase, cathepsin G)
and play several different roles in metabolic control.
Proteases determine the lifetime of other proteins
playing important physiological role like hormones,
antibodies, or other enzymes; this is one of the
fastest “switching on” and “switching off” regulatory
mechanisms in the physiology of an organism. By
complex cooperative action the proteases may
proceed as cascade reactions, which result in rapid
and efficient amplification of an organism’s response
to a physiological signal. Proteases are part of many
laundry detergents5.
Protease Inhibitors
Protease inhibitors are a class of drugs used to
treat or prevent infection by viruses, including HIV
and Hepatitis C. Protease inhibitors prevent viral
replication by inhibiting the activity of proteases,
e.g. HIV-1 protease, enzymes used by the viruses
to cleave nascent proteins for final assembly of new
virions. Protease inhibitors have been developed or
are presently undergoing testing for treating various
viruses: HIV/AIDS antiretroviral protease inhibitors
(saquinavir, ritonavir, indinavir, nelfinavir, amprenavir
etc.)6.
HIV protease inhibitors were first invented
between 1989 and 1994 by researchers working for
the pharmaceutical companies of Hoffmann- La Roche
Inc. (in Nutley, New Jersey), Abbott Laboratories
and Merck & Co., Inc. HIV protease inhibitors are
used in the treatment of patients with AIDS and were
considered the first breakthrough in over a decade
of AIDS research. HIV protease inhibitors can lower
the viral load carried by AIDS patents. There are five
HIV protease inhibitors approved by US FDA for the
treatment of HIV infection. These medications work at
the final stage of viral replication and attempt to prevent
HIV from making new copies of itself by interfering
with the HIV protease enzyme. As a result, the new
copies of HIV are not able to infect new cells7. The
United States Food and Drug Administration (US FDA)
approved the drug invirase in December 1995; and
norvir and crixivan in March, 19968, 9, 10. Invention of
protease inhibitors are summarized in (Table I)11, 12.
HIV PROTEASE
Human immunodeficiency virus (HIV) is a
lentivirus that has two major species13, HIV-1 which
causes the majority of the epidemic, and HIV-2, a
close relative whose distribution is concentrated in
western Africa14. HIV infection was first described
in 1981 in San Francisco and New York City. In
1985, HIV was identified as the causative agent of
acquired immune deficiency syndrome (AIDS) and
its complete genome was immediately available.
This knowledge paved the way for the development
of selective inhibitors15. HIV-2 carries a slightly lower
risk of transmission than HIV-1 and infection tends
to progress more slowly to AIDS. In common usage
HIV usually implies HIV-1. HIV-1 protease is one of
the best known aspartic proteases, and an attractive
target for the treatment of AIDS16.

Table I: Invention of protease inhibitors are summarized in tabulated form as below11, 12

Name Trade name Company Patents Notes

Saquinavir Fortovase, Hoffmann–La U.S. Patent It was the first protease inhibitor approved by the
Invirase Roche 5196438 FDA (December 6, 1995).
Ritonavir Norvir Abbott U.S. Patent
Laboratories 5541206
Indinavir Crixivan Merck & Co. U.S. Patent
5413999
Nelfinavir Viracept Agouron U.S. Patent
Pharmaceuticals 5484926
Amprenavir Agenerase GlaxoSmithKline U.S. Patent The US FDA approved it April 15, 1999, making
5585397 it the sixteenth FDA-approved antiretroviral.
It was the first protease inhibitor approved for
twice-a-day dosing instead of needing to be
taken every eight hours. The convenient dosing
came at a price, as the dose required is 1,200
mg, delivered in eight very large gel capsules.
Production was discontinued by the manufacturer
December 31, 2004 as it has been superseded
by fosamprenavir.
Lopinavir Kaletra Abbott - Is only marketed as a combination, with
ritonavir.
Atazanavir Reyataz Bristol-Myers The FDA approved it on June 20, 2003. Atazanavir
Squibb was the first PI approved for once- daily dosing.
It appears to be less likely to cause lipodystrophy
and elevated cholesterol as side effects. It may
also not be cross-resistant with other PIs.

INDIAN DRUGS 50(02) February 2013 7

 After the discovery of HIV protease, it only took
10 years for its first inhibitor to reach the market17. The
first reports of highly selective antagonists against the
HIV protease were revealed in 1987. Phase I trials
of saquinavir began in 1989 and it was the first HIV
protease inhibitor to be approved for prescription
use in 1995. Four months later, two other protease
inhibitors, ritonavir and indinavir, were approved. In
2009, ten protease inhibitors had reached the market
for treatment against HIV but one protease inhibitor,
amprenavir, was withdrawn from the market in 2004
because it produced resistance and showed side
effects18.

Life cycle of HIV

HIV belongs to the class of viruses called
retroviruses, which carry genetic information in the
form of RNA. The mRNA is then translated into viral
proteins and the third virally encoded enzyme, namely
HIV protease, is required to cleave a viral polyprotein
precursor into individual mature proteins. The viral
RNA and viral proteins assemble at the surface of
the cell into new virions. The virions bud from the cell
and are released to infect other cells. All infected cells
are eventually killed because of this extensive cell
damage, from the destruction of the host’s genetic
system to the budding and release of virions shown
in (Fig. 1) 19, 20.

Mechanism of Action for Protease Inhibitors

There are several steps in the HIV life cycle that
may be interfered with, thus stopping the replication of
the virus. A very critical step is the proteolytic cleavage
of the polypeptide precursors into mature enzymes
and structural proteins catalyzed by HIV protease21.
HIV protease inhibitors are peptide-like chemicals that
competitively inhibit the action of the virus aspartyl
protease. These drugs prevent proteolytic cleavage
of HIV Gag and Pol polyproteins that include essential
structural and enzymatic components of the virus.
This prevents the conversion of HIV particles into their
mature infectious form22, 23. Protease inhibitors can
alter adipocyte metabolism causing lipodystrophy, a
common side effect associated with the use of most
HIV protease inhibitors. Many mechanisms have
Fig. 1: Life Cycle of HIV19, 20
been proposed, for example inhibition of adipocyte
differentiation, triglyceride accumulation and
increased lipolysis. Theories considering the effect
of protease inhibitors on insulin-stimulated glucose
uptake have also been linked to the lipodystrophic
syndrome. It is possible that protease inhibitors
can cause a decrease in insulin-stimulated tyrosine
phosphorylation of IRS-1, representing inhibition of
early steps in insulin signaling. Decreased adiponectin
secretion and induced expression of interleukin-6
associated with HIV protease inhibitors may also
contribute to inhibition of insulin-stimulated glucose
uptake24, 25.

8 INDIAN DRUGS 50(02) February 2013

Design of Protease inhibitors non scissile junction, present in inhibitors other than
those containing the reduced peptide bond isosteres,
Protease inhibitors were designed to mimic the
is positioned between the Asp25/Asp25´ carboxyls
transition state of the protease’s actual substrates. A
of the protease, within hydrogen-bonding distance to
peptide linkage consisting of –NH-CO- is replaced by
at least one carboxylate oxygen of each aspartate. A
hydroxyethylene group (-CH2-CH (OH)-) which the
feature common to almost all complexes of HIV-1 PR
protease is unable to cleave. HIV protease inhibitors fit
is a buried water molecule that bridges the P2 and
the active site of the HIV aspartic protease and were
P10 CO groups of the inhibitor and Ile50 and Ile150
rationally designed utilizing knowledge of the aspartyl
NH groups of the flaps. This water is approximately
protease’s mode of action. The most promising
tetrahedrally coordinated and is completely separated
transition state mimic was hydroxyethylamine, which
from the bulk solvent33. The functional substitution
lead to the discovery of the first protease inhibitor,
of this water has led to the design of urea-based
saquinavir. Following that discovery, other HIV
inhibitors (Fig. 2).
protease inhibitors were designed using the same
principle26, 27, 28.

Co crystals of HIV-1 PR with a variety of inhibitors

have been grown in a number of different crystal
forms. The structures of these complexes have
been used to investigate the binding of substrate-
based modified oligopeptide inhibitors as well as
of the inhibitors of non-peptidic nature. Binding of
an inhibitor introduces substantial conformational
changes to the enzyme. The overall movement of
the subunits can be described as a rotation of up to
about 2± around a hinge axis located in the subunit
sheet interface. This motion, which slightly tightens
the cavity of the active site, is also accompanied by
a very large motion of the flap region as much as
7°A for the tips of the flaps29. However, the enzyme
structure is well conserved among the different Fig. 2: Hydrogen binding between HIV-PIs and molded
complexes, with rms deviations between the C atoms structure32
seldom exceeding 0.6A°. Such differences are well Binding site
within the agreement range for protein structures
refined independently or crystallized in different The HIV protease is a C2-symmetric homodimeric
space groups30. Most of the inhibitors co-crystallized enzyme consisting of two 99 amino acid monomers.
with HIV PR, including all peptidomimetic inhibitors, Each monomer contributes an aspartic acid residue
are bound in the enzyme active site in an extended that is essential for catalysis21. Asp-25 and Asp-25´
conformation so that when they are superimposed is shown in (Fig. 3)34. The HIV protease has the
upon one another, their functional elements align quite sequence Asp-Thr-Gly, which is conserved among
well overall31. The contacts between the main chain other mammalian aspartic protease enzymes. An
of the peptidomimetic inhibitors and the protease extended beta-sheet region on the monomers shows
are almost uniform for all the complexes in (Fig. 2)32. by an arrow in (Fig. 3), known as the flap, constitutes
Following a similar pattern, the hydrogen bonds are in part the substrate binding site with the two aspartyl
made mostly between the main-chain atoms of both residues lying on the bottom of a hydrophobic
the enzyme and the inhibitor. The hydroxyl group at the cavity35, 36, 37. Each flexible flap contains three

INDIAN DRUGS 50(02) February 2013 9

characteristic regions: side chains that extend outward
(Met46, Phe53), hydrophobic chains extending inward
(Ile47, Ile54), and a glycine rich region (Gly48, 49,
51, 52). Ile50 remains at the tip of the turn and when
the enzyme is unliganded a water molecule makes
hydrogen bonds to the backbone of Ile50 on each
monomer shows by an arrow in (Fig. 3).
Fig. 3: schematic diagram showing structure
of a HIV-1 protease.34
HIV proteases catalyze the hydrolysis of peptide
bonds with high sequence selectivity and catalytic
proficiency. The mechanism of the HIV protease
shares many features with the rest of the aspartic
protease family although the full detailed mechanism
of this enzyme is not fully understood38. The water
molecule seems to play a role in the opening and
closing of the flaps as well as increasing the affinity
between enzyme and substrate. The aspartyl
residues are involved in the hydrolysis of the peptide
bonds39. The preferred cleavage site for this enzyme
is the N-terminal side of proline residues, especially
between phenylalanine and proline or tyrosine and
proline40, 41.
Development of Protease Inhibitors
Classification of PIs
1) First generation protease inhibitor therapy
a) Saquinavir
b) Ritonavir
c) Indinavir
d) Nelfinavir of inhibitors. The most marked improvements in
10 INDIAN DRUGS 50(02) February 2013
2) Second-generation protease inhibitor therapy;
boosting of protease inhibitors
a) Amprenavir/fosamprenavir
b) Lopinavir
c) Atazanavir
d) Tipranavir
e) Darunavir
First generation protease inhibitor therapy
Saquinavir
The first approved protease drug. The discovery
and development of the Hoffmann–La Roche
drug Saquinavir, the first inhibitor of HIV-1 PR
to be approved by the US FDA, proved to be a
classic example of serendipity coupled with hard
work. Initial inhibitors created in that study were
peptide derivatives utilizing transition-state mimetic
concepts42, 43. The basic design criterion relied on the
observation that HIV-1 PR, unlike other proteases,
is able to cleave Tyr-Pro or Phe-Pro sequences in
the viral polyprotein. Because the amide bonds of
proline residues are not susceptible to cleavage by
mammalian endopeptidases, the design of HIV-1 PR
inhibitors based on this criterion was expected to bring
potential advantages of higher selectivity. Reduced
amides and hydroxyethylamine isosteres most readily
accommodate the imino acid moiety characteristic
of a Phe-Pro or Tyr- Pro retroviral substrate and,
therefore, were chosen for further studies. As
was shown later, the reduced amide isosteres
were relatively poor inhibitors but, in contrast, the
compounds incorporating the hydroxyethylamine
moiety were very potent and highly selective inhibitors
of HIV PR. A minimum sequence required for potent
inhibition, as well as the unexpected preference for
R stereochemistry at the hydroxyl-bearing carbon,
was determined by enzymatic studies of a series of
related compounds. The minimum length inhibitor
included three residues on the N-terminal side of
the isostere and two residues on the C-terminal side.
Varying the side chains of the residues in all subsites
did not lead to dramatic improvement of the potency
potency were achieved by varying the amino acid Ritonavir (Norvir)
at subsite P10, via replacement of proline by (S, S,
The development of the Abbott drug Ritonavir
S)-decahydroisoquinoline- 3-carbonyl (DIQ). The
shows the shift of the thinking of the designers from
resulting compound (later designated as Ro 31-8959)
the creation of symmetric inhibitors of this inherently
had a Ki value of 0.12 at pH 5.5 against HIV-1 PR and
symmetric enzyme to their ultimate conversion to
an even better inhibition constant against HIV-2 PR
asymmetric compounds. The concept was first tested
(Ki < 0.1). It was also shown to be highly selective,
with the synthesis and characterization of symmetric
causing less than 50% inhibition of the human aspartic
inhibitors of HIV-1 PR49, 50, 51, which were designed to
proteases. The compound was subsequently used
match the C2 symmetry of the homodimeric HIV PR.
for further clinical trials and was finally approved
Although symmetry was not thought to be an absolute
by the US FDA in 1995 under the name Saquinavir
requirement for the design of HIV PR inhibitors, it
(Invirase). This design effort was accompanied by only
was expected to be useful in tightly constraining the
limited structural studies. The first crystal structure
rather rigid ligands52. It was also expected that the
to be solved was of a complex with another peptidic
less peptidic nature of the inhibitors might enhance
inhibitor, Ro 31-8588, which exhibited the expected
their stability in vivo. Such symmetric inhibitors were
S-configuration of the carbon bearing the central
expected to confer higher specificity for retroviral
hydroxyl group44, 45. The subsequent crystallographic
proteases over the related mammalian proteases,
study of Ro 31-8959 showed that, as predicted, this
whose substrate binding sites are less symmetric. A
inhibitor bound in an extended conformation, forming
hypothetical tetrahedral intermediate of the cleaved
a characteristic set of hydrogen bonds with the
peptide divided by the C2 axis of the enzyme was
enzyme46, 47. With the exception of the flap, the two
taken as the template for the development of inhibitors.
fold symmetry of the enzyme was preserved, allowing
This hypothetical axis was placed on the carbonyl ‘O’
the S and S0 subsites to be essentially equivalent. A
atom or between the C-N atoms of the cleaved peptide
comparison of the HIV-1 PR/Ro 31-8959 structure with
bond respectively, and one half of the template was
the structure of HIV-1 PR complexed with a longer
deleted. The C2 operation was then implemented
inhibitor, JG-365, showed that the conformation of
on the remaining template, generating symmetric
the inhibitor in the binding cavity critically depended
inhibitors in either the mono-ol or diol form. The first
on the nature of the P10 residue and on the presence
of these symmetric inhibitors showed good kinetic
or absence of the extension beyond subsite S20 48.
profiles, and the concept of symmetry seemed to
It was also established that short inhibitors preferred
the R-configuration of the central carbon. Moreover, work remarkably well in cell cultures.
the carbonyl ‘O’ atom of the DIQ group was able
to maintain the hydrogen bond between the water
molecules connecting the inhibitor with the flap
regions (Wat 301).

Ritonavir

Indinavir (Crixivan)
The discovery and development of the Merck drug
Indinavir was a long and complicated research project.
Saquinavir Similarly to the approach taken at Roche, the original

INDIAN DRUGS 50(02) February 2013 11

design strategy began with a compound based on the
transition-state mimetic concept. This approach was
successfully implemented in an earlier design of renin
inhibitors, although no approved drugs resulted from
the project. One important structural feature present
in most tight-binding aspartic protease inhibitors is
a critical hydroxyl group that hydrogen bonds to the
carboxyl groups of the catalytically active aspartic
acids. Incorporation of a hydroxyethylene isostere as
a dipeptide mimic resulted in compounds that were
potent and selective inhibitors of HIV PR. The available
structure-activity data indicated that an S-hydroxyl
was the preferred configuration. The constituent
hydroxyethylene isostere was extensively examined
in a series of peptidomimetic inhibitors of different
length and with particular residues occupying subsites
on both sides of the nonscissile isostere. Residues
in these positions were systematically changed
to establish the relationships between particular
modifications and the efficiency of the inhibitors53.
The important questions of bioavailability were also
addressed by these modifications. Some of the useful
directions of synthesis were based on the results of
molecular modeling and calculations54.
Indinavir
Nelfinavir (Viracept)
The pathway of the discovery and development
of Viracept by Agouron Pharmaceuticals is the least
completely described in the literature of the four
US FDA-approved protease inhibitor drugs. Little is
known about the basis of its synthesis, and no crystal
structure of the complex with HIV-1 PR has been
released. This inhibitor is the only compound among
these four that does not utilize a peptidomimetic
concept, although its development pathway may
have utilized some peptidomimetics. Two inhibitors
12 INDIAN DRUGS 50(02) February 2013
developed by Agouron, AG-1002 and AG-1004, had
a statine isostere instead of a normal peptide bond52.
These compounds were peptidomimetic inhibitors
with the flanking amino acid sequences naturally
recognized by HIV-1 PR and were 272 WLODAWER &
VONDRASEK bound to the active site in an extended
conformation. They could form 16 and 18 hydrogen
bonds, respectively, due to the presence of hydrophilic
side chains. Despite the large number of hydrogen
bonds, the inhibitors had relatively low potency, with
binding constants of 0.55 M for AG-1002 and 0.32
¹M for AG-1004. A possible explanation for this low
potency was the absence of a P10 group, as well as
the large free energy required for desolvating of the
hydrophilic side chains53.
Nelfinavir
Second-generation protease inhibitor therapy
Amprenavir/fosamprenavir
Amprenavir reached the market in 199954. It is
an N,N-disubstituded amino-sulfonamide nonpeptide
HIV protease inhibitor and shares some common
features with previous protease inhibitors55. It has
a core similar to that of saquinavir but with different
functional groups on both ends. On one end it has
a tetrahydrofuran carbamate group and on the
other end is an isobutylphenyl sulfonamide with
an added amide. This structure results in fewer
chiral centers, that makes it easier to synthesize
and gives it enhanced aqueous solubility. That in
turn gives better oral bioavailability56. However,
amprenavir was withdrawn from the market in 2004
since fosamprenavir, its prodrug, proved superior
in many aspects.

fosamprenavir
Lopinavir
Lopinavir was marketed in 200057. It was originally
designed to diminish the interactions of the inhibitor
with Val82 of the HIV-1 protease, a residue that is
often mutated in the drug resistant strains of the
virus58. It is a peptidomimetic HIV protease inhibitor
and its core is identical to that of ritonavir. Instead
of the 5-thiazolyl end group in ritonavir, lopinavir has
a phenoxyacetyl group and the 2-isopropylthiazolyl
group in ritonavir was replaced by a modified valine in
which the amino terminal had a six-membered cyclic
urea attached. Fosamprenavir was marketed in 2003,
and is a phosphoester prodrug that is rapidly and
extensively metabolized to amprenavir. The solubility
and bioavailability is better than of amprenavir which
results in reduced daily pill burden59.
Lopinavir
Atazanavir
Atazanavir was marketed in 200360. It is an
azapeptide protease inhibitor designed to fit the C2-
symmetry of the enzyme binding site. Atazanavir
showed better resistant profiles than previous HIV
INDIAN DRUGS 50(02) February 2013 13
protease inhibitors. It is unique among the other
protease inhibitors as it can only be absorbed in an
acidic environment61, 62.
Atazanavir
Tipranavir
Tipranavir is a nonpeptidic HIV-1 protease
inhibitor and reached the market in 2005. Unlike other
HIV protease inhibitors on the market, tipranavir was
developed from a nonpeptidic coumarin template
and its antiprotease activity was discovered by high-
throughput screening. This sulfonamide containing
5, 6-dihydro-4-hydroxy-2-pyrone had emerged
from screenings of 3-substituted coumarins and
dihydropyrones. It possesses broad antiviral activity
against multiple protease inhibitor resistant HIV-163, 64.
Tipranavir
Darunavir
Darunavir reached the market in 200665. It is a
nonpeptidic analogue of amprenavir, with a critical
change at the terminal tetrahydrofuran (THF) group.
Instead of a single THF group, darunavir contains
two THF groups fused in the compound, to form
a bis-THF moiety which makes it more effective
than amprenavir. With this structural change, the
stereochemistry around the bis-THF moiety confers
orientational changes, that allow for continued binding
with the protease which has developed a resistance
for amprenavir66.
Darunavir
Structure-activity relationship
All the HIV protease inhibitors on the market
contain a central core motif consisting of a
hydroxyethylene scaffold, with the only exception
being the central core of tipranavir, which is based on
a coumarin scaffold67. A very important group on the
HIV protease inhibitors is a hydroxyl group on the core
motif which forms a hydrogen bond with the carboxylic
acid on the Asp-25 and Asp-25´ residues in the binding
site shows in (Fig. 4)68, 69. Hydrogen bonds between
the water molecule, which is linked to Ile50 and Ile50’,
and carbonyl groups of the peptidomimetic inhibitors
seem to connect them with the flap regions70. On the
other hand, on the nonpeptidic inhibitors, there is a
proton acceptor which replaces the tetracoordinated
water molecule and interacts directly with the two Ile50
residues on the flap of the enzyme. Specific pockets
in the binding site of the HIV protease, often referred
to as S1,S1’,S2 and S2’, recognize hydrophobic
amino acids on natural substrates shows in (Fig. 4).
The potency of inhibitors bearing hydrophobic groups
complementing these areas is therefore increased71.
Some residues in the enzyme binding site are capable
of forming hydrogen bonds with hydrophilic groups
on the inhibitor, for example with the THF moieties
on amprenavir and darunavir. Since darunavir has a
14 INDIAN DRUGS 50(02) February 2013
bis-THF moiety, instead of a single THF moiety like
on amprenavir, it can form more hydrogen bonds and
increase binding energy72.
Development of Drug Resistance
In common with other retroviral polymerases, HIV
RT is unable to edit transcription errors during nucleic
acid replication, and thus enhances the mutational
rates of the virus73, 74. As one of the results, divergent
viral populations are present during infection and
the sequences of the proteases from these different
strains differ, sometimes substantially. Many of these
differences do not affect the activity of the enzyme,
and the structures of fully active proteases that differ
in a number of positions in their sequences, from
strains such as NY5 or SF2, have been reported.
However, high rates of viral turnover in HIV infection
and the inability of HIV reverse transcriptase to correct
transcriptional errors also mean that populations of
resistant virus will eventually emerge during any
antiviral therapy as a result of drug selection of viral
strains75. It has previously been shown that the clinical
use of RT inhibitors has led to the rapid development
of resistance and cross-resistance against different
RT inhibitors. Similarly, drug-induced mutations
in HIV PR alter the susceptibility of the enzyme to
inhibition by specific inhibitors. A recent survey of the
mutations associated with drug resistance has shown
the modifications of 45 residues in HIV-1 PR, almost
half of the total, for enzyme isolated from samples
obtained with the help of 21 drugs. An obvious
and expected mechanism for the development of
resistance to HIV PR inhibitors is caused by the
changes of specificity-determining residues that can
directly interfere with the binding of the inhibitor to the
enzyme. The mutations that do not directly change
the shape or character of the binding cavity can
indirectly influence inhibitor binding via long range
structural perturbations of the active site, or they can
change the efficiency of catalysis and the stability of
the enzyme. A characterization of resistant variants
isolated from patients undergoing therapy with the
protease inhibitor MK-639 (Indinavir) was published
in 199576.
 Some of these variants exhibited cross-resistance 8. Nephrolithiasis
to all members of a panel of six structurally diverse
9. Photosensitivity
protease inhibitors. This study provided the first
evidence that inhibition of HIV-1 PR can also lead Current status
to the emergence of drug-resistant mutants in vivo
In November 2006 darunavir was still the most
and that combination therapy with multiple protease
recent HIV protease inhibitor78 to reach the market.
inhibitors may not preclude the loss of antiviral activity
In 2006, GlaxoSmithKline discontinued the phase II
resulting from resistance selection. A similar study
using clinical isolates was also reported by Winslow
et al. Five noncontiguous regions of HIV-1 PR, as
described originally by Fontenot et al, were found to
be conserved across all examined isolates. These
regions include residues 1–9 (N terminus), 21–32
(the sequence surrounding the catalytic aspartate
found in the active site), 47–56 (flap region), 78–88
(substrate-binding region), and 94–99 (C terminus
and the dimerization region). The mutational analyses
have shown that diversity is allowed at some positions,
where as mutations in these conserved regions often
result in abnormal gag processing in vitro77.

Application of Protease Inhibitors

1) Tissue culture drug resistance analysis of a novel
HIV-1 protease Inhibitor termed PL-100 in non-B
HIV-1 sub types. Fig. 4: A simplified image of protease inhibitors binding
to the active site of the HIV-168.
2) The new and less toxic protease inhibitor
saquinavir now maintains anti-HIV-1 properties in
vitro indistinguishable from those of the parental
compound saquinavir.
3) Prediction of drug-resistance in HIV-1 subtype C
based on protease sequences from ART naive
and first-line treatment failures in North India
using genotypic and docking analysis.

Adverse effect of Protease Inhibitors

1. Gastrointestinal intolerance
2. Asthenia
3. Headache and dizziness
Fig. 5: Trends in annual rates of death due to 7 leading
4. Limb and facial tingling causes among persons 25-44 years old in the United
5. Numbness and rashes States during period 1987-2011. Dramatic decrease
in the rate of death due to AIDS coincides with the
6. Dyslipidaemia introduction of HIV protease inhibitors (source:
7. Crystalises in urine increase risk of urinary National Vital Statistics, Centers for Disease Control
and Prevention, Atlanta)81.
calculi

INDIAN DRUGS 50(02) February 2013 15

 Table II: Inhibitors currently used in clinical practice81
Summarized data including antiviral activity, side-effects, and pharmaceutical boosting

Generic Commonly Most common side effects Position in the present

name and recommended dosage and off-target activities therapeutic arsenal
abbreviation
Ritonavir 100–200 mg p.o. BID as nausea, diarrhea, practically only
(RTV)a pharmacokinetic booster abdomenalgia, hyperlipidemia, pharmacoenhancing of
of various PIs (original lipodystrophy syndrome, various PIs
pharmacodynamic dose inhibition of the cytochrome
was 600 mg p.o. BID) P450 3A4
Saquinavir 1000 mg + diarrhea, hyperlipidemia, second-line HAART therapy
(SQV) RTV 100 mg p.o. BID lipodystrophy syndrome
Indinavir (IDV) 800 mg + RTV 100 mg nephrolithiasis, lipodystrophy second/third-line HAART
p.o. BID syndrome, hyperlipideamia, therapy in case of resistance
hepatotoxicity or intolerance
Nelfinavir (NFV) 1250 mg p.o. BID diarrhea, hyperlipidemia, second/third-line HAART
lipodystrophy syndrome therapy in case of resistance
or intolerance, approved for
therapy of children
Lopinavir 400 mg + RTV 100 mg diarrhea, hyperlipideamia, first line option for PI based
(LPV/r)b p.o. BID lipodystrophy syndrome HAART regimen
Amprenavir 600 mg + RTV 100 mg diarrhea, toxoallergic rash, replaced by its prodrug
(APV) p.o. BID hyperlipidemia, lipodystrophy fosamprenavir
Fosamprenavir 700 mg p.o. + diarrhea, toxoallergic rash, first-line option for PI
(FPV) RTV 100 mg p.o. BID hyperlipidemia, lipodystrophy based HAART
syndrome regimen
Atazanavir 300 mg + RTV 100 mg hyperbilirubinemia, first-line option for PI
(ATV) p.o. q24h or ECG abnormalities based HAART
400 mg p.o. q24h (1° atrioventricular block) regimen
Tipranavir 500 mg + toxoallergic rash, second-line HAART
(TPV) RTV 200 mg p.o. BID hepatotoxicity, therapy in case of
intracranial hemorrhage, resistance
lipodystrophy syndrome,
diarrhea
Darunavir 600 mg + nausea, diarrhea, recently approved for
(DRV) RTV 100 mg p.o. BID hyperlipidemia, first-line HAART
or 800 mg + RTV 100 headache, toxoallergic rash
mg p.o. q24h

Abbreviations: BID - twice per day, q24h - every 24 hours

a
Ritonavir is at present used in therapy of HIV infection practically only as a pharmacokinetic booster.
b
Lopinavir is marketed by the manufacturer only as (Kaletra), in co-formulation together with low doses of
ritonavir which acts as a pharmacokinetic booster.

16 INDIAN DRUGS 50(02) February 2013

clinical development of brecanavir, an investigational
protease inhibitor for the treatment of HIV, due to
insurmountable issues regarding formulation. In
the summer of 2009, GlaxoSmithKline and Concert
Pharmaceuticals announced their collaboration to
develop and commercialize deuterium-containing
medicines79. One of them is CTP-518, a protease
inhibitor for the treatment of HIV, expected to
enter phase I clinical trials in the second half of
2009. CTP-518 is a novel HIV protease inhibitor
developed by replacing certain key hydrogen atoms
of atazanavir with deuterium80. Pre-clinical studies
have demonstrated that this modification fully retains
the antiviral potency but can evidently slow hepatic
metabolism and thereby increase the half life and
plasma trough levels. CTP-518, therefore, has the
potential to be the first HIV protease inhibitor to
eliminate the need to co-dose with a boosting agent,
such as ritonavir. Protease Inhibitors currently used in
clinical practice are shows in tabulated form in Table
II. Also due to the use of protease inhibitors it was
observed that dramatic decrease in the rate of death
due to AIDS occurs, as shown in Fig. 581.
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 original research articles

METHOD DEVELOPMENT AND VALIDATION OF OLANZAPINE IN PURE AND

PHARMACEUTICAL DOSAGE FORM BY RP-HPLC METHOD
Mastiholimath V.S.*, Dandagi P.M., Gadad A.P., Murali Krishna N.V. and Mannur V.

(Received 11 April 2012) (Accepted 23 January 2013)

Abstract

A simple and reliable reverse phase high-performance liquid chromatography method was developed and
validated for Olanzapine in pure and pharmaceutical dosage form. The method was developed on BDS
Hypersil C18, (150 mm x 4.6 mm, 3μm) with a mobile phase of 0.01M tetra butyl ammonium hydrogen
sulphate : methanol (80:20 v/v). The effluent was monitored by SPD-M20A, prominence UV-VIS detector
at 234 nm. Calibration curve was linear over the concentration range of 10 –60μg/ml. For inter–day and
intra–day precision % relative standard deviation values were found to be 0.18% and 0.24% respectively.
Recovery of olanzapine was found to be in the range of 99.93 -100.00%. The limit of detection (LOD)
and quantitation (LOQ) were 0.39275 and 1.1901μg/ml, respectively. The retention time and run time
was very short; hence it is cost effective, making it more economical and rapid. Also this method can be
used for the analysis of large number of samples.

Keywords: Olanzapine, RP-HPLC, Validation, estimation of olanzapine which includes HPLC3-9,

Pharmaceutical dosage form.
INTRODUCTION
Olanzapine1, 2 is official in Indian Pharmacopoeia.
It is chemically known as 2-methyl-4-(4-methyl-1-
piperazinyl)-10Hthieno [2,3-b][1,5]benzodiazepine
(Fig.1). The molecular formula is C 17 H 20 N 4 S
and molecular weight is 312.4. It is used as an
antipsychotic drug due to antagonism at dopamine
and drugs serotonin type 2 receptors, with greater
activity at serotonin 5-HT2 receptors than at dopamine
type 2 receptors. Antagonism at muscarinic receptors,
H1-receptors, and alpha (1)-receptors also occurs
with Olanzapine.
stability indicating method10, HPTLC11-13, HPLC-
MS14,15 and spectroscopic method16-20. The present
paper describes a new quantitative reversed-phase
high-performance liquid chromatographic method,
coupled with Shimadzu Prominence UV-VIS detector,
as an alternative technique for quality control of OLN
products. The purpose of this investigation was to
develop and validate a method using a simple, rapid,
sensitive, precise, accurate and specific reversed
phase HPLC assay.

Extensive literature survey reveals that several

analytical methods have been reported for the

*For correspondence
Department of Quality Assurance
KLE University’s College of Pharmacy
Belgaum-590 010, Karnataka
E-mail: mastiholimath@rediffmail.com Fig. 1: Chemical structure of Olanzapine

20 INDIAN DRUGS 50(02) February 2013

MATERIALS AND METHODS
Chemicals and solvents
The reference sample of olanzapine was supplied
by Chandra Labs Pvt Ltd., Hyderabad. HPLC grade
water and methanol were purchased from E. Merck
(India) Ltd., Mumbai. Tetra butyl ammonium hydrogen
sulphate of AR Grade was obtained from S.D. Fine
Chemicals Ltd., Mumbai. Tablet formulations of
Oleanz® were procured from a local pharmacy with
labeled amount of 5 mg per tablet.
Instrument
The HPLC system is of Shimadzu, LC-20AT,
a SPD-M20A prominence UV-VIS detector,
HPLC Pump LC-20AT pump and Injector loop
rheodyne, model No. 2767, 20 µl volume loop.
Data acquisition was performed by the Spinchrom
software.
Chromatography condition
Chromatographic analysis was performed on
a BDS Hypersil reversed phase C-18 column with
150 x 4.6mm internal diameter and 3μm particle
size. The mobile phase consisted of 0.01M tetra
butyl ammonium hydrogen sulphate : methanol
(80: 20 v/v) and that was set at a flow rate of 1 ml/
min. The mobile phase was degassed and filtered
through 0.45 μ filter under vacuum before pumping
into HPLC system. The effluent was monitored by
UV detection at 234 nm.
Preparation of 0.01M tetra butyl ammonium
hydrogen sulphate
Dissolve 0.33954 g of tetra butyl ammonium
hydrogen sulphate in small amount of distilled water
transferred in to 100 mL volumetric flask and make
up to final volume with water.
Preparation of mobile phase and diluents
800 mL of the 0.01M tetra butyl ammonium
hydrogen sulphate was mixed with 200 mL of
methanol. The solution was degassed in an ultrasonic
water bath for 5 minutes and filtered through 0.45 μ
filter under vacuum.
INDIAN DRUGS 50(02) February 2013 21
RESULTS
Method development and optimization
A mixture of 0.01M tetra butyl ammonium hydrogen
sulphate and methanol in the ratio of 80:20v/v was
found to be the most suitable mobile phase for ideal
separation of olanzapine. The solvent mixture was
filtered through a 0.45 μ membrane filter and sonicated
before use. It was pumped through the column at a
flow rate of 1ml/min. The column was maintained at
ambient temperature. The pump pressure was set at
800 psi. The column was equilibrated by pumping the
mobile phase through the column for at least 30 min
prior to the injection of the drug solution. The detection
of the drug was monitored at 234 nm. The run time was
set at 6 min. Under these optimized chromatographic
conditions the retention time obtained for the drug
was 2.61 min. A typical chromatogram showing the
separation of the drug is given in Fig. 2.
Validation procedures
The method validation was carried out according
to the recommendations for analytical method
validation21,22.
System suitability
System-suitability tests are an integral part
of method development and are used to ensure
adequate performance of the chromatographic
system. Retention time (tR), number of theoretical
plates (N) and tailing factor (T) were evaluated for
six replicate injections of the drug at a concentration
of 50 μg/mL. The results which are given in Table I
were within acceptable limits.
Fig. 2: Chromatogram of Olanzapine at 234 nm
Linearity
Several aliquots of standard stock solution(1mg/
ml) (0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 ml) of olanzapine
drug were taken in different six standard 10.0 mL
volumetric flasks and diluted up to mark with mobile
phase. Evaluation was performed with UV detector
at 234 nm. The peak area was recorded for all the
peaks and calibration graph was obtained by plotting
peak area versus concentration of olanzapine.
The plot of peak area against concentration of Concentration µg/mL
olanzapine was found to be linear in the range
of 10 to 60μg/mL with correlation coefficient of Fig. 3: Calibration curve of Olanzapine
0.9996. The calibration data of olanzapine is given
Inter-day precision
in Table II and the calibration curve of olanzapine
is shown in Fig. 3. In the inter-day variation studies, six injections of
standard solution (50μg/mL) were injected at different
Accuracy days. % RSD was calculated and is presented in
Accuracy was performed in triplicate after spiking Table V.
pure drug equivalent to 80, 100, and 120% of the Table I: Results from system suitability studies
standard concentration of olanzapine (50 μg/mL). The
results obtained (Table III) indicate that recovery was Required
Property Values ±SD* %RSD
limits
excellent, not less than 100% ± 2. The results indicate
the method is highly accurate for determination of Retention
2.55±0.0066 0.26 RSD ≤ 1%
the olanzapine. time (tR)

Sensitivity Theoretical
2672±14.88 0.56 N > 2000
plates (N)
Limit of detection (LOD) and limit of quantitation
(LOQ) were determined from standard deviation and Tailing
1.568±0.0223 1.421 T≤2
factor(T)
slope method as per ICH guideline, for olanzapine
LOD was found to be 0.39275μg/mL and LOQ was *Average of six determinations.
found to be 1.1μg/mL.
Table II: Calibration data of Olanzapine by RP-
Precision HPLC method
The precision of the method was demonstrated S.No Concentration Peak Area
by intra-day and inter-day variation studies. (μg/mL) (m V.s)
Intra-day precision 1 10 729.418

In the intra-day studies, six injections of 2 20 1419.661

standard solution (50μg/mL) were injected into the 3 30 2205.335
chromatographic system in different time interval
4 40 2949.615
within a day. %RSD was calculated and is presented
in Table IV. 5 50 3636.578
6 60 4314.348

22 INDIAN DRUGS 50(02) February 2013

 Table III: Results from recovery studies

Sample Area Initial Amount Amount %Recovery ± SD* %RSD

(m V.s) amount added recovered*
(μg/mL) (μg/mL) (μg/mL)
80% 3329.88 5 40 44.97 99.93 ±1.98 0.06
100% 4072.52 5 50 55 100.00 ±3.97 0.097
120% 4811.37 5 60 64.98 99.97±3.93 0.081
*Average of three determinations.

Table IV: Intra-day precision results for Olanzapine

S. No Concentration (μg/mL) Retention time (min) Area (m V.s)

1 50 2.563 3713.551
2 50 2.550 3699.609
3 50 2.567 3696.849
4 50 2.563 3700.417
5 50 2.567 3703.568
6 50 2.54 3710.65
AVG 2.5583 3704.10
SD 0.0109 6.6153
%RSD 0.43 0.18

Table V: Inter-day precision results for Olanzapine

S. No Concentration (μg/mL) Retention time (min) Area (m V.s)

1 50 2.567 3674.252
2 50 2.557 3649.501
3 50 2.567 3664.827
4 50 2.553 3657.62
5 50 2.553 3668.155
6 50 2.555 3665.12
AVG 2.558667 3663.2458
SD 0.006623 8.6234408
%RSD 0.26 0.24

Robustness rate. It was observed that there were no marked

Robustness of the method was determined changes in the chromatograms, which demonstrated
by making slight changes in the chromatographic that the RP-HPLC method developed is robust. The
conditions, such as change in wavelength and flow results are shown in Table VI.

INDIAN DRUGS 50(02) February 2013 23

 Table VI: Robustness studies of Olanzapine by RP-HPLC method

Mean % RSD
S.No Condition Modification Mean Peak area ± SD Mean Rt ± SD*
(for AREA)

1 Flow rate 0.9 4195.7726±15.06 2.813±0.015 0.359

(ml/min)
1.1 3232.094±29.694 2.313±0.0057 0.249

2 Wavelength 232 4125.60567±44.5 2.81±0.01 1.079

(nm) 236 4323.51±8.727 2.54±0.0057 0.201

*Average of three determinations.

Table VII: Ruggedness studies of Olanzapine by RP-HPLC method

Injection
S. No Analyst-1 Analyst-2
Number
Area Retention Theoretical Area Retention Theoretical
(m V.s) time (min) Plates(N) (m V.s) time (min) Plates(N)
1 1 3780.498 2.55 2647 3786.926 2.537 2619
2 2 3767.652 2.56 2633 3783.783 2.561 2625
AVG 3774.075 2.555 2640 3785.355 2.549 2622
SD 9.083 0.007 9.899 2.222 0.0169 4.242
%RSD 0.240 0.276 0.374 0.058 0.6657 0.161

Table VIII: Assay studies of Olanzapine by RP-HPLC method

Label claim Standard Area* Sample Area* Amount found (%)Recovery

Sample
(mg) (m V.s) (m V.s) (mg)

Oleanz® 5 3789.1933 3665 4.99 99.73

*Average of three determinations

Ruggedness to the mark with the same solvent (Stock solution).

It was checked by determining precision on the
same instrument, but by a different analyst. Results
of reproducibility are shown in Table VII.
Assay
Preparation of Standard Solution
Accurately weigh and transfer 25mg of
Olanzapine working standard into a 25 mL volumetric
flask, add about 15 mL of mobile phase and sonicate
to dissolve it completely and make up the volume
Further pipette out 0.5 mL of the above stock solution
into a 10 mL volumetric flask and dilute up to the mark
with the same solvent to obtain the concentration of
50 μg/mL. Mix well and filter through Whatman filter
paper (No. 41).
Preparation of Sample Solution
Weigh 5 Olanzapine Tablets and calculate the
average weight. Accurately weigh and transfer the
sample equivalent to 5 mg of Olanzapine into a 10

24 INDIAN DRUGS 50(02) February 2013

 Table IX: Characteristic parameters of
Olanzapine for the RP-HPLC method
Parameters RP-HPLC
Calibration range (µg / ml) 10-60
Detection wavelength 234
Mobile phase(Buffer : methanol)
80:20
(V/V)
Regression equation (Y) 72.455x+5.6381
RT 2.61
Slope (b) 72.455
Intercept (a) 5.6381
Correlation coefficient(r )
2
0.9996
LOD (µg / ml) 0.3927
LOQ (µg / ml) 1.1901
mL volumetric flask. Add about 7 mL of diluent and
sonicate it to dissolve completely and make up the
volume up to the mark with diluent. Mix well and filter
through Whatman filter paper (No.41). Further pipette
out 1 mL of the above stock solution into a 10mL
volumetric flask and dilute up to the mark with the
same solvent. Mix well and filter through Whatman
filter paper (No.41).The results of assay are shown
in Table VIII.
DISCUSSION
A rapid and simple RP-HPLC method for
determination of olanzapine has been developed and
validated and the results are shown in Table IX. The
RP-HPLC method involved a mobile phase consisting
of 0.01M tetra butyl ammonium hydrogen sulphate:
methanol in the ratio of 80:20% (V/V) accomplished at
234 nm. The retention time was 2.61 min at a flow-rate
of 1.0 ml/min and the injection volume was 5 μl. This
chromatographic assay fulfilled all the requirements
to be identified as a reliable and feasible method,
including linearity, accuracy, sensitivity, precision,
ruggedness and robustness. The chromatographic
system allows the analysis of a large number of
samples in a short period of time. Therefore, the
method is suitable for analysis of large samples during
routine analysis of formulations and raw materials.
INDIAN DRUGS 50(02) February 2013 25
ACKNOWLEDGEMENT
The authors wish to thank Chandra Labs Ltd,
Hyderabad for providing the necessary facilities to
carry out work.
REFERENCES
1. Indian Pharmacopoeia. Ghaziabad: The Indian
pharmacopoeia commission, 2007, 3, 857-858.
2. http://en.wikipedia.org/wiki/Olanzapine
3. Patel S, Patel N. J.: Simultaneous RP-HPLC and HPTLC
Estimation of Fluoxetine Hydrochloride and Olanzapine
in Tablet Dosage Forms, Indian J Pharm Sci.  2009,
71(4), 477–480.
4. Eswarudi M.M., Sushma M, Sushmitha M, Yamini K.:
Validated spectroscopic method for the determination of
salbutamol sulphate in bulk and pharmaceutical dosage
forms, Int Res. J.Pharma. 2012, 3(4), 310-313.
5. Rao R.A., Delhi raj N, Jagadeesh.: RP-HPLC method
for quantitative estimation of olanzapine in tablet dosage
forms, Intl J. Pharma. Bio. Sci. 2012, 3(3), 267-272.
6. Karpinska J, Sokół Y, Bernatowicz A , Szulecka A, Kotowska
U.: Studies on photodegradation of levomepromazine and
olanzapine under simulated environmental conditions,
Photochem. Photobiol. Sci., 2012, 11, 1575-1584.
7. Rani P, Sekaran B.: Development of HPLC method for
the determination of Olanzapine in bulk and dosage forms,
Inter J of Pharmtech research. 2009, 1(3), 654-657.
8. Basavaiah K, Rangachar U.A., Tharpa K.: Quantitative
Determination of Olanzapine in Pharmaceutical
Preparations by HPLC, J. Mex. Chem. Soc. 2008,
52( 2), 120-124.
9. Goyal S, Sharma D, Srinivas S.K.: Simultaneous
estimation of risperidone, olanzapine and quetiapine
and their degradation products by HPLC, Acta
Pharmaceutica Sciencia. 2010, 52, 345-352.
10. Krishnaiah C.H., Murthy M.V., Kumar R.: Development
of a stability-indicating UPLC method for determining
Olanzapine and its associated degradation products
present in active pharmaceutical ingredients and
pharmaceutical dosage forms, J of Pharm and Biomed
Anal. 2011, 54, 667-673.
11. Patel R.B., Patel M.R..: Development and validation of
an HPTLC method for determination of olanzapine in
formulations, JAOAC int. 2010, 3, 811-819.
12. Shah C.R., Shah N.J., Patel D.R.: Stability-indicating
simultaneous HPTLC method for olanzapine and
fluoxetine in combined tablet dosage form, 2008, 70(2),
251-255.
13. Raggi M.A., Sabbioni C, Mandrioli R, Albers L.: Rapid
analysis of Olanzapine and Desmethyl Olanzapine
in human plasma using high-performance liquid
chromatography with coulometric detection, J of
Analytica Chemica Acta. 2004, 51, 111-117.
14. Nirogi R.V.S., Kandikere V.N., Mauryaa S.: Development
and validation of a sensitive liquid chromatography/
electrospray tandem mass spectrometry assay for the
quantification of Olanzapine in human plasma, J of
Pharm and Biomed Anal. 2006, 41, 935-942.
15. Josefsson M, Roman M, Skogh E.: Liquid
chromatography/tandem mass spectrometry method for
determination of Olanzapine and N-desmethylOlanzapine
in human serum and cerebrospinal fluid, J of Pharm
and Biomed Anal. 2010, 53, 576-582.
16. Gopinath S, Kumar R.S., Alexander S, Danabal P.:
Development of a rapid and sensitive SPE-LC-MS/MS
method for the simultaneous estimation of fluoxetine
and olanzapine in human plasma, Biomed chromatogr.
2011.
17. Rubesh S, Kiran C.H., Duganath N.: Simultaneous
Estimation of Fluoxetine HCl and Olanzapine in Bulk Drug
and Pharmaceutical Formulation by Using UV-Visible
Spectroscopy Method, Intern. J. of Pharma. Sci. and
Drug Research. 2011, 3(1), 52-55.
18. Basavaiah K, Tharpa K, Vinay K.B.: Spectrophotometric
Determination of Antipsychotic Drug Olanzapine in
Pharmaceuticals, Jordan Journal of Chemistry. 2009,
4(1), 65-76.
19. Basavaiah K, Rajendraprasad N.: Determination
of Olanzapine by spectrophotometry using
Permanganate, Brazillian J of Pharm Sci. 2009,
45(3), 540-550.
20. ICH: Q2B, Analytical validation-methodology; 1996.
21. ICH: Q2A, Text on validation of analytical procedure;
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22. ICH: Q2 (R1), Validation of analytical procedures:text
and methodology; 2005.

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26 INDIAN DRUGS 50(02) February 2013

 QUANTITATIVE DETERMINATION OF DEFERASIROX IN BULK AND
PHARMACEUTICAL FORMULATION BY UV SPECTROPHOTOMETRIC METHOD
Marathe G. M., Pande V. V.*, Patil P. H., Mutha R. E. and Bari S. B.

(Received 25 October 2012) (Accepted 23 January 2013)

ABSTRACT

A simple, fast and reliable zero order spectrophotometric method was developed for determination of
deferasirox in bulk and pharmaceutical dosage forms. Beer’s law was obeyed in concentration range of
2–12 µ/ml deferasirox at 245.6 nm wavelength. The correlation coefficient was found to be (r2 = 0.999),
precision (repeatability % RSD 1.29), percentage recovery 100.054±0.271. The detection limit (DL) and
quantitation limit (QL) were 0.247 µg/ml and 0.75 µg/ml respectively. The proposed method was found
to be simple, accurate, precise, reproducible and gave an acceptable recovery of the analyte, which could
be directly and easily applied to analysis of bulk and pharmaceutical tablet formulations of deferasirox.

Keywords: Deferasirox, UV spectrophotometry, who are receiving long term blood transfusions
Area under curve for conditions such as beta-thalassemia and other
chronic anemias.
INTRODUCTION
Literature survey revealed terbium-sensitized
Deferasirox is 4-[3, 5-bis-(2-hydroxyphenyl)-[1, fluorescence method for the determination of deferasirox
2, 4]-triazol-1-yl]benzoic acid, molecular formula in biological fluids and tablet formulations3. It also
C21H15N3O4 molecular weight: 373.4, deferasirox) revealed method development using UV spectroscopy4-5.
(Fig. 1) is an orally active iron chelating agent. RP-HPLC methods have been developed for the
Deferasirox is a white to slightly yellow powder and estimation of Deferasirox6-8. The aim of this study
is a non-chiral compound. At the physiological pH of is to develop a simple, sensitive and validated, UV
the intestine, the solubility is about 40 mg/L. In an spectrophotometric method for the determination of
iron balance metabolic study in iron overloaded adult deferasirox as per ICH guidelines9-11.
thalassaemic patients, deferasirox reduced chronic
iron overload in adult and paediatric patients (aged
2 years and older) due to blood transfusions. The
underlying conditions requiring transfusion include
beta-thalassaemia, sickle cell disease, and other
congenital and acquired anaemias (myelodysplastic
syndromes, Diamond-Blackfan syndrome, aplastic
anaemia and other very rare anaemias). Deferasirox
(ICL670) is a novel once-daily, orally administered
iron chelator to treat chronic iron overload in patients
with transfusion-dependent anemias1-2. Its main
use is to reduce chronic iron overload in patients

*For correspondence
H.R. Patel Institute of Pharmaceutical Education &
Research,
Karwand Naka, Shirpur, Dist. Dhule (M.S.) 425405
E-mail: drvishalpande@gmail.com Fig. 1: Structure of Deferasirox

INDIAN DRUGS 50(02) February 2013 27

EXPERIMENTAL
Instrument
Shimadzu UV-Visible double beam
Spectrophotometer (model-1700) was used for
spectral studies and validation.
Reagents and solutions
Methanol (HPLC grade) was procured from
Merck Fine Chemicals (Mumbai, India), the pure
drug deferasirox was obtained as a gift sample from
Hetero Drugs Limited, Hyderabad.
Standard solution
Stock Standard Solution: Accurately weighed
quantity of deferasirox (10 mg) was dissolved in 10
mL of methanol to get 1000 µg/mL, from that 1mL
diluted upto 10 mL with methanol (concentration
100 µg/mL).
Preparation of working standard solution:
12µg /mL of deferasirox solution was prepared by
diluting 1.2 mL of stock solution with water in 10 mL
volumetric flask up to the mark. Working standard
solution of deferasirox was scanned between
200-400 nm on Shimadzu double beam UV visible
spectrophotometer. The wavelength maximum
exhibited for deferasirox was at 245.6 nm (Fig. 2).
Fig. 2: Spectrum of Deferasirox
28 INDIAN DRUGS 50(02) February 2013
Construction of calibration curve
Aliquot of the standard stock solution (0.2, 0.4,
0.6, 0.8, 1.0, 1.2 mL) was transferred into a series of
volumetric flasks (10 mL) and volume was adjusted
up to the mark with water to get desired concentration
(2 – 12 µg/mL). The absorbance of the prepared
solutions was measured at 245.6 nm
Assay
The assay of the proposed method was
ascertained by performing assay of the standard drug
with reference to the sample drug and finding out the
absorbance. From the absorbance percentage purity
was calculated (Table I).
Validation
Linearity: To establish linearity of the proposed
methods, separate series of solutions of deferasirox
(2-12 µg/mL) in water were prepared from the stock
solutions and analyzed. Least square regression
analysis was performed on the obtained data. The
absorbance of the prepared solutions was measured
at 245.6 nm (Fig. 3 and Table II, III).
Precision
Repeatability: Repeatability expresses the
precision under the same operating conditions over
a short interval of time. Six determinations of same
concentration were performed. Percentage RSD was
found to 1.2 (Table IV).
Conc (µg/mL)
Fig. 3: Linearity of Deferasirox
 Table I: Assay of Deferasirox

Formulation Claim of tablet (mg/tablet) % Label claim [n=3] % RSD

Desirox ® 250mg 99.49667 0.446356

Table II: Linearity of Deferasirox by Zero Order Spectroscopic Method

Sr. No. Conc. (µg/mL) Absorbance % RSD

1 2 0.111 1.37
2 4 0.234 0.65
3 6 0.349 0.72
4 8 0.469 0.56
5 10 0.591 0.33
6 12 0.715 0.42

Table III: Results of Linearity Study by Zero Order Spectroscopic Method

Beer’s law limit Correlation Regression

Slope (m) Y-Intercept (c)
(µg/mL) coefficient (R2) equation (y)
2-12 0.999 y=0.060x- 0.009 0.060 0.009

Table IV: Repeatability of Deferasirox by Zero Order Spectroscopic Method

Concentration (µg/mL) Mean of absorbance (n= 6) S.D % R.S.D

6 0.3475 0.0045 1.29

Table V: Results of Intra-Day and Inter-Day precision Study by Zero Order Spectroscopic Method

Concentration Intra-day [n=3] Inter-day [n=3]

% RSD % RSD
[µg/mL] Amount found [µg/mL] Amount found [µg/mL]
6 5.97 0.422 5.97 0.421
8 7.98 0.433 8.02 0.826
10 10.00 0.237 10.01 0.502

Table VI: Accuracy by Zero Order Spectroscopic Method

Percentage Percentage recovery [n=3] % RSD Mean ± SD

80% 100.3667 0.943869
100% 99.91667 0.641985 100.054±0.271
120% 99.88 0.820558

INDIAN DRUGS 50(02) February 2013 29

Table VII: Summary of validation parameter by
Zero Order Spectroscopic Method

Parameter Result

Beer’s law limit (µg/mL) 2-12

Regression equation (y) y=0.060x- 0.009

Correlation coefficient (R2) 0.999

Repeatability (% R.S.D) 1.29

Accuracy (Mean±SD) 100.054±0.271

Assay (Mean± R.S.D) 99.49667±0.446

DL and QL (µg/mL) 0.247 and 0.75

Fig. 5: Second order derivative of Deferasirox

Fig. 4: First order derivative of Deferasirox Fig. 6: Area under curve of Deferasirox

Intra-day and inter-day precision Accuracy

Intra-day and inter-day precision were determined
by analyzing three different solutions of deferasirox
within the same day and three different days over
a period of one week. Intra-day precision was
estimated by analyzing 6 µg/mL, 8 µg/mL, 12 µg/mL
for three times within same day. Inter-day precision
was estimated by analyzing above mentioned
concentration of deferasirox for three different days
over a period of one week (Table V).
Standard addition and recovery experiments were
conducted to determine the accuracy of the proposed
method (Table VI).
Detection limit (DL) and quantitation limit (QL)
The DL and QL of deferasirox by the proposed
methods were determined using calibration standards.
DL and QL were calculated as 3.3r/S and 10r/S,
respectively, where S is the slope of the calibration

30 INDIAN DRUGS 50(02) February 2013

curve and r is the standard deviation of y-intercept of
regression equation. DL and QL of deferasirox was
found to be 0.247 and 0.75 respectively.
First-Derivative spectrometry
The entire above obtained zero-order spectrums
were transformed to get first-order derivative spectra
and the minima values of the derivative spectra were
recorded. First order derivative spectrum (D1) of (12 µg/
mL) at delta lambda 2 with scaling factor 1 (Fig. 4).
Second order-Derivative spectrometry
The above obtained zero-order spectra were
transformed to get second-order derivative spectra
and the minima values of the derivative spectra were
recorded. First order derivative spectrum (D2) of
(12 µg/mL) at delta lambda 4 with scaling factor 1 is
shown in Fig. 5.
Area Under Curve
Area Under Curve (AUC) method involves the
calculation of integrated value of absorbance with
respect to the wavelength between two selected
wavelength 240.6nm and 250.6 nm. Area calculation
processing item calculates the area bound by the
curve and the horizontal axis. The horizontal axis is
selected by entering the wavelength range over which
the area has to be calculated. The wavelength range
is selected on the basis of repeated observations so
as to get the linearity between area under curve and
concentration. Area bound by the curve was 0.190
units (Fig. 6).
RESULTS AND DISCUSSION
A simple, fast and reliable zero order
spectrophotometric method was developed
for determination of deferasirox in bulk and
pharmaceutical dosage forms. Beer’s law was obeyed
in concentration range of 2–12 µg/mL deferasirox at
245.6 nm wavelength. The correlation coefficient was
found to be (r2 = 0.999), precision (repeatability%
RSD 1.29), percentage recovery 100.054±0.271.
The detection limit (DL) and quantitation limit (QL)
were 0.247µg/mL and 0.75 µg/mL, respectively. The
INDIAN DRUGS 50(02) February 2013 31
proposed method was found to simple, accurate,
precise, reproducible and gave an acceptable
recovery of the analyte, which can be directly and
easily applied to analysis of bulk and pharmaceutical
tablet formulations of deferasirox. The summary of
validation parameters by zero order spectroscopic
method is mentioned in Table VII.
CONCLUSION
The method described here is direct method for
the analysis of deferasirox without any extraction
process to eliminate the excipients; it does not use
time consuming procedure such as standard addition
method and also any expensive equipment. Due
to the results of the present study the developed
spectrophotometric method is accurate, sensitive,
precise, reproducible and can be easily and directly
applied to the bulk pharmaceutical preparations.
Additionally, the short analysis time and low costs are
the other advantages of these methods for routine
analysis. Its advantages over other existing methods
are its simplicity, speed and low cost.
ACKNOWLEDGMENT
The authors are thankful to Hetero limited,
India for providing gift sample of deferasirox and
Department of Pharmaceutical Analysis, H. R. Patel
Institute of Pharmaceutical Education and Research,
Shirpur, Dist. Dhule for support.
REFERENCES
1. Waldmeier F., Bruin G.J., Glaenzel U., Hazell K., Sechaud
R., Warrington S., Porter J.B.: Pharmacokinetics,
metabolism, and disposition of deferasirox in beta-
thalassemic patients with transfusion-dependent iron
overload who are at pharmacokinetic steady state, Drug
Metab Dispos. 2010, 38(5), 808-816.
2. Piga A., Galanello R., Forni G.L., Cappellini M.D., Origa R.,
Zappu A., Donato G., Bordone E., Lavagetto A., Zanaboni
L., Sechaud R., Hewson N., Ford J.M, Opitz H., Alberti
D.:Randomized phase II trial of deferasirox Exjade®,
ICL670), a once-daily, orally-administered iron chelator,
in comparison to deferoxamine in thalassemia patients
with transfusional iron overload, Haematologica. 2006,
91(7), 873-880.
3. Manzoori J.L., Abolghasem J., Mohammad A; Vahid P.,
Elnaz T, Jalil V., Terbium-sensitized fluorescence method
 for the determination of deferasirox in biological fluids
and tablet formulation. Luminescence. 2011, 26(4),
244-250.
4. Ananda kumar K., Ramu C., Subhash V., Jayamariappan
M.:Development and validation of newer analytical
methods for the estimation of deferasirox in bulk and in
tablet dosage form by UV spectroscopy and RP-HPLC,
J Pharm Res.2011, 4(9), 2998-3000.
5. Sambasivarao V., Ashok kumar G.: Spectrophotometric
determination of deferasirox in formulations using Folin-
ciocalteu and ferric chloride reagents, IJRRPAS. 2011,
1(2), 62-71.
6. Chakravarthy V.K., GowriSankar D.: LC Determination
of Deferasirox in Pharmaceutical Formulation J Global
Trends PharmaSci.2010, 1(1), 42-52.
7. Suman A., Naveen Babu K., RatnaPrasad G., Ramana
M.V.:Development and validation of LC method for the
estimation of deferasirox in pharmaceutical dosage
form, AJPI’s J of Analytical Chem.2011, 1(6),
9-15.
8. Chauzit E., Bouchet S., Micheau M., Mahon F.X., Moore
N., Titier K., Molimard M.: A method to measure deferasirox
in plasma using HPLC coupled with MS/MS detection and
its potential application, Ther Drug Monit. 2010, 32(4),
476-481.
9. Beckett A.H., Stenlake J.B.:Practical Pharmaceutical
Chemistry. CBS Publishers and Distributors New Delhi,
1986.
10. ICH Harmonized tripartite guidelines, Text on Validation of
Analytical Procedures Q2A, (ICH, Geneva, Switzerland,
1994).
11. ICH Harmonized tripartite guidelines, Validation of
Analytical Procedures: MethodologyQ2B, (ICH, Geneva,
Switzerland, 1996).

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32 INDIAN DRUGS 50(02) February 2013

 Preclinical evaluation of nootropic activity of Glabridin Rich
Extract of Glycyrrhiza glabra using Passive Avoidance Paradigm
in rats
Desai S. K.*, Pandey C. H. and Mulgaonkar S.M.
(Received 18 September 2012) (Accepted 23 January 2013)

ABSTRACT

The study was aimed at establishing the nootropic potential of Glycyrrhiza glabra using appropriate
experimental model. Nootropic activity evaluation of Glabridin Rich Extract (GRE) of roots and stems of
Glycyrrhiza glabra was carried out in rats using passive avoidance paradigm. In this model, scopolamine
induced imbalance in the level of biochemical parameters like increase of lipid peroxidation and
acetylcholinesterase levels and decrease of superoxide dismutase, reduced glutathione and dopamine
levels leading to impairment in cognitive functions were normalized following administration of GRE. An
enhancement in step down latency was also seen in scopolamine induced memory impairment following
GRE treatment. The mechanism of such protection of Glycyrrhiza glabra may be due to reduction in
anticholinesterase levels, monoamino oxidase inhibition and augmentation of cellular antioxidants. Our
data suggests that Glycyrrhiza glabra is a screened candidate that deserves to be investigated further
as an herbal alternative for treatment of Alzheimer’s disease (AD).

Keywords: Glycyrrhiza glabra, Glabridin Rich Extract,
Passive avoidance paradigm, Acetylcholinesterase,
Dopamine, Antioxidant parameters, Alzheimer's
disease.
INTRODUCTION
Normal ageing is known to deteriorate memory
in human beings. Oxygen free radicals, the harmful
by-products of oxidative metabolism, are known
to cause organic damage to the living system
that may be responsible for the development of
Alzheimer’s disease (AD) in the elderly 1, 2. AD
is a neurodegenerative disorder characterized
by a progressive loss of memory and cognition.
Pathology of AD includes defective beta-amyloid
(Aβ) protein metabolism, abnormalities of adrenergic,
dopaminergic neurotransmission and the potential
involvement of inflammatory and oxidative pathways3.
Reducing oxidative stress by anti-oxidants,
protecting brain inflammatory lesions using anti-
*For correspondence
Department of Pharmacology & Toxicology
Prin. K. M. Kundnani College of Pharmacy, 23-Jote Joy
Building, Rambhau Salgaonkar Marg, Cuffe Parade,
Colaba, Mumbai- 400 005.
E-mail: sandhyakdesai@yahoo.com
inflammatory drugs and facilitation of brain cholinergic
neurotransmission with anti-cholinesterases are some
positive approaches for management of AD4.
Nootropic agents such as piracetam are used
primarily for improving memory, mood and behaviour.
However, the side-effects like mental confusion,
muscle tremors, shivering, headaches that are
associated with these agents have limited their use5.
Since the cholinesterase inhibitors confer only modest
benefits, additional non-cholinergic AD therapies are
urgently needed. Multipotent agents aiming at diverse
targets are expected to act better than the single target
aiming counterparts in the fight against AD3.
Glycyrrhiza glabra Linn. of the family Leguminosae,
is a genus of perennial herbs and shrubs distributed
in the subtropical and warm temperate regions of
the world, chiefly in the Mediterranean countries and
China6. In the traditional system of medicine, the
roots and rhizomes of Glycyrrhiza glabra have been
used clinically for centuries. Roots have demulcent,
antacid, antiulcer, antiinflammatory, expectorant,
tonic, diuretic, laxative and sedative properties. They
also possess antipyretic, antimicrobial, antiherpes,
anxiolytic and memory enhancing activity7.

INDIAN DRUGS 50(02) February 2013 33

 Liquorice roots contain many bioactive
components, such as glycyrrhizin, liquiritin, liquiritenin
and glabridin. Among these functional components,
glabridin, a major polyphenolic flavonoid, is found in
the cork layer and decayed part of its thickening roots8.
Glabridin is an isoflavone which has a wide variety of
pharmacological activities, such as cytotoxic activity
and antimicrobial activity against Helicobacter pylori
and has shown beneficial effects in the treatment
of cardiovascular and central nervous system
disorders9, 10. It has shown promising memory
enhancing activity in various behavioural models of
memory11. The present investigation was undertaken
to evaluate the nootropic activity of Glabridin rich
extract from Glycyrrhiza glabra.
MATERIALS AND METHODS
Drugs and Chemicals
Scopolamine hydrobromide, dopamine hydro-
chloride, serotonin hydrochloride, acetylthiocholine
iodide were purchased from Sigma Aldrich Chemicals,
U.S.A., Glabridin was gifted by Pravinchandra & Co,
Mumbai and piracetam was gifted by IPCA Labora-
tory, Mumbai, India. All the other chemicals were
obtained from local sources and were of analytical
grade. Volume of oral administration and intraperi-
toneal injection was 1 mL/100g of rat.
Plant Materials
Fresh roots and stems of Glycyrrhiza glabra L.
were collected from Sheetal Herbal Products. The
plant was authenticated at Agharkar Research
Institute, Pune, India (Certificate reference no:
47083).
Preparation of Glabridin Rich Extract (GRE)
The method of Jiao et al. 12 was followed with
necessary modification.
In brief, 30g dried powder of liquorice roots
and stems were sonicated with 80mL ethyl acetate
for 30min. The extract was dried over water bath
maintained at 40°C and vacuum dried to obtain a
crude extract. The extract was subjected to preliminary
34 INDIAN DRUGS 50(02) February 2013
phytochemical screening by the methods previously
described by Kokate 13.
Column Chromatography and Characterization
The crude extract, dissolved in 15mL of ethyl
acetate-hexane (1:4 v/v), was subjected to column
chromatography using various concentrations of silica
gel of 230-400 mesh size (30g) and ethyl acetate-
hexane (1:4, 1:3, 1:2 & 1:1 v/v respectively). Fractions
showing UV spectrum similar to standard Glabridin
were combined to obtain Glabridin Rich Extract (GRE).
GRE (100 ug/mL) in ethanol was scanned in UV range
of 200-400nm and compared with standard Glabridin
(20 ug/mL) by using UV/VIS spectrophotometer
(Jasco - UV-550 spectrophotometer).
Animals
Albino wistar rats of either sex weighing between
120-170g were used. Animals were housed in
polypropylene cages under standard conditions of
humidity (50± 5%), temperature (25± 2ºC) and light
(12 h light/ 12 h dark cycle) and fed with standard
pellet diet and water ad libitum. Experimental protocol
was reviewed and approved by the Institutional Animal
Ethics Committee and care of laboratory animals was
taken as per CPCSEA guidelines (Protocol no: 1011
02, Reg. No. 25/1999/CPCSEA).
Passive avoidance paradigm 14, 15
Albino wistar rats of either sex were divided into
5 groups (n=6), Group I: normal control (negative
control), Group II: toxicant control (Scopolamine
hydrochloride, 0.4mg/kg, i.p.), Group III: standard
treatment group (piracetam 200mg/kg), Group IV:
GRE5 treatment group (5mg/kg) and Group V: GRE10
treatment group (10mg/kg). The standard drug and
the test drugs were administered to the respective
groups (p.o.) once daily for 8 days. Selection of
test treatment doses was done based on the data
obtained from acute toxicity studies and preliminary
dose range finding studies. The apparatus consisted
of a box (27 cm×27cm×27 cm) having three walls of
plastic and front wall of plexiglass, featuring a grid
floor (made up of 3 mm stainless steel rods set 8
mm apart), with a wooden platform (10 cm×7 cm×1.7
cm) in the centre of the grid floor. Electric shock
(6 mA) was delivered to the grid floor. Training (on 08th
day of drug treatment) was carried out in two similar
sessions. Each rat was gently placed on the wooden
platform set in the centre of the grid floor. When the
rat stepped-down placing all its paws on the grid floor,
shocks were delivered for 15sec and the step-down
latency (SDL) was recorded. SDL was defined as
the time (in seconds) taken by the mouse to step
down from the wooden platform to grid floor with
all its paws on the grid floor. Animals showing SDL
in the range of 2– 15 seconds during the first test
were used for the second session and the retention
test. The second session was carried out 90min after
the first test. During second session, if the animals
stepped down before 60sec, electric shocks were
delivered once again for 15sec. During the second
test, animals were removed from shock free zone,
if they did not step down for a period of 60sec and
were subjected to retention test. Retention memory
was tested after 24 hr (i.e., 09th day, 24 hr after
last dose) in similar manner, except that the electric
shocks were not applied to the grid floor observing
an upper cut-off time of 300sec and immediately after
rats were humanely sacrificed using ether.
The whole brain was removed aseptically and
divided in two equal parts. 5% (w/v) homogenate
was prepared in HCl-butanol solution from the first
half of the brain which was utilized for estimation of
dopamine16. A 10% (w/v) homogenate was prepared
in phosphate buffer solution (pH 7.4) from the
second half of the brain which was further utilized
for estimation of various biochemical parameters
like acetylcholinesterase 17 (AChE), superoxide
dismutase18 (SOD), reduced glutathione19 (GSH),
lipid peroxidation20 (LPO) and total protein21.
STATISTICAL ANALYSIS
The results are expressed as mean ± SEM from
6 animals in each group. Results were statistically
analyzed using one-way ANOVA followed by Tukey
Kramer test; P < 0.05 was considered significant.
GraphPad InStat version 4.00 of Graph Pad Software
INDIAN DRUGS 50(02) February 2013 35
Inc., San Diego, USA was the software used for
statistical analysis.
RESULTS
GRE Characterization
UV scanning in the range of 200-400nm showed
an absorbance peak at 228nm that coincided with the
peak record for standard Glabridin22, confirming the
presence of glabridin in GRE. The yield of GRE was
found to be 0.25%w/w. Preliminary phytochemical
screening of GRE has shown presence of saponin
glycosides, flavonoids and phenolic compounds.
Passive avoidance paradigm
Scopolamine significantly decreased SDL as
compared to control group indicating impairment
of memory (amnesia). GRE administration at both
doses reversed the amnesia induced by scopolamine
(Table I).
MDA, the lipid peroxidation marker was
significantly elevated in toxicant group when compared
with the normal group. Treatment with GRE5 and
GRE10 to scopolamine intoxicated rats depleted
significantly (P<0.01 and P<0.001 respectively) the
increased levels of MDA. Significant decline in brain
GSH and SOD levels was observed in the toxicant
scopolamine treated group when compared with
normal control group of rats. Treatments with GRE5
and GRE10 significantly restored the GSH and SOD
levels depleted by scopolamine (Table I).
The AChE activity in brains of toxicant scopolamine
group was found to be significantly higher as compared
to the control group (P<0.001). GRE10 treatment
decreased the AChE activity that was comparable
to the standard treatment (Fig. 1).
A significant decrease in dopamine level was
observed in the toxicant scopolamine group when
compared with the normal control group of rats
(Fig. 2). Treatments with GRE5 and GRE10 restored
significantly the dopamine levels depleted by
scopolamine (P<0.05 and P<0.001).
 Fig. 1: Effect of GRE on brain AChE level in passive
avoidance paradigm
All values expressed as mean ± SEM, n = 6. One-way
ANOVA followed by Tukey-Kramer test is applied for statistical
analysis, P values: x < 0.001, z < 0.05 where treatment groups
compared with toxicant control and a < 0.001 where toxicant
control compared with normal control
DISCUSSION AND CONCLUSIONS
In the present study, passive avoidance behaviour
based on negative reinforcement was used to examine
the long-term memory using piracetam as a standard
for comparison. Increase in SDL following treatment
with GRE5 and GRE10 is suggestive of animal
retaining the memory of the shock once it entered
the shock free zone. So, long term treatment with
GRE exhibited pronounced effect in reversal of the
scopolamine induced amnesia.
Many clinical studies have reported strong
evidence that oxidative stress is involved in the
pathogenesis of Alzheimer’s disease. Oxygen-free
radicals, implicated in the process of age related decline
in the cognitive performance may be responsible for
the development of Alzheimer’s disease in elderly
persons 23. Brain tissue homogenates of toxicant
control group showed a depletion in the level of SOD,
GSH, dopamine and serotonin and a marked rise
in LPO and AChE levels. An increased oxidation of
lipids, proteins and deoxyribonucleic acid, alterations
in mitochondrial function and a possible role of
amyloid beta and its precursor protein in oxidative
reaction in experimental models of Alzheimer’s
36 INDIAN DRUGS 50(02) February 2013
Fig. 2: Effect of GRE on brain dopamine level in
passive avoidance paradigm
All values expressed as mean ± SEM, n = 6. One-way
ANOVA followed by Tukey-Kramer test is applied for statistical
analysis, P values: x < 0.001, z < 0.05 where treatment groups
compared with toxicant control and a < 0.001 where toxicant
control compared with normal control.
disease are demonstrated. Moreover, strong evidence
supporting the involvement of oxidative damage in
neurodegenerative disease has been suggested by
various clinical studies24. The drugs with antioxidant
effects might be beneficial for preserving brain
function. Antioxidant enzymes such as superoxide
dismutase (SOD), glutathione peroxidase (GPx)
and catalase as well as glutathione reductase and
ascorbate are involved in the reduction of oxidative
stress. Antioxidant enzymes display reduced activities
in the affected brain region of patients of Alzheimer’s
disease. Augmentation of endogenous antioxidants
by therapeutic substances has recently evoked
scientific interest because any such property of a
therapeutic agent can be expected to cause significant
improvement in the endogenous defense against
oxidative stress25. These agents also reduce the
oxidative damage and promote a functional recovery
in degenerative disorders.
Our results are in accordance with El-Sherbiny
who has reported increased oxidative stress within
rat brain with altered brain levels of SOD and
GSH26. Administration of scopolamine significantly
increased the MDA level, an important marker for
LPO and reduced both GSH and SOD activities in rat
 Table I: Effect of GRE on various biochemical parameters in passive avoidance paradigm

Parameters / Groups SDL (sec) LPO GSH SOD

(nmol MDA/g tissue) (nmol/mg protein) (U/mg protein)
Normal Control (Group I) 178.72 ± 3.04 348.10 ± 14.93 3.64 ± 0.04 12.91 ± 0.34
Scopolamine (Group II) 143.72 ± 4.94a 443.72 ± 3.99 a 1.75 ± 0.05 a 6.45 ± 0.45 a
Piracetam (Group III) 210.35 ± 6.17 x 333.03 ± 10.05 x 4.66 ± 0.26 x 16.24 ± 0.60 x
GRE5 (Group IV) 161.20 ± 1.62 z 401.12 ± 4.80 y 2.32±0.1269 z 9.35 ± 0.52 y
GRE10 (Group V) 210.43 ± 4.59 x 338.87 ± 1.42 x 4.33± 0.07 x 14.06 ± 0.49 x

All values expressed as mean ± SEM, n=6. One-way ANOVA followed by Tukey-Kramer test is applied for
statistical analysis.
P values : x<0.001, y<0.01, Z<0.05 where treatment groups compared with toxicant control and a<0.001 where
toxicant control compared with normal control.

brain. Treatment with GRE10 for 8 days produced a
significant fall in MDA and restored the GSH and SOD
activities in rat brain. GRE may exert a protective effect
against oxidative damage induced by scopolamine
by augmenting the activities of GSH and SOD in rat
brain. GRE treatment groups have shown a reduction
in AChE and increase in dopamine levels in brain.
The presence of flavonoids and phenolic
compounds in GRE as seen in preliminary
phytochemical analysis might have been responsible
for the antioxidant effects. The present study
demonstrates that GRE has therapeutic potential
in improving memory as seen in passive avoidance
paradigm model that may be attributed to reduced
levels of brain AChE, increased levels of dopamine
and supplemented by its antioxidant properties. As
a result, the susceptible brain cells might have been
exposed to less oxidative stress resulting in reduced
brain damage and improved neuronal function
probably due to acetylcholinesterase and monoamine
oxidase inhibitory activity translating into improved
memory function 25.
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Pharmacol Biochem Behav. 2003, 76(3-4), 525-
533.

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38 INDIAN DRUGS 50(02) February 2013

 ANTIHYPERGLYCEMIC AND IN VITRO ANTIOXIDANT ACTIVITIES OF PUNICA
GRANATUM LINN. IN ALLOXAN INDUCED DIABETIC RATS
Patil U. S., Bandawane D. D.*, Bibave K. H. and Chaudhari P. D.

(Received 10 September 2012) (Accepted 23 January 2013)

ABSTRACT

The present study was undertaken to evaluate antihyperglycemic and antioxidant activities of aqueous
and methanolic extracts of leaves of Punica granatum. The aqueous and methanolic extracts (200 and
400 mg/kg) of the leaves were tested for their efficacy in alloxan induced diabetic rats. Glibenclamide
(4 mg/kg), p.o. and insulin (5 unit/kg s.c.) were used as standard drugs. The maximum reduction in
fasting blood glucose in diabetic rats was observed with aqueous extract as compared to methanolic
extract. Oral glucose tolerance test in normal rats showed reduction in fasting blood glucose level at 60
min of extract administration. Aqueous and methanolic extracts of leaves of Punica granatum exhibited
scavenging effect in concentration dependent manner on 2, 2- diphenyl-1-picrylhydrazyl (DPPH), nitric
oxide and reducing power assay. Ascorbic acid was used as a standard. The findings of the present study
suggested that Punica granatum possess significant antihyperglycemic and antioxidant activity.

Keywords: Punica granatum, Alloxan monohydrate,
Antihyperglycemic, Antioxidant.
INTRODUCTION
Diabetes mellitus is a common endocrine
disorder characterized by chronic hyperglycemia
due to either insufficient insulin production by
pancreatic beta cells or by cellular resistance to
insulin. It leads to disturbances of carbohydrate,
fat and protein metabolism1. Diabetes is now one
of the most common non-communicable diseases
globally and is the fifth leading cause of death in
most of the developed countries. Complications
from diabetes such as coronary artery, peripheral
vascular disease, diabetic neuropathy, nephropathy
and retinopathy result in increasing disability, reduced
life expectancy and enormous health costs virtually
in every strata of society. Currently over 150 million
people are diabetic worldwide and it is estimated
that diabetes mellitus will affect more than 300
*For correspondence
Department of Pharmacology,
Progressive Education Society’s Modern
College of Pharmacy,Sector No. 21, Yamunanagar
Nigdi, Pune- 411044, Maharashtra
E-mail : rspatil_pharma@yahoo.co.in
million people by the year 20252. The prevalence
of diabetes mellitus is increasing with ageing of the
population and lifestyle changes associated with rapid
urbanization and westernization3,4. Currently available
pharmacotherapy for the treatment of diabetes
mellitus includes oral hypoglycemic agents and
insulin. However these current drugs do not restore
normal glucose homeostasis and they are not free
from side effects. It is therefore become necessary to
make use of vast reserves of plant origins for medical
purposes which will help to search effective as well
as safer drug remedy for diabetes mellitus5.
Oxidative stress and oxidative damage to
the tissue are common end points of chronic
diseases, such as atherosclerosis, diabetes and
rheumatoid arthritis6. Oxidative stress is currently
suggested as mechanism underlying diabetes and
diabetic complications7. During diabetes, persistent
hyperglycemia causes increased production of
free radicals, especially reactive oxygen species
(ROS), for all tissues from glucose auto-oxidation
and protein glycosylation. Oxidants are generated
as a result of normal intracellular metabolism in
mitochondria and peroxisomes, as well as from a
variety of cytosolic enzyme systems8. It has been
reported that the natural antioxidants present in

INDIAN DRUGS 50(02) February 2013 39

many plants reduce such damage and help to
prevent mutagenesis, carcinogenesis and aging due
to their radical scavenging activities9. These natural
antioxidants have been isolated from various fruits10,
vegetables and medicinal plants11,12, and the identified
compounds cover a wide range of phytochemicals
including phenolics (e.g., cinnamic acid derivatives
and flavonoids) and carotenoids (e.g., lycopene).
Punica granatum Linn. is commonly known as
pomegranate in English and Anar in Hindi belonging
to family Punicaceae. It is a fruit-bearing deciduous
shrub or small tree, native to Asia13. Pomegranates
are drought-tolerant, and can be grown in dry areas
with either a mediterranean winter rainfall climate
or in summer rainfall climates. Today, it is widely
distributed throughout the world. P. granatum has
been extensively used as a traditional medicine in India
for the treatment of dysentery, diabetes, diarrhea,
helminthiasis, acidosis, hemorrhage and respiratory
diseases. The leaves are used in hepatoprotection,
microbial infection, inflammation, lipid regulation,
gastroprotection, helminthes14 and diarrhea15. The
leaves are reported to contain apigenin, flavones,
apigenin 4’-O-β glucopyranoside, luteolin 4’-O-β
glucopyranoside, luteolin 3’-O-β glucopyranoside,
luteolin 3’-O-β xylopyranoside flavones glycoside,
and tannins like punicalin and punicafolin13. In
Unani system of medicine flower extract of P.
granatum is used for the treatment of diabetes16. To
our knowledge, there are no available reports on
antihyperglycemic effect of the leaves of this plant.
Hence, the present study was carried out to evaluate
acute antihyperglycemic activity of aqueous and
methanolic extract of P. granatum leaves in alloxan
induced diabetes in Wistar albino rats and its in
vitro antioxidant activity through various analytical
methods.
MATERIALS AND METHODS
Collection of plant material
Fresh leaves of Punica granatum were collected
from regions of Nasik district in the months of
September and October. The leaves of the plant
40 INDIAN DRUGS 50(02) February 2013
were identified by the botanist of Botanical Survey of
India, Pune and a voucher specimen (V.No. USP-1)
was deposited for future reference.
Preparation of plant extracts
Freshly collected Punica granatum leaves were
dried in shade and coarse powder was extracted with
water and methanol by maceration17. The extracted
mixture was filtered through muslin cloth and
evaporated at 60ºC up to one third of initial volume.
Remaining solvent was completely evaporated at 40ºC
and concentrated to dryness. The yield (13.6% w/w)
of the powdered plant material was collected dried
and stored at 5ºC in air tight container.
Animals
Male albino Wistar rats weighing 150-200 g
were used for present study. The animals were
maintained under standard environmental conditions
and were fed with standard pellet diet and water
ad libitum. The study was approved by Institutional
Animal Ethics Committee (Reg. no. 884/OP/05/ac/
CPCSEA). CPCSEA guidelines were adhered during
the maintenance and experiment.
Preliminary phytochemical screening
The aqueous and methanolic extract of Punica
granatum were screened for the presence of
various phytoconstituents like alkaloids, glycosides,
flavonoids, tannins, carbohydrates, amino acids and
proteins18.
Thin layer chromatomatography
The aqueous and methanolic extracts of Punica
granatum were subjected to thin layer chromatography
using plates of 250 µm thickness (TLC Silica gel 60
F254 Merck, Germany).The spots were developed in
two different solvent systems as follows:
Solvent system A- Ethyl acetate: glacial acetic
acid: formic acid: water (100:11:11:27) (V/V).
Solvent system B- Ethyl acetate: methanolic:
water (200:33:7) (V/V)19.
Acute toxicity study
Healthy adult Wistar albino rats of either sex,
starved overnight were divided into two groups
consisting of five rats and were orally fed with the
extract in increasing dose levels of 300, 500, 2000
and 5000 mg/kg body weight. The acute toxicity study
was carried out according to OECD guidelines no.
423. The animals were observed continuously for
2 h under the following profiles.
Behavioural profile: Alertness, restlessness,
fearfulness and irritability.
Neurological profile: Spontaneous activities,
reactivity, touch response, pain and gait.
Autonomic profile: Defecation and urination.
After period of 24 h, 72 h and 14 days they were
observed for any lethality or death20.
Oral glucose tolerance test (OGTT)
The oral glucose tolerance test was performed in
overnight fasted (18 h) normal rats. Rats divided into six
groups, each consisting of six rats were administered
0.9% (w/v) saline, glibenclamide 4 mg/kg, Punica
granatum aqueous extracts (AEPG) 200 and 400
mg/kg and Punica granatum methanolic extracts
(MEPG) 200 and 400 mg/kg, respectively. Glucose
(3 g/kg) was fed 30 min after the administration of
extracts. Blood was withdrawn from the tip of the tail
vein under ether inhalation at 0, 30, 60, 90 and 120
min of glucose administration and glucose levels
were estimated using glucose oxidase–peroxidase
reactive strips and a glucometer21.
Induction of diabetes
The animals were fasted for 12 h prior to the
induction of diabetes as described by Prince and
Kamalakkannan (2004) with slight modification.
Alloxan freshly prepared in saline was administered
intraperitoneally (i.p.) at single dose of 150 mg/
kg. Development of diabetes was confirmed by
measuring blood glucose concentration at 3 days
after the administration of alloxan. Rats with blood
INDIAN DRUGS 50(02) February 2013 41
glucose level of above 200 mg/dl were considered
to be diabetic and used for the studies. Control rats
received similar volume of vehicle, normal saline
(2ml/kg body weight) alone.
Acute antihyperglycemic activity
Animals were divided into seven groups, each
consisting of six rats. The extracts were administered
orally in single dose.
Group I: Normal control rats, administered saline
(0.9% w/v),
Group II: Diabetic control rats administered saline
(0.9% w/v),
Group III: Diabetic rats administered glibenclamide
(4 mg/kg),
Group IV: Diabetic rats administered AEPG
(200 mg/kg),
Group V: Diabetic rats administered AEPG
(400 mg/kg),
Group VI: Diabetic rats administered MEPG
(200 mg/kg),
Group VII: Diabetic rats administered MEPG
(400 mg/kg).
The effects of administration of AEPG and MEPG
in diabetic rats were observed by measuring fasting
blood glucose level. Fasting blood glucose levels
were obtained from the tail tip of rats estimated by
using glucometer (Accu-check sensor-comfort).
Blood glucose levels were determined before (0 hr)
and then at 2, 4, 6 and 8 hrs. after administration of
plant extract.
In vitro antioxidant activity
Determination of reducing power
The reducing power was determined according
to the method of Oyaizu. Various concentrations of
AEPG and MEPG (100- 1000 µg/ml) were mixed with
1ml of 200 mmol/l sodium phosphate buffer (pH 6.6)
and 1ml of 1% potassium ferricyanide. The mixture
was incubated at 50ºC for 20 min. After incubation
1ml of 10% trichloroacetic acid (w/v) was added and
Fig.1: Hypoglycemic effect of AEPG and MEPG on fasting
blood glucose level (FBG) of normal rats during OGTT.
Each value shown in mean±S.D. n (number of animals in each
group) = 6. AEPG: Aqueous extract of Punica granatum leaves,
MEPG: Methanolic extract of Punica granatum leaves, *p <
0.05 vs. glucose control,**p < 0.01 vs. glucose control.
Fig. 3 : DPPH radical scavenging activity of AEPG
and MEPG.
AEPG: Aqueous extract of Punica granatum leaves, MEPG:
Methanolic extract of Punica granatum leaves
the mixture was centrifuged at 2000 rpm for 10min.
The upper layer solution (2.5 ml) was mixed with 2.5
ml of deionised water and 0.5 ml of ferric chloride
(0.1%) solution. The absorbance was measured at
700 nm. A higher absorbance indicates a higher reducing
power. Ascorbic acid used as a standard22.
Determination of DPPH (1-1-diphenyl 2-picryl
hydrazyl) radical scavenging activity
The free radical scavenging activity of Punica
granatum leaves were measured in terms of hydrogen
donating or radical scavenging ability using the stable
42 INDIAN DRUGS 50(02) February 2013
Fig.2 : Reducing power of aqueous and methanolic
extract of Punica granatum leaves.
AEPG: Aqueous extract of Punica granatum leaves, MEPG:
Methanolic extract of Punica granatum leaves, EAECA: Ethyl
acetate extract of Punica granatum leaves.
Fig. 4 : Nitric oxide scavenging activity of AEPG
and MEPG.
AEPG: Aqueous extract of Punica granatum leaves, MEPG:
Methanolic extract of Punica granatum leaves.
radical DPPH. AEPG and MEPG of concentrations
200-1000 µg/ml (0.1 ml) were added to 3 ml of a
0.004% methanolic solution of DPPH. Absorbance at
517 nm was determined after 30 minutes. Ascorbic
acid was used as the reference compound. Lower
absorbance of the reaction mixture indicated higher
free radical scavenging activity. Radical scavenging
activity was expressed as the inhibition percentage of
free radical by the sample and was calculated using
the following formula22
% inhibition = (A0 - At) /A0 X 100)
 Table 1. Effect of Aqueous (AEPG) and methanolic (MEPG) extract of leaves of Punica granatum on
fasting blood glucose level in alloxan induced diabetic rats.

Experimental
Fasting blood glucose level (mg/dl)
groups (n=6)
0h 2h 4h 6h 8h
Normal Control
77.33 ± 2.29 75.66 ± 2.98 77.16 ± 2.54 77.66 ± 2.15 75.5 ± 2.487
(NC)
Diabetic Control
490.83 ± 44.49## 488 ± 43.25## 493 ± 27.06## 506.84 ± 20.96## 504.84 ± 18.30##
(DC)
DC+AEPG
376.00 ± 32.27 349.16 ± 65.04 281 ± 60.78** 248.5 ± 77.83* 187 ± 65.20**
(200 mg/kg)
DC+AEPG
436.00 ± 16.15 311.5 ± 44.26* 251.34 ± 43.37** 275.67 ± 49.65* 159.34 ± 55.21**
(400 mg/kg)
DC+MEPG
476.16 ± 67.83 405.17 ± 60.48 380 ± 70.12* 340.83 ± 50.50* 318.34 ± 41.25*
(200 mg/kg)
DC+MEPG
438.00 ± 41.46 456.5 ± 38.50 398.17 ± 54.42* 326.84 ± 67.98* 302.67 ± 76.32*
(400 mg/kg)
DC+GL (4 mg/kg) 481.16 ± 41.23 470.33 ± 25.37 265 ± 29.15** 283.16 ± 31.96* 313.34 ± 11.66*
DC+Insulin
499.29 ± 45.32 139.66 ± 41.24** 90.34 ± 6.3** 161.5 ± 8.9** 243 ± 23.18**
(5 units/kg)

n=6, Values are mean ± S.E.M., #p<0.05, ##p<0.01 as compared to normal control group; *p<0.05, **p<0.01 as
compared to diabetic control group. AEPG: Aqueous extract of Punica granatum leaves; MEPG: Methanolic
extract of Punica granatum leaves; GL: Glibenclamide, Data analysed by one way Analysis of Variance (ANOVA)
followed by Dunnet’s multiple test for comparison.

where A0 was the absorbance of the control
(blank, without extract) and At was the absorbance
in the presence of the extract. All the tests were
performed in triplicate and the graph was plotted with
the mean values.
Determination of nitric oxide radical scavenging
activity
Nitric oxide was generated from sodium
nitroprusside and measured by the Greiss reaction.
Sodium nitroprusside in aqueous solution at
physiological pH spontaneously generates nitric
oxide, which interacts with oxygen to produce nitric
ions that can be estimated by using Greiss reagent.
Scavengers of nitric oxide compete with oxygen
leading to reduce production of nitric oxide. Sodium
nitroprusside (5 mM) in phosphate buffer saline (PBS)
was mixed with 3.0 ml of different concentrations
(10–100 µg/ml) of AEPG and MEPG and incubated
at 250C for 150 min. The samples were added to
Greiss reagent (1% sulphanilamide, 2% H3PO4 and
0.1% napthylethylenediamine dihydrochloride).
The absorbance of the chromaphore formed during
the diazotization of nitrite with sulphanilamide and
subsequent coupling with napthylethylenediamine
was measured at 546 nm and referred to the
absorbance of standard solutions of ascorbic acid
treated in the same way with Greiss reagent as a
positive control. The percentage of inhibition was
measured by the following formula23.
% inhibition = (A0 - At) /A0 X 100)
where A0 was the absorbance of the control
(blank, without extract) and At was the absorbance

INDIAN DRUGS 50(02) February 2013 43

in the presence of the extract. All the tests were
performed in triplicate and the graph was plotted with
the mean values.
Statistical analysis
Values were represented as mean ± S.D. for six
animals in each group. Data were analyzed using
one-way analysis of variance (ANOVA) followed by
Dunnet’s multiple test for comparison.The values
were considered significant when p < 0.05.
RESULTS
Acute toxicity study
Acute toxicity study revealed the non-toxic nature
of the extracts. There was no lethality or any toxic
reactions found among the tested animals when
administered as a single dose.
Thin layer chromatography
The results of thin layer chromatography showed
the presence of three spot in the solvent system A
which is for flavonoid glycosides. Brownish-yellow
coloured single spot in solvent system B indicated
the presence of flavone and/or flavonol in AEPG
and MEPG.
Oral glucose tolerance test (OGTT)
Fig. 1 depicts the hypoglycemic effect of single
oral administration of AEPG (200 and 400 mg/kg)
and MEPG (200 and 400 mg/kg) on OGTT of normal
rats. The aqueous extract at the dose of 200 and
400 mg/kg produced maximum fall in blood glucose
level at 30 min while methanolic extract at the dose
of 200 and 400 mg/kg produced maximum fall at 90
min after glucose administration.
Acute antihyperglycemic activity of AEPG and
MEPG in alloxan induced diabetic rats
The effect of AEPG and MEPG on the blood
glucose levels of normal and diabetic rats is given in
Table I. After oral administration of alloxan, there is
a significant elevation of the plasma glucose levels
by 2-3 folds during experimental time periods when
44 INDIAN DRUGS 50(02) February 2013
compared to normal group. The blood glucose levels
of diabetic untreated rats were significantly higher
than those of normal rats. Alloxan induced diabetic
rats treated with AEPG (200 and 400 mg/kg) showed
reduction in blood glucose level by 50.26% and
63.45% at 8 h respectively. Diabetic rats treated with
MEPG (200 and 400 mg/kg) showed reduction in blood
glucose level by 33.28% and 28.42% at 8 h. Alloxan
induced diabetic rats treated with glibenclamide
(4 mg/kg) showed reduction in blood glucose level
by 44.90 % at 4 h respectively.
Reducing power
Fig. 2 depicts total reducing power of AEPG
and MEPG. Reducing power increased with an
increase in extracts concentration as evident by the
concentration dependant increase in optical density
at 700 nm. The highest reducing power of AEPG,
MEPG was shown at a concentration of 1000 μg/
ml in the following order: ascorbic acid (2.1097)
> AEPG (1.8689) > MEPG (1.4561). The results
obtained were comparable with standard antioxidant
ascorbic acid.
DPPH (1-1-diphenyl 2-picryl hydrazyl) radical
scavenging activity
Fig. 3 depicts DPPH radical scavenging activity
of AEPG and MEPG. AEPG, MEPG and EAEPG
showed a concentration dependent increase
in scavenging of DPPH. The highest DPPH
scavenging activity of AEPG, MEPG was shown
at a concentration of 1000 μg/ml in the following
order: ascorbic acid (94.2± 0.02) > AEPG (90.02±
0.02) > MEPG (66.75± 0.005). The results obtained
were comparable with standard antioxidant ascorbic
acid.
Nitric oxide radical scavenging activity
Fig. 4 depicts nitric radical scavenging activity of
AEPG and MEPG. The highest nitric oxide radical
scavenging of AEPG and MEPG was found to be
56.24 ± 0.02 and 51.02± 0.02 percent respectively
at concentration of 100 μg/ml. Ascorbic acid was
used as standard compound.
DISCUSSION
The present study is the preliminary assessment
of the antihyperglycemic activity of the aqueous and
methanolic extracts of Punica granatum leaves. The
extract shows dose dependant fall in fasting blood
glucose level in alloxan induced diabetic rats. The
diabetogenic agent alloxan is a hydrophilic and
chemically unstable pyrimidine derivative which is
toxic to pancreatic β - cells because it can generate
toxic free oxygen radicals during redox cycling in the
presence of reducing agents such as glutathione
and cysteine24.
When AEPG and MEPG were administered to
glucose loaded normal rats (OGTT) fasted for 18 h,
reduction in blood glucose levels was observed after
60 and 90 min respectively. The decline reached its
maximum at 2 h. In the present study, the difference
observed between the initial and final fasting blood
glucose levels of different groups under investigation
revealed a significant elevation in blood glucose level
in diabetic control group. Administration of AEPG
and MEPG to diabetic rats showed a significant
decrease in fasting blood glucose level in alloxan
induced diabetic rats.
In our present experiment, we studied the in vitro
scavenging activity of different concentrations of
AEPG and MEPG. Reactive oxygen species (ROS)
are capable of damaging biological macromolecules
such as DNA, carbohydrates or proteins25. The
reducing power assay measures the electron-donating
ability of antioxidants using potassium ferricyanide
reduction method. The reducing properties are
generally associated with the presence of reductones,
which have been shown to exert antioxidant action by
breaking the free radical chain by donating a hydrogen
atom26. 2, 2-dipheny-l-picrylhydrazyl (DPPH) is
a stable free radical at room temperature, which
produces a violet solution in ethanol. The decrease
of DPPH molecules by the action of test extracts can
be correlated with the number of available hydroxyl
groups27. DPPH scavenging activity of AEPG and
MEPG may be probably due to the presence of
substance with an available hydroxyl group in the
INDIAN DRUGS 50(02) February 2013 45
extract such as phenolic groups such as flavonoids
and tannins. Excess levels of nitric oxide are found
in several diseases including diabetes. AEPG
showed maximum nitric oxide scavenging activity
than MEPG which may be the greater presence of
flavonoids in the particular extract. AEPG showed
more potent antioxidant activity compared to MEPG
extracts. Ascorbic acid was used for comparing the
antioxidant activity.
The present study showed that aqueous and
methanolic extract of Punica granatum leaves
showed significant antihyperglycemic activity
in alloxan induced diabetic rats. The possible
mechanisms by which Punica granatum brings about
its antihyperglycemic action may be through the
insulinomimetic action of phytoconstituents or may
be through potentiation of pancreatic secretion of
insulin from the intact β-cells of islets28,29. It could be
due to free radical scavenging activity of plant extract
as evident from in vitro antioxidant study30.
CONCLUSION
It is concluded from the data that Punica granatum
leaves possesses significant antihyperglycemic and
in vitro antioxidant activity than and it may prove to
be effective for the treatment of diabetes mellitus.
However, longer duration studies on chronic models
are necessary to elucidate the exact mechanism of
action so as to develop it as a potent antihyperglycemic
and antioxidant drug.
ACKNOWLEDGEMENT
The author would like to thank Dr. Diwakar,
Director, Botanical Survey of India, Pune for
authentication of plant material and Mr. Bhushan
Pimple for his help during the project work.
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Bangladeshi medicinal plants, S. J. Pharm. Sci. 2008,
1, 57-62.
24. Szkudelski T.: The mechanism of alloxan and
streptozotocine action in B-cells of the Rat pancreas,
Physiol Res. 2001, 50, 536.
25. Bagchi D., Garg A. and Krohn R. L.: Protective
effects of grape seed proanthocyanidins and selected
antioxidants against TPA-induced hepatic and brain lipid
peroxidation and DNA fragmentation, and peritoneal
macrophage activation in mice, Gen Pharmac. 1998,
30, 771–776.
26. Gordon M. H.: The mechanism of antioxidant action in
vitro. In: BJF Hudson (Ed.), Food antioxidants, Elsevier
Applied Sci., London. 1990, 1–18.
27. Annie S., Rajagopal P. L. and Malini S.: Effect of Cassia
auriculata linn. root extract on cisplatin and gentamicin-
induced renal injury, Phytomedicine. 2005, 12, 555-
560.
28. Prince P. S. M., Kamalakkannan N. and Menon V.
P.: Antidiabetic and antihyperlipidaemic effect of
alcoholic Syzigium cumini seeds in alloxan induced
diabetic albino rats, J. Ethnopharmacol. 2004, 91,
209-213.
29. Ohno T., Horio F., Tanaka S., Terada M., Namikawa T.
and Kitoh J.: Fatty liver and hyperlipidemia in IDDM of
streptozotocin treated shrews. Life Sci. 2000, 66, 125-
131.
30. Grover J. K., Yadav S. and Vats V.: Medicinal plants of
India with anti diabetic potential. J Ethanopharmacol.
2002, 81, 81.
 Short Notes

A NOVEL METHOD FOR ISOLATION OF MANGIFERIN FROM MANGIFERA

INDICA L. BARK

Abstract
A simple and practical method for the isolation of mangiferin from Mangifera indica Linn. (Anacardiaceae)
(Mango) bark has been developed. The method involves solvent extraction of the dried bark powder
using a single solvent and further purification by recrystallization to yield pure mangiferin. Purity of the
compound was ascertained using HPLC. The structure of the compound was elucidated by IR, MS and
NMR spectral analysis.

Key words: Mangiferin, solvent extraction, Mangifera
indica.
Mangifera is a large genus of evergreen trees,
distributed in tropical and sub-tropical parts of South-
East Asia. Three to four species are found in India,
of which Mangifera indica is cultivated extensively
for its fruit1.
M. indica bark majorly contains tannins
(16-20%) such as protocatechuic acid and catechin.
Other constituents present are mangiferin, alanine,
glycine, α-aminobutyric acid, kinic and shikimic
acid2. Mangiferin (2-β-D-glucopyranosyl-1,3,6,7-
tetrahydroxy-9H-xanthen-9-one, fig. 1 found in the
bark and leaves of Mangifera indica, is also found in
leaves of Bombax ceiba, Canscora decussata and
Swertia chirata. It is also found in many angiosperms
and ferns3. Mangiferin is currently of interest because
of its activities such as antidiabetic, hypolipidemic,
gastroprotective, antibacterial, chemopreventive,
antiallergic, antiviral, anti-inflammatory and is also
a potent antioxidant 3,4,5,6,7. Mangiferin isolation has
earlier been reported to be done using the column
chromatography and hydroalcoholic solvent extraction
techniques.
The bark of M. indica were collected from the
campus of the Institute of Chemical Technology,
Matunga, Mumbai and authenticated by Dr. H. M.
Pandit, Khalsa College, Mumbai; a voucher specimen
was deposited in Medicinal Natural Products
Research Laboratory, ICT, Mumbai. The barks were
dried at a temperature of 60°C and powdered. The
chemicals used were of analytical grade. HPLC-
grade Acetonitrile was sourced from Merck (India).
Distilled water was filtered through 0.45 µm filter.
Mass spectrum was recorded on Micromass Q-TOF
MS Mass Spectrometer. All 1H and 13C NMR spectra
were recorded on a JOEL 400-MHz instrument with
an internal standard of tetramethylsilane (TMS).
UV/Vis Spectrum was recorded on a Jasco V-530
Spectrophotometer.
TLC was performed on 0.20 mm silica gel G60 F254
(E. Merck) aluminium-supported plates. Compound
was visualized either under UV light at 254 nm or with
Ferric chloride reagent; reagent grade solvents were
used for extraction. HPLC analysis was performed
with a Jasco (Hachioji, Tokyo, Japan) system,
using 250 mm × 4.6 mm i.d., RP-18 (5-μm particle
size) column, with a flow rate of 1.00 ml/min, Injection
volume loop: 20 μl., monitoring at 257 nm (UV-1575),
and elution program: 15 min isocratic, acetonitrile :
water containing 0.1% v/v of o-phosphoric acid
(15 : 85). Chromatographic data were processed with
Borwin Software.
50 g of powdered M. indica bark was subjected to
solvent extraction with methanol for 26 h using Soxhlet
apparatus. The methanolic extract was reduced to
1/5th of its original volume. Crude mangiferin was
obtained as a yellow precipitate. Yield of crude
mangiferin been 8.1 % w/w of dry bark powder.
Filtered precipitate was further recrystallized using
methanol as solvent. For 1 g of crude mangiferin

INDIAN DRUGS 50(02) February 2013 47


Fig. 1: Mangiferin (2-β-D-glucopyranosyl-1,3,6,7-
tetrahydroxy-9H-xanthen-9-one)
600ml of methanol was used. Crude mangiferin was
refluxed with methanol, solution filtered, concentrated
to 1/5th the volume and left overnight for crystallization.
Light yellow coloured, needle-shaped crystals of pure
mangiferin were obtained. Yield of pure mangiferin
from 50g of bark powder was found to be 3.30g
(6.6%w/w). The quantity of mangiferin in the dried
bark of M. indica was found to be 10.2% w/w using
the HPLC method.
Thin layer chromatography was performed on
pre-coated silica gel G60 F254 (E. Merck) using ethyl
acetate: methanol: water: formic acid (10:1:1:0.1)
as mobile phase, a single band was seen at Rf 0.41
which gave a green colour with ferric chloride reagent
(5% alcoholic Ferric chloride). Infra red (IR) spectrum
of the isolated compound showed a broad peak
at 3407 cm-1(hydroxyl), 1618 cm-1 (aryl aldehyde),
1094 cm-1 (C-O stretching of ethers) and 1350 cm-1
(C-O stretching of phenols). Molecular ion peak at
423 m/e gave the molecular weight of the compound.
The UV/Vis maxima in methanol were found to be at
208, 241, 257, 315, 360 and 365 nm. The 1H NMR
and 13C NMR was performed and the compound
was confirmed to be mangiferin by comparing with
the published data8. The isolated compound melts
at 269°C. Mangiferin showed 94% purity by HPLC.
Medicinal Natural Products Research Laboratory, Gholkar M. S * and Laddha K. S
Department of Pharmaceutical Sciences and Technology
Institute of Chemical Technology, Nathalal Parikh Marg
Matunga, Mumbai – 400019
E-mail: mpnabar@gmail.com
*For correspondence
(Received 29 November 2012) (Accepted 11 January 2013)
48 INDIAN DRUGS 50(02) February 2013
Based on the chemical and spectral studies, the
isolated compound was identified as mangiferin.
The method described above employing solvent
extraction method and subsequent purification by
recrystallization was found to be efficient on laboratory
scale for isolation of mangiferin. However, further
optimization of this method is necessary to make it
suitable for commercial extraction.
REFERENCES
1. The Wealth of India, Council of Scientific & Industrial
Research, New Delhi, 2005, Vol VI, 265-285
2. The Ayurvedic Pharmacopoeia of India, Government of
India, Ministry of Health and Family Welfare, Department
of ISM&H, New Delhi, 2004, Part-I, Vol-III, 9-10
3. Dineshkumar B., Mitra A. and Manjunatha M.; Studies on
the anti-diabetic and hypolipidemic potentials of mangiferin
(Xanthone Glucoside) in streptozotocin-induced Type 1
and Type 2 diabetic model rats, International Journal of
Advances in Pharmaceutical Sciences, 2010, 1, 75-85
4. Saveri J. A., Lima Z. P., Kushima H., Brito A. R., Santos
L. C., Viegas W. and Hirumalima C. A.; Polyphenols
with antiulcerogenic action from aqueous decoction of
mango leaves (Mangifera indica L.), Molecules, 2009,
14, 1098-1110
5. Rasool M., Sabina E. P., Mahinda P. S. and Gnanaselvi B.
C.; Mangiferin, a natural polyphenol protects the hepatic
damage in mice caused by CCl4 intoxication, Comp Clin
Pathol, 2012, 21, 865-872
6. Yoshimi N., Matsunaga K., Katayama M., Yamada
Y., Kuno T., Qiao Z. et al; The inhibitory effects of
mangiferin, a naturally occurring glucosylxanthone, in
bowel carcinogenesis of male F 344 rats, Cancer Lett.,
2001, 163(2), 163-170
7. Garrido G., Gonzalez D., Lemus Y., Garcia D., Lodeiro L.,
Quintero G., et al; In vivo and in vitro anti-inflammatory
activity of Mangifera indica L. extract (Vimang),
Pharmacological Research, 2004, 50(2), 143-149
8. Shahat A. A., Hassan A. A., Nazif N.M., Van Miert S.,
Pieters L., Hammuda F. M., et al; Isolation of mangiferin
from Bombax malabaricum and structure revision of
shamimin, Planta Medica, 2003, 69, 1068-1070.
ANTI-INFLAMMATORY EFFECT OF SEEDS OF TAMARINDUS INDICA IN WISTAR
RATS

Abstract

Tamarindus indica L. is highly valued in Indian medicine for management of painful inflammation,
diabetes and constipation. In the present study, the anti-inflammatory potential was evaluated using
carrageenan induced rat paw edema model. The coarse seed powder was extracted with distilled water,
ethyl acetate and methanol and submitted to chemical tests which revealed the presence of alkaloids,
tannins, flavonoids, glycosides, phenolic compounds and steroids. All the three extracts i.e. aqueous,
ethyl acetate and methanolic extract of Tamarindus indica seeds at 500 mg/kg showed significant
anti-inflammatory activity in the second phase of inflammation. For comparison purpose, we used
indomethacin (10 mg/kg) as a reference drug. The results showed that methanolic extract exhibited more
promising anti-inflammatory activity compared to aqueous and ethyl acetate extracts.

Keywords: Tamarindus indica seeds, Anti- Tamarindus indica L. (family: Leguminosae),

inflammatory, Carrageenan.
INTRODUCTION
Inflammation is a part of the complex biological
response of vascular tissues to harmful stimuli,
such as pathogens, damaged cells or irritants.
It is the defence reaction to limit the spread of
these injurious agents. This initiates the healing
process. Inflammation is considered as a protective
mechanism and the progressive destruction of the
tissue would compromise the survival of the organism.
However, chronic inflammation can also lead to a
host of diseases, such as hay fever, periodontitis,
atherosclerosis, rheumatoid arthritis and even cancer
(e.g. gallbladder carcinoma). Therefore inflammation
is normally closely regulated by the body1.
Currently, synthetic drugs like non-steroidal
anti-inflammatory agents (indomethacin, aspirin,
diclofenac, etc.) and steroids are used in the treatment
of inflammation. All these drugs possess immense
side effects; also they are very expensive to develop.
Moreover in developing countries like India, most of
the rural population relies on traditional remedies,
being cost effective and easily available2. It is
therefore essential to study the traditionally used plant
materials in order to provide a scientific base to the
plant remedies by the people especially in the rural
area, where cost effectiveness and easy availability
is of utmost importance.
commonly known as tamarind, grows naturally in
the tropical and sub-tropical regions of India3. It is
widely found throughout the tropical belt, from Africa
to south Asia, northern Australia, Southeast Asia,
Taiwan and China4. The healing power of tamarind is
first mentioned in the traditional Sanskrit literatures5.
In the Indian system of medicine, tamarind has wide
therapeutic application including inflammation 6,
diabetes7, constipation, indigestion and flatulency8.
The seeds of Tamarindus indica are reported
to possess pharmacological activities such as
antidiabetic and hypoglycemic, antioxidant, anti-ulcer,
anti-venom, hepatoprotective, antibacterial, inhibition
of nitric oxide production and serin proteinase
inhibitor9. Fruits and leaves of Tamarindus indica are
reported with antiasthamtic, hepatoprotective and
antimicrobial activities10.
Several studies have demonstrated that various
flavonoids like quercetin, luteolin, apigenin and tannins
are responsible for analgesic and anti-inflammatory
activity11, 12. The seeds of Tamarindus indica are
reported to contain polymeric tannins and flavonoids
like (+) catechin, procyanidin B2, (-) epicatechin,
procyanidin trimer, procyanidin tetramer, procyanidin
pentamer, procyanidin hexamer, taxifolin, apigenin,
eriodictyol, luteolin and naringenin13. Thus it may be
possible that seeds of T. indica may possess anti-
inflammatory potential due to the presence of above
stated phytoconstituents. This forms the basis of the

INDIAN DRUGS 50(02) February 2013 49

current study which was designed to evaluate the
anti-inflammatory effect of different extracts of seeds
of Tamarindus indica in Wistar rats.
MATERIAL AND METHODS
Experimental Animals
Wistar rats (180-200 g) of either sex were
used for the study. The animals were procured from
the National Institute of Biosciences (NIB), Pune.
The animals were kept under standardized animal
house conditions with free access to standard pellet
diet and water ad libitum. The study was approved
by Institutional Animal Ethical Committee (IAEC)
and experimental procedures were conducted in
accordance with the regulations of CPCSEA (884/
PO/ac/05/CPCSEA).
Plant Materials
The seeds of Tamarindus indica were purchased
from the local market in the Pune region of Maharashtra
in India in the month of September 2011 and identified
and authenticated from Botanical Survey of India,
Pune. Voucher specimen (TAMIMAG6) has been
deposited in the herbarium of BSI, Pune.
Preparation of Extract
All the extracts were prepared by maceration
technique14. The coarse powder of tamarind seeds
(100 g) was extracted separately with 500 mL distilled
water, ethyl acetate and methanol for 72 h at room
temperature. The extracts were filtered and the
solvents were evaporated under vacuum to obtain
reddish-brown powdered residues.
Preliminary Phytochemical Screenings
The preliminary phytochemical analysis of
aqueous, ethyl acetate and methanolic extract of
Tamarindus indica seeds was performed as per
the standard methods. A series of chemical tests
were carried out viz. Molisch’s, Fehling’s, Benedict’s
test for carbohydrates; Biuret and Million’s test for
proteins; Salkowski and Liebermann-Buchard’s test
for steroids; Borntrager’s test for anthraquinone
glycosides; foam test for saponins; Shinoda and
50 INDIAN DRUGS 50(02) February 2013
alkaline test for flavonoid glycosides; Dragendorff’s,
Mayer’s, Hagger’s and Wagner’s test for alkaloids;
ferric chloride, lead acetate and potassium dichromate
test for tannins and phenolics15.
Acute Toxicity Study
The aqueous, ethyl acetate and methanolic
extracts of Tamarindus indica seeds were tested
for their acute toxicity in rats. Acute toxicity studies
were performed according to OECD (Organization
for Economic Co-operation and Development)
guidelines OECD 423. To determine short term
toxicity, the adult female Wistar rats were starved
overnight and were administered with aqueous, ethyl
acetate and methanolic extract of Tamarindus indica
seeds orally in increasing dose levels of 100, 300,
500, 2000, 5000 and 8000 mg/kg body weight. The
mortality and general behavior of the animals were
observed periodically for 48 h. The animals were
observed individually after dosing periodically for
24 h with special attention during first two hours and
then intermittently thereafter, for a total period of 14
days. The animals were observed for the signs of
toxicity which include changes in eyes and mucous
membrane, skin and fur and behavior pattern.
Attention was given to parameters like grooming,
convulsions, tremors, salivation, lethargy, diarrhea,
loss of righting, reflex, sleep and coma16.
Anti-inflammatory Study
Wistar rats of either sex were selected for the
study. The animals were randomly divided into 8
groups. Animals from group I received distilled water
(1 mL) and group II received indomethacin (10 mg/
kg) orally. Group II, IV and V received aqueous, ethyl
acetate and methanolic extract of Tamarindus indica
seeds at 500 mg/kg body weight, per oral.
Groups I : Control
Groups II : Standard
Groups III : Aqueous Extract
Groups IV : Ethyl acetate Extract
Groups VI : Methanolic Extract
 Table I. Anti-inflammatory activity of different extracts of Tamarindus indica seeds

Treatment Dose Increase in paw volume in mL

1h 2h 3h 4h 6h
Control 1 mL/rat 1.65±0.10 1.95±0.07 2.46±0.20 2.66±0.20 2.96±0.25
Indomethacin 10 mg/kg 1.41±0.09* 1.64±0.08* 1.48±0.15** 1.79±0.17** 1.70±0.15**
(14.54) (15.89) (39.83) (32.70) (42.56)
Aqueous 500 mg/kg 1.38±0.04* 1.61±0.17* 1.92±0.30* 2.05±0.21* 2.39±0.22*
Extract (16.36) (17.43) (21.95) (22.93) (18.98)
Ethyl acetate 500 mg/kg 1.55±0.05ns 1.86±0.13* 1.98±0.14* 2.07±0.07* 2.35±0.18*
Extract (6.06) (15.38) (19.51) (15.85) (20.60)
Methanolic 500 mg/kg 1.57±0.03ns 1.51±0.04** 2.03±0.04** 1.87±0.09** 2.10±0.12**
Extract (4.84) (22.56) (23.10) (29.69) (29.05)

n=6,*p<0.05 and **p<0.01, considered significant, by Dunnet Multiple Comparison Test (One way analysis of
variance, ANOVA). Value expressed as mean±S.E.M.

0.1 mL of 0.1 % w/v of carrageenan suspension
in 0.9% normal saline was injected subcutaneously
into the sub-planter of the left hind paw. All the
test groups were treated with their respective
treatments, 1 h before the carrageenan injection.
The paw volumes were measured immediately after
carrageenan injection and then at 1, 2, 3, 4 and 6 h
using plethysmometer.
In the above model, the degree of edema formation
was expressed as an increase in paw thickness. The
% inhibition in paw volume was calculated by using
following formula,
% inhibition in paw volume = 100 × (1 – Vt/Vc)
where,
Vt = mean paw volume in the drug treated group.
Vc = mean paw volume in control group17.
Statistical Analysis
The results are expressed as the mean ± S.E.M
of six animals. p<0.05 was considered as statistically
significant when all groups were compared with the
control gruop. The difference between experimental
groups was compared by One-way Analysis of
Variance (ANOVA) followed by Dunnet Multiple
comparison test.
RESULTS
The 100 g powder of Tamarindus indica seeds
gave a percent yield of 24.1 % w/w for aqueous
extract, 10.8% w/w for ethyl acetate extract and
17.9 % w/w for methanolic extract. The aqueous
and methanolic extracts were found to be crystalline,
powdered reddish-brown residue, whereas ethyl
acetate extract was found to be brown in colour with
sticky consistency. The preliminary phytochemical
analysis of these extracts revealed the presence of
secondary metabolites like alkaloids, anthraquinone
glycosides, tannins, flavonoids, phenolic compounds,
steroids and saponins.
As suggested by OECD guidelines, the test
animals were observed individually, after dosing at
once, during the first 24 h periodically with special
attention during first 2 h. The test animals did not
exhibit any visible change and survived beyond
recommended duration of observation i.e., 48 h with
8000 mg/kg. Hence, all the three extracts were found
to be safe up to 8000 mg/kg. Therefore, dose of 500
mg/kg less than submaximal dose (800 mg/kg) was
selected for the study.

INDIAN DRUGS 50(02) February 2013 51

 Table 1 indicates the anti-inflammatory activity
of aqueous, ethyl acetate and methanolic extract
of Tamarindus indica seeds. The aqueous extract
at 500 mg/kg shows significant activity at 4 h with
percentage inhibition of 22.93, while the ethyl
acetate extract showed significant activity at 6 h with
percentage inhibition of 20.60. Similarly, methanolic
extract showed inhibition of paw edema in second
phase (at 4 h) with percentage inhibition of 29.69
at 500 mg/kg. The standard indomethacin showed
significant activity from 3 h onwards, maximum at
6 h with inhibition of 42.56%.
DISCUSSION
In the present study, the anti-inflammatory activity
was evaluated using carrageenan induced paw
edema model. This model has been widely used
to screen natural products with anti-inflammatory
potentials18,19. Inflammation induced by carrageenan
involves the release of platelet activation factor and
pro-inflammatory mediators such as prostaglandins,
kinins, tumor necrosis factor and nitric oxide20. Sub-
planter injection of carrageenan in the rat hind paw
induces inflammation in two distinct phases: the first
phase (0-2 h) which involves release of histamine and
5-hydroxytryptamine21,22 and second phase (2-6 h)
which involves release of the inflammatory mediators
like prostaglandins, leukotrienes, polymorphonuclear
cells and bradykinins. These two phases are
linked with kinin release23. The knowledge of these
mediators is important for interpreting the mode of
drug action.
It can be concluded that aqueous, ethyl acetate
and methanolic seed extract have moderate to
significant anti-inflammatory activity, among which
the methanolic extract is much more efficient than
aqueous and ethyl acetate extract. Tannins and
flavonoids are reported to inhibit prostaglandin
synthesis12. As the anti-inflammatory effect of the
seed extracts was more significant during later phase
of inflammation, perhaps there might be inhibition of
prostaglandins, leukotrienes, polymorphonuclear cells
52 INDIAN DRUGS 50(02) February 2013
or bradykinins which is due to presence of alkaloid,
tannins and flavonoids. Since methanolic extract was
found to be more potent that other two extracts, we
can say that the active constituents responsible for
anti-inflammatory effect have been extracted to more
extent in methanol. However, isolation of chemical
constituents and mechanism responsible for the
anti-inflammatory activity of Tamarindus indica seeds
remain to be investigated.
ACKNOWLEDGEMENT
The authors are thankful to the Management and
the Principal, Modern College of Pharmacy, Nigdi,
Pune, for providing the necessary facilities to carry
out research work. The authors are also thankful to
Mr. P. G. Diwakar, Joint Director, Botanical Survey
of India, Pune, for authentification of plant and Mrs.
S.S. Nipate and Mr. B. P. Pimple for their valuable
suggestions.
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R., Carvalho J. T. C., Bastos J, K. and DaSilva M. L.
A., Analgesic and anti-inflammatory activity of a crude
root extract of Pfaffia glomerata (Sperng) Pedersen. J.
Ethnopharmacol. 2005, 96, 87-91.
23. Kuashik M. L. and Jalalpure S. S.: Evaluation of anti-
inflammatory effect of ethanolic and aqueous extracts of
Curcuma zedoaria Rosc root. Int. J. Drug Dev. & Res.
2011, 3(1), 360-365.

Department of Pharmacology, Hivrale M. G., Mali A. A. and Bandawane D. D.*

PES’s Modern College of Pharmacy
Nigdi, Pune, Maharashtra 411044
E-mail : rspatil_pharma@yahoo.co.in

*For correspondence

(Received 22 August 2012) (Accepted 11 January 2013)

have you renewed your membership for the current year 2012-2013?
if not, please do so: Kindly Contact: Ms. Prachi
Tel.: 022 - 2494 4624 / 2497 4308 Ext.: 103 / Fax: 022 - 2495 0723
E-mail: ppr@idmaindia.com

INDIAN DRUGS 50(02) February 2013 53

 IDMA takes pleasure in announcing the much awaited
“PAC-2013”
Kindly block your dates

INDIAN DRUG MANUFACTURERS’ ASSOCIATION

IDMA-APA-PAC 2013
“16th PHARMACEUTICAL ANALYSTS’ CONVENTION”
on
27th & 28th September 2013
At
Hotel Hyatt Regency, Mumbai

We are pleased to inform you that Indian Drug Manufacturers’Association (IDMA)

and Association of Pharmaceutical Analysts (APA) have pleasure in announcing the
16th Pharmaceutical Analysts’ Convention (PAC) 2013 on Friday & Saturday, 27th & 28th September
2013, at Hotel Hyatt Regency, Sahar Airport Road, Andheri, Mumbai.

Excellent opportunity for the Professionals working in the Pharmaceutical industry, R&D Institutions,
Government Laboratory and Approved Testing Laboratories, Academicians, Machine Manufacturers,
API, Excipients & Intermediates Manufacturers.

Registration fees, Exhibits, sponsorship details etc. will be informed shortly:   

 
For more details, please contact:
Ms. Prachi
Sr. Manager- Publications & PR,
Cell: +91 9867634383,
E-mail: ppr@idmaindia.com

INDIAN DRUG MANUFACTURERS’ ASSOCIATION (IDMA)

102/B, A Wing; Poonam Chambers; Worli; Mumbai 400 018, India.
Tel: +91 22-24974308 / 24944624; Fax: +91 22-24950723
email: ppr@idmaindia.com, web: www.idma-assn.org

54 INDIAN DRUGS 50(02) February 2013

 INDIAN DRUGS
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CONTACT: PUBLICATIONS DIVISION. Ms. Prachi

indian Drug Manufacturers' Association
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Tel: 24944624/24974308 Extn: 103 Fax: 24950723
Email: ppr@idmaindia.com
Website: www.idma-assn.org

INDIAN DRUGS 50(02) February 2013 55

 Our expertise has been preferred around the world for 35 years.
Become a part of our exciting new growth story by joining the Calyx family.
Calyx has been a preferred CRAMs & API supplier to customers around the world. Now more t han ever before, we
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Development (Dombivli, Mumbai) (20)
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M.Sc. / BE.(Chem.) with PGDMM, with 15-18 yrs. in imported RM
procurement, vendor development/negotiation, analysis of market General Manager/ Dy. Gen. Manager – Productivity (Tarapur)
intelligence and competitive pricing. M.Tech.(IIT) & MBA, expertise in Six Sigma (Black Belt), TPM,
Automation, etc..
Dy. Gen Manager/ Manager – Purchase Engineering (2)
BE(Mech./Elect.) with PGDMM, with 8 -15 yrs. in procurement of high Dy. Gen. Manager – Audit Compliance (Tarapur) (2)
value engineering equipments from Engineering, Chemical and API M.Sc/ B.Tech & MBA, handled customers & regulatory audits, CAPA
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Dy. Gen Manager - Finance Analyst

Managers/ Assistant Managers/ Executives – Production,
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(Dombivli & Tarapur) (45)
Ratio / Profitability / Industry Analysis, Costing, Budgets, Investments,
M.Sc.(Org)/ BE/ Dip./ B. Sc (Chem.), with 5 – 12 yrs in a reputed API
Treasury & Capex monitoring.
Company.
General Manager - R&D/Organic Synthesis (Dombivli, Mumbai)
Ph. D / Post Doc (Org.), with 15 -20 yrs. in process chemistry with Dy. Gen Manager – Quality Assurance (Tarapur, Mumbai)
proven capabilities in developing and delivering synthetic molecules. M.Sc.(Org./Analy.), with 12 -15 yrs. in QA with expertise in QMS and
cGMP.
Asst. Gen Managers / Managers/ Executives - R&D/Organic
Synthesis (Dombivli, Mumbai) (28) Dy. Gen Manager - Environment, Health & Safety (Dombivli &
Ph. D / Post Doc / M.Sc. (Org.), with 5 -15 yrs in leading CRAMS/ APIs Tarapur) (2)
projects. M.Sc.(Org) / BE (Chem.) with Dip. in Safety, 12 - 15 yrs in API
/Chemical Industry, HAZOP, HAZAN studies & ETP management.
Dy. Gen Manager/ Managers/ Executives - R&D/Analytical
Development ( Dombivli, Mumbai) (9) Executive – Projects (Civil / Mech. / Elec./ Chem) (Tarapur,
Ph. D / M.Sc (Org./Analy.), with 5 -15 yrs in method development of Mumbai) (4)
custom synthesis / API Compounds using HPLC, GC-MS, UV, IR, BE/Dip.(Civil / Mech. / Elec./ Chem), with 3-6yrs. in new projects in a
NMR. API Company

Manager/ Executive - R&D/Information Scientist (Dombivli,

Manager- Administration (On Contractual terms)
Mumbai) (2)
Graduation in Management / Law, with 10+ yrs in Corporate Admin.
Ph. D / Post Doc (Org.), with 5– 10 yrs in literature search, route
scouting and patents.

Interested candidates are requested to e-mail their resume to hrd@calyxindia.com or hrad@calyxindia.com. Visit our 'Job Opportunities' Section at www.calyxindia.com
Calyx Chemicals and Pharmaceuticals Ltd., Andheri (E), Mumbai. Tel. 022- 66077597/8 Fax: 022 - 66466401
Calyx Chemicals and Pharmaceuticals Limited (the“Company”) is proposing to make, subject to receipt of requisite approvals, market conditions and other considerations, an Initial Public Offering of its
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www.sebi.gov.in, the website of the BRLMs,i.e.PLCapital Markets Private Limited at www.plindia.com and YES Bank Limited at www.yesbank.in and is also available on the website of the Company at
www.calyx-pharma.com. Potential investors should note that investment in equity shares involves a degree of risk. For details, please refer to the DRHP, including the section titled “Risk Factors” of the
DRHP. This publicity material does not constitute an offer of securities in any jurisdiction, including the United States of America (“USA”). Securities may not be offere d or sold in the USA without
registration under the U.S.Securities Act of1933 as amended, or an exemption there from. The Company has not and does not intend to offer any securities to the public in the USA

56 INDIAN DRUGS 50(02) February 2013

INDIAN DRUGS 50(02) February 2013 57
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INDIAN DRUGS 50(02) February 2013 59
 RNI REGN.NO.11547/63
REGD.NO.MH/MR/WEST/98/2012-2014
Published on 28th of Every Month
This issue posted at Mumbai Patrika Channel Sorting Office on 28.02.2013

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60 INDIAN DRUGS 50(02) February 2013

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