Indian Drugs March PDF 09 Final

Vol.
50
INDIAN DRUGS 50(03) march 2013
Issue No.03
march 2013
Vol. 50
No. 03
March 2013
review article
- Melt Granulation: An Alternative to Traditional Granulation Techniques
Desai U.S., Chaudhari P.D., Bhavsar D.B. and Chavan R.P................................................ 5
original research articles

- Effectiveness of Microwave Drying in Improving Granule Characteristics in Tablets
Tarkase K. N., Tarkase M. K., Deshpande A. P., Wagh V. S. and Dokhe M. D.................. 14
- Development and Validation of Uv Spectrophotometric Method for Estimation of
Prochlorperazine Maleate and Pyridoxine Hydrochloride in Tablet Dosage Form by
Uv Using Multi-Component Mode of Analysis
Bhagwat G. B., Wate S. P. and Mundhey A. S................................................................... 20
- A Simple and Rapid Hplc Method for Estimation of Dimethicone from Formulations
Jadhav J. J., Mungekar S., Velada J. V., Doshi H. A. , Gajbe V. and Raunak K................. 26
- Simultaneous Estimation of Montelukast Sodium and Desloratadine
in Bulk and in Tablet Formulation by Uv-Spectophotometry
Jain R. R., Patil P. O. and Bari S. B..................................................................................... 30
- Anti Atherosclerotic Effect of Ethanolic Stem Extract of Cissus Quadrangularis Linn
Soumya V.S., and Dominic S.............................................................................................. 36
short notes
- Immunomodulatory Activity of Methanolic Extracts of Seeds and Bark of Pongamia
Glabra Vent. on Human Neutrophils
Heroor S.S., Beknal A.V. and Mahurkar N.K. ..................................................................... 46
Indian Drug Manufacturers' Association

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Tel : 022-2494 4624 / 2497 4308 Fax: 022-2495 0723
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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
Prof. K. G. Akamanchi, Ph.D. (Tech.)
Prof. Y. K. Agrawal, Ph.D., F.I.C., F.R.M.S.
Dr. Evans Coutinho, Ph.D. (Tech.)
Prof. H. L. Bhalla, Ph.D.
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.
Dr. K. N. Ganesh, Ph.D.
Dr. C. L. Kaul, Ph.D.
Dr. (Mrs.) Gopa Ghosh, Ph.D.
Dr. S. P. S. Khanuja, Ph.D.
Dr. Parthajyoti Gogoi, Ph.D.
Prof. J. K. Lalla, Ph.D.
Dr. Nirmala D. Grampurohit, Ph.D.
Dr. D. B. Anantha Narayana, Ph.D.
Dr. (Mrs.) S. S. Mahajan, M.Sc. (Tech.), Ph.D.
Dr. Nitya Anand, Ph.D.
Prof. Bhushan Patwardhan, Ph.D.
Dr. Harish Padh, Ph.D.
Dr. Ashwinikumar Raut, M.D.
Dr. M. K. Raina, Ph.D.
Dr. Sanjay Singh, M.Pharm, Ph. D.
Dr. A. V. Rama Rao, Ph.D. (Tech.), D.Sc.
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
review article
Melt Granulation: An alternative to traditional granulation
techniques
Desai U.S.*, Chaudhari P.D., Bhavsar D.B. and Chavan R.P.
(Received 20 September 2012) (Accepted 23 February 2013)
Abstract
Melt granulation is a size enlargement process in which the addition of a binder that melts or softens
at relatively low temperatures (about 600C) is used to achieve agglomeration of solid particles in the
formulation. The process utilizes materials that are effective as granulating agents when they are in the
softened or molten state. This process can be used for the preparation of sustained released dosage
forms by using lipophilic polymers, such as glycerol monostearate, a combination of a hydrophobic
material such as a starch derivative and stearic acid. It also can be used to prepare fast release melt
granules by utilizing water-soluble polymers and surfactants, such as PEG and poloxomers. Melt
granulation is one of the most widely applied processing techniques in the array of pharmaceutical
manufacturing operations.
Introduction
Fluidised hot melt granulation
Industrial application of extrusion processes dates

back to 1930s. Hot-melt extrusion (HME) is one of
the most widely applied processing technologies in
the plastic, rubber and food industry. Currently, more
than half of all plastic products, including plastic
bags, sheets and pipes are manufactured by this
process. Recently, melt extrusion has found its place
in the array of the pharmaceutical manufacturing
operations. Several research groups have evaluated
this technology to achieve enhancement in
dissolution rates for poorly water soluble drugs, to
modify drug release and for transdermal passage
of the drug. Extrusion is the process of converting
a raw material into a product of uniform shape
and density by forcing it through a die under
pressure 1-8.
Fluidised hot melt granulation (FHMG) has

received considerable attention in recent years with
the majority of these processes involving the spraying
of molten binder onto a bed of fluidised particles.
Schaefers group has shown that the granule growth
mechanism is dependent on the ratio of binder
droplet size to powder particle size2. A low ratio led
to a nucleation mechanism, which then gave rise to
coalescence and further granule growth. Kidokoro and
co-authors have shown that the increase in granule
size during FHMG is influenced by viscosity of the
binder melt. they further showed that the physical
properties of tablets pressed from the fluidized hot
melted granules were influenced by the properties
of the binder material9.
*For correspondence
Department of Pharmaceutics
Modern College of Pharmacy, Sector No.21
Yamuna Nagar , Nigdi, Pune 411044
E-mail: ujudesai@gmail.com
Advantages
l Neither solvent nor water used in this process.
Therefore good alternative for water sensitive
drugs.
l Fewer processing steps needed since wetting
& drying phases eliminated, thus less time

consuming.
5
l There are no requirements on compressibility
of active ingredients, entire procedure simple,

continuous and efficient.
l Uniform dispersion of fine particle occurs.
l Good stability at varying pH and moisture levels.
l Safe application in humans due to their non-
swellable and water insoluble nature .

1, 3
l Clinically advantaged dosage forms, such as drug
abuse and dose dumping deterrent technology.

l Sustained, modified and targeted release
capabilities.
l Better content uniformity was obtained from
HME process among granules of different size

ranges.
l Reduced number of unit operations.
l Production of a wide range of performance dosage
forms1-4,10,11.
Disadvantages
l Thermal process (drug/polymer stability).
l Requires high energy input.
l The melt technique is such that the process cannot
be applied to heat-sensitive materials owing to

the elevated temperatures involved.
l lower-melting-point binder risks situations where
melting or softening of the binder occurs during

handling and storage of the agglomerates
l Higher-melting-point binders require high melting
temperatures and can contribute to instability

problems especially for heat-labile materials.
l Flow properties of the polymer are essential to
processing.
l Limited number of available polymer1,4.
Applications in the pharmaceutical industry

In pharmaceutical industry, melt extrusion has
been used for various purposes, such as
1. Improving the dissolution rate and bioavailability
of the drug by forming a solid dispersion or solid
solution.
6
2. Controlling or modifying the release of the

drug.
3. Masking the bitter taste of an active drug.
4. Enhancing the tabletting compactability of poorly
compactible high dose drugs.
5. Enhancing the chemical stability of highly water
soluble drugs.
6. preparation of fast dissolving tablets of poorly
water soluble drugs.
7. preparation of sustained release floating
tablets1,4,6,11-15.
Materials used in melt granulation
Binders or matrix carriers
In hot-melt extruded drug delivery systems,
the active compound is embedded in a carrier
formulation, often comprised of one or more meltable
substances and other functional excipients. The
meltable substance is generally a polymer or low
melting point wax16.
Lipids are considered as an alternative to
polymer in the design of sustained drug delivery
system due to their advantages such as low melt
viscosity (thus avoiding the need of organic solvents
for solubilization), absence of toxic impurities
such as monomer catalysis and initiator, potential
biocompatibility and biodegradability. The various
meltable binders used for sustained drug delivery
system are mentioned in following tables I & II.
Plasticizers
Plasticizers are typically low molecular weight
compounds capable of softening polymers to make
them more flexible. The use of polymeric carriers
in melt granulation often requires the incorporation
of a plasticizer into the formulation to improve the
processing conditions during the manufacturing of
the extruded dosage form or to improve the physical
and mechanical properties of the final product.
Plasticization of the polymer is generally attributed
to the inter-molecular secondary valence forces
Table I : Hydrophilic Meltable Binders in the

Melt Granulation Technique10,17
Sr.
No.
1
2
3
4
5
6
7
8
9
Hydrophilic Meltable Typical Melting

Binders
Range(oC)
Gelucire 50/13
44-50
Poloxamer 188
50.9
Polyethylene glycol 2000
42-53
48-63
49-63
54-63
57-64
53-66
Stearate 6000 WL 1644
46-58
Table II : Hydrophobic Meltable Binders in the

Melt Granulation Technique4,16
Sr.
No.
1
2
3
4
5
6
7
8
9
10
11
12
Hydrophobic Meltable
Binders
Beeswax
Carnuba wax
Cetyl palmitate
Glycerin monostearate
Paraffin wax
Stearic acid
Glyceryl behenate
Glyceryl palmitostearate
Glyceryl stearate
Hydrogenated castor oil
Microcrystalline wax
Stearyl alcohol
Typical Melting
Range(oC)
56-60
75-83
47-50
47-63
47-65
46-69
67-75
48-57
54-63
62-86
58-72
56-60
between the plasticizer and the polymer. Plasticizers

are able to decrease the glass transition temperature
and the melt viscosity of a polymer by increasing the
free volume between polymer chains. In doing so,
the ease of movement of polymer chains with respect
to each other is dramatically reduced. Plasticizers
were also found to facilitate the fusion process of
semicrystalline polymers. Less energy is usually
required to melt semi-crystalline polymers following
the addition of one or more plasticizers. With the
addition of a plasticizer, a melt granulation process

can be conducted at lower temperatures and with
less torque. Generally, both the active ingredient
and the polymer will be more stable during the
granulation process due to these improved processing
conditions11. Materials commonly used as plasticizers
that are approved by the Food and Drug Administration
for use in pharmaceutical dosage forms are listed in
Table III .
Table III : Plasicizers used in Melt Granulation
Technique18
Sr. Type
Examples
No.
1 Citrate esters triethyl citrate, tributyl citrate,
acetyl triethyl citrate, acetyl
tributyl citrate
2 Fatty acid
butyl stearate, glycerol
esters
monostearate, stearyl alcohol
3 Sebacate
Dibutyl sebacate
esters
4 Phthalate
diethyl phthalate, dibutyl
esters
phthalate, dioctyl phosphate
5 Glycol
Polyethylene glycol,
derivatives
propylene glycol
6 Others
triacetin, mineral oil, castor oil
7 Vitamin E
D--tocopheryl polyethylene
TPGS
glycol 1000 succinate
OTHER PROCESSING AIDS

Anti-oxidants
The excessive temperatures needed to process

unplasticized or under plasticized polymers may lead
to polymer degradation. The stability of polymers that
are susceptible to degradation can be improved with
the addition of antioxidants, acid receptors and or
light absorbers during HME.
Antioxidants are classified as preventive
antioxidants or chain-breaking antioxidants based
upon their mechanism. Preventive antioxidants
include materials that act to prevent initiation of free
radical chain reactions. Reducing agents, such as
7
ascorbic acid, are able to interfere with autoxidation

in a preventive manner since they preferentially
undergo oxidation. The preferential oxidation of
reducing agents protects drugs, polymers, and other
excipients from attack by oxygen molecules. Chelating
agents such as edetate disodium (EDTA) and citric
acid are another type of preventive antioxidant that
decrease the rate of free radical formation by forming
a stable complex with metal ions that catalyze these
reduction reactions.
Hindered phenols and aromatic amines are the
two major groups of chain breaking antioxidants
that inhibit free radical chain reactions. Commonly
used antioxidants such as butylated hydroxyanisole,
butylated hydroxytoluene and vitamin E are hindered
phenols. Because the O-H bonds of phenols and the
N-H bonds of aromatic amines are very weak, the rate
of oxidation is generally higher with the antioxidant
than with the polymer18.

Table IV : Examples of drug substances

processed by Melt Granulation Technique
Sr.
No.
1
Drug
TM(0C)
Diclofenac
sodium
284
Theophyllin
255
Nifedipine
175
Carbamazepine
192
Lubricants
Lubricants such as magnesium stearate, Aerosil

1000 etc. are used. Waxy materials like glyceryl
monostearate have been reported to function as
lubricant in hot-melt-processing18.
Active pharmaceutical ingredient
The properties of the active drug substance often
limit the formulation and processing choices available
to the pharmaceutical scientist in the development of
dosage forms. Hydrolysis, solvolysis, and oxidation
are three primary mechanisms of drug degradation.
Drugs containing carboxylic acids, phosphoric acids
or carbonyl functional groups are vulnerable to
hydrolysis. Water or a solvent must be present for
hydrolysis or solvolysis to occur.
Melt granulation is an anhydrous process, which
avoids potential hydrolytic degradation pathways.
In addition, poorly compactable materials can be
prepared as tablets without a compression process
by cutting an extruded rod to the desired dimensions.
The only restriction is that it is not suitable for the drug
substances which melt under the HME processing
8
conditions18. The examples of drug substances which

has been processed by melt granulation technique
are given in Table IV.
References
Bhavsar D.B. et
al., 2010; Lyons.
J.G., 2006; Sato
et al., 1997
Bhavsar D.B. et
al., 2010; Henrist,
D. et al., 1999,
1999a 1999b;
Sprockel et al.,
1997; Young Cr,
2005; Young et
al., 2002; Zhang
et al., 2000
Forster et al.,
2001a, 2001c;
Nakamichi et al.,
2002
Perissutti et al.,
2002
Effect of Process Variables on Granulation

1. Effect of initial particle size and size distribution:
It has been shown that a narrow distribution
of feed particles to the granulator increases
the sphericity and decreases the porosity of
the granular products. Porosity, and its shape,
orientation and size distribution have a large
effect on the breakage behaviour and strength of
granules. A high granule porosity results in lower
the granule strength. The pores can be regarded
as crack release zones, where the highest local
tensile stress is generated and where fracture
initiates.
2. Effect of binder content: Correlations have been
developed to estimate the required binder content,
but these have proved unsuccessful for powder
mixtures with more than one component, or with

powders that have varying diameter and particle
shape. Moreover, during granulation, some solid
may dissolve partially in the liquid, which leads
to very complicated binding forces.
3. Effect of binder viscosity: proposed the extent of
the granulation is dependent upon the viscosity
of the binder solution within the granulation
vessel. In general, a higher binder viscosity
leads to a higher degree of granulation. The
viscosity of binders may be controlled by varying
the temperature within the granulation vessel
indicating that the binder viscosity affected both
the rate of size enlargement and the mechanism
of size enlargement.
4. Effect of particle deformability during granulation:
The Ennis theory postulates that the probability
of successful coalescence between colliding
granules is a function of the granule deformability
and inertia have studied the impact deformation
of wet granules and correlated granule formation
to solid liquid ratio, particle size, binder viscosity
and granulation time.
5. Effect of granulation time: As one would expect, a
longer granulation time produces larger granules.
However, the rate at which the average particle
size increases is very dependent on the binder
content of the feed and deformability of the
granulation mix19.
Melt Agglomeration
Melt agglomeration is a process by which the solid
fine particles are bound together into agglomerates, by
agitation, kneading and layering in the presence of a
molten binding liquid. Dry agglomerates are obtained
as the molten binding liquid solidifies by cooling.
Typical examples of melt agglomeration processes
are melt pelletization and melt granulation. During the
agglomeration process, a gradual change in the size
and shape of the agglomerates would take place. It
is usually not possible to clearly distinguish between
granulation and pelletization. Thus granulation is
considered a pelletization process when highly
spherical agglomerates of narrow size distribution are
produced. Conversely, an unsuccessful pelletization

process may be classified as granulation15.
The equipment for melt agglomeration include
rotating drums or pans, fluid-bed granulators, lowshear mixers such as Z-blade, planetary mixers,
and high-shear mixers. Presently, the more popular
agglomeration equipment for industrial-scale
production are high-shear mixers and fluid-bed
granulators. In both methods, a gradual buildup of
agglomerates occurs during the process. The marked
difference between the methods is the absence of
shearing forces in the fluid-bed process, whereas
very high shearing forces are generated in high-shear
mixing20,21.
During a melt agglomeration process, the meltable
binder may be added as molten liquid, or as dry
powder or flakes. In the latter, the binder may be
heated by hot air or by a heating jacket to above the
melting point of the binder. Alternatively, the melt
agglomeration process exploits an extremely high
shear input, of a high-shear mixer, where the heat
of friction alone raises the temperature of the binder
and effects melting. Typically, the melting points of
meltable binders range from 50 to 80C. A lowermelting-point binder risks situations where melting
or softening of the binder occurs during handling and
storage of the agglomerates22.
In assessing the influence of meltable materials
on the formative and growth processes of melt
agglomerates, it is imperative to have a thorough
understanding of the melt agglomeration process.
The mechanism of melt agglomeration is similar to
that of wet agglomeration.
The agglomerate formation and growth
mechanisms for a melt agglomeration process have
been studied in high shear mixers and conventional
fluid bed granulators. The formation mechanism has
been described as a distribution or an immersion
mechanism. By the distribution mechanism, small
agglomerates (nuclei) are formed by initial solid
particles being wetted by distribution of molten binder
on their surface enabling agglomerate formation by
9
coalescence between the wetted solid particles.

Further agglomerate growth occurs by coalescence
between the nuclei, provided that the liquid saturation
is sufficiently high. By the immersion mechanism,
initial solid particles become immersed in the surface
of a molten binder particle, and further immersion
of initial particles gives rise to agglomerate growth.
Agglomerate growth by coalescence between
agglomerates will primarily occur when no more
initial particles are available. This is because the
tendency for coalescence between initial particles
or between initial particles and agglomerates is
larger than the tendency for coalescence between
two agglomerates since the tendency is inversely
proportional to the size. Agglomerate growth by
coalescence will only occur until a critical agglomerate
size is reached. This size depends on a balance
between coalescence and breakage and can be
increased by e.g. using smaller powder particles,
by increasing the binder viscosity, and by reducing
the energy input. For melt agglomeration in high
shear mixers and fluid bed granulators, it has been
found that a binder particle size being smaller than
that of the solid particles promotes the distribution
mechanism. On the other hand, a binder particle size
being larger than that of the solid particles, promotes
the immersion mechanism. Furthermore, it has been
found for high shear mixers that a low viscosity of the
binder and/or high shearing forces will promote the
distribution mechanism whereas a high viscosity of
the binder and/or low shearing forces will promote
the immersion mechanism. Consequently, the
immersion mechanism can be promoted by keeping
the process temperature within or slightly below the
melting range of the meltable binder since this will
increase the viscosity of the binder markedly. In
a melt agglomeration process, both mechanisms
might be active simultaneously, although one of
the mechanisms will normally be dominant. In high
shear mixers, the dominant mechanism will usually
be the distribution mechanism because of the
high shearing forces. In fluid bed granulators, the
agglomerate formation occurs to a larger extent by
the immersion mechanism due to the lower shearing
forces. A rotary processor, which is a fluid bed
10
granulator equipped with a rotating friction plate, has

been shown to be suitable for a melt agglomeration
process. For optimum use of a melt agglomeration
process in the rotary processor, it is necessary to be
able to control the agglomerate size. This requires an
understanding of the influence of process conditions
on agglomerate properties and of the fundamental
mechanisms of agglomerate formation and growth.
process conditions such as binder concentration,
massing time, friction plate rotation speed, and
surface of the friction plate significantly influenced the
agglomerate size. However, no fundamental studies
of the mechanisms of agglomerate formation and
growth in a melt agglomeration process in a rotary
processor have been carried out23.
Modes of melt agglomeration
Fig. 1 : Modes of melt agglomeration :

(a) Distribution and (b) Immersion
These are based on the elementary mechanisms

distribution and immersion. In agglomeration by the
distribution mode, a distribution of molten binding
liquid on the surfaces of primary particles will occur,
and agglomerates are formed via coalescence
between the wetted nuclei (Fig. 1). In agglomeration by
the immersion mode, nuclei are formed by immersion
of the primary particles onto the surface of a droplet
of molten binding liquid (Fig. 1). The distribution of
molten binding liquid to surfaces of nuclei has to
be effected by densification prior to coalescence
between the nuclei. Depending on the relative size
between the solid particles and the molten binding
liquid droplets, the distribution will be a dominant mode
when the molten binding liquid droplets are smaller

than the solid particles or of a similar size. On the
other hand, the immersion mode will dominate when
the molten binding liquid droplets are larger than the
solid particles. The distribution mode is promoted by
a low molten binding liquid viscosity. In the case of
immersion, it is more favorable for molten binding
liquid of high viscosity, which could resist breakup
by dispersive forces4.
Techniques for Melt Granulation
Spray Congealing
Spray congealing is a melt technique of
high versatility. In addition to manufacture
multiparticulate delivery system, it can be applied
to process the raw meltable materials into particles
of defined size and viscosity values for the melt
agglomeration process. Processing of meltable
materials by spray congealing involves spraying a
hot melt of wax, fatty acid, or glyceride into an air
chamber below the melting point of the meltable
materials or at cryogenic temperature. Spraycongealed particles (103000 m in diameter) are
obtained upon cooling. The congealed particles are
strong and nonporous as there is an absence of
solvent evaporation. Ideally, the meltable materials
should have defined melting points or narrow melting
ranges. Viscosity modifier, either meltable or nonmeltable at the processing temperature, may be
incorporated into the meltable matrix to change the
consistency of the molten droplets24.
Tumbling Melt Granulation
A newer melt agglomeration technique, i.e.,
tumbling melt granulation, for preparing spherical
beads has been reported. A powdered mixture of
meltable and non-meltable materials is fed onto the
seeds in a fluid-bed granulator (Fig. 2). The mixture
adheres onto the seeds with the binding forces of a
melting solid to form the spherical beads. In preparing
the spherical beads, both viscosity and particle size of
the meltable materials should be kept at an optimum
value. The particle size of a meltable material should
be 1/6 or lower than the diameter of the seeds. HighINDIAN DRUGS 50(03) march 2013
viscosity meltable materials should not be employed

to avoid agglomeration of seeds and producing beads
of low sphericity25.
Both particle size and viscosity of the meltable
materials play a significant role in the melt
agglomeration process. The control of the melt
agglomeration process is best initiated by using
meltable materials of controlled properties. For the
melt pelletization and melt granulation processes,
it is desirable that meltable materials have a high
viscosity to improve the mechanical strength of the
agglomerates, but a reduced particle size to prevent
uncontrollable agglomerate growth. In tumbling melt
granulation, small meltable particles with sufficient
viscous binding forces are obligatory for the production
of spherical beads26,27.
Fig. 2: Process of Tumbling Melt Granulation
Marketed Products
The interest in HME is growing rapidly. The
US and Germany hold approximately more than
half (56%) of all issued patents. In spite of this
increased interest, there are few commercialized HME
pharmaceutical products currently marketed. There
are a number of companies using HME as a drug
delivery technology including Pharma Form (TX, USA)
and SOLIQS (Germany). SOLIQS has developed
a proprietary Meltrex formulation and redeveloped
a protease inhibitor combination product, Kaletra,
for the treatment of human immunodeficiency virus
(HIV). Moreover, HME Kaletra tablets were shown to
have significant advantages for the patient compared
11
with the previous soft gel capsule formulation, such

as reduced dosing frequency and improved stability.
SOLIQS has also developed a fast-onset ibuprofen
system and a sustained release formulation of
verapamil (Isoptin SRE) that was the first directly
shaped HME product on the market. There are a
number of products which have been developed
by several companies using HME as drug delivery
technology and this technology is gaining importance
in the field of pharmaceuticals19.
Future Trends
The application of HME technology in the
pharmaceutical industry has tended to focus on
the development of bio-enhanced formulations
to increase the efficacy of poorly water soluble
compounds. There has also been an increase in the
application of HME for the development of controlled
release formulations, in the form of pellets, beads
or minimatrices, and as a means to facilitate the
continuous processing of products to reduce the
number of manufacturing unit operations. Moreover,
there have been several articles investigating the
application of HME technology for the production
of bio adhesive hot-melt extruded film for topical
and mucosal adhesion applications and drug
delivery. Recent studies have also demonstrated
the production of biocompatible shape-memory
polymers for use in biomedical applications, using
HME as a manufacturing process. The production of
multiparticulate dosage forms using HME has been
investigated using hot melt pelletization and, lately,
the use of die, face-cutting the polymer extrudate
to produce HME pellets shows the continuing
utilization of technology from the plastics industry
for pharmaceutical manufacturing. The scope of the
technology has also been broadened to expand the
range of polymers and APIs that can be processed
through application of HME. The growing market in
medical devices, including those that incorporate
drugs such as biodegradable stents and drug
loaded catheters, might require HME manufacturing
processes to be commercialized, and may lead to
new areas of collaboration across pharmaceutical,
medical device and biotechnology research. HME is
12
a versatile processing technology for pharmaceutical

industry and has broad prospectus for future19.
CONCLUSION & OUTLOOK
Today, melt extrusion technology represents an
efficient pathway for manufacture of drug delivery
systems resulting in products mainly found among
semi-solid and solid preparations. The potential
of the technology is reflected in the wide scope of
different dosage forms including oral dosage forms,
implants, bioadhesive ophthalmic inserts, topical films,
and effervescent tablets. In addition, the physical
state of the drug in an extrudate can be modified
with the help of process engineering and the use
of various polymers. The drug can be present in
crystalline form for sustained release applications or
dissolved in a polymer to improve dissolution of poorly
water-soluble drugs. The possible use of a broad
selection of polymers starting from high molecular
weight polymers to low molecular weight polymers
and various plasticizers has opened a wide field of
numerous combinations for formulation research.
Drawbacks of the technology are often related
to high energy input mainly related to shear forces
and temperature. This is where process engineering
becomes significant. The design of screw assemblies
and extruder dies are two major areas which have
significant impact on product quality and degradation
of drug and polymers. Drugs which are sensitive to
elevated temperatures can be processed successfully
when the residence time is short compared to
conventional processes like sterilization. Work in this
field is increasing and the literature published reveals
many novel and interesting aspects of this technology
such as in-situ salt formation, fast dispersing systems
with foam like structures, complex formation in the
melt and nanoparticles released from molecular
dispersions manufactured by melt extrusion.
REFERENCES
1. Chokshi R. and Zia H., Hot melt extrusion technique: a
review, Iranian J. Pharm. Res. 2002, 3, 3-16.
2. Schafer T., Holm P., Kristensen H.G., Melt granulation
in a laboratory scale high shear mixer, Drug Devl. Ind.
Pharm.1990, 16 (8), 1249 1277.
3. Crowley M.M. and Zhang F., Pharmaceutical Applications

of Hot-Melt Extrusion: Part II, Drug Devl. Ind. Pharm.
2007, 33, 909926.
4. http://www.pharmainfo.net/reviews/melt-granulationtechnique-review
5. Sundaramoorthy K., Kavimani S., Vetrichelman T., Manna
P.K., and Venkappayya D., Formulation and evaluation of
extended release dosage form of metformin hydrochloride
using combined hydrophobic and hydrophilic matrix,
ijper, 2008, 232-241.
6. Campisi B., Vojnovic D., Chicco D. and Phan-Tan-Luu
R., Melt granulation in a high shear mixer: optimization
of mixture and process variables using a combined
experimental design , Chemom. Intell. Lab. Syst. 1999,
48, 5970.
7. Brabander C.D., Vervaet C. and Remon J.P.,
Development and evaluation of sustained release minimatrices prepared by hot melt extrusion, J. Control Rel.
2003, 89, 235 247.
17. Singh C., Jain A.K. and Agrawal K., Formulation and
evaluation of extended release tablet of pioglitazone
by melt granulation, Indian Drugs. 2008, 45(6),
461-468.
18. Crowley M.M. and Zhang F., Pharmaceutical Applications
of Hot-Melt Extrusion: Part I, Drug Dev. Ind. Pharm.
2007, 33, 909926.
19. Singhal S., Lohar V.K., Arora V., Hot Melt Extrusion
Technique, Webmed Central Pharmaceutical Sciences
2011; 2,1.
20. Al-Suwayeh A. A., El-Shaboury H. M., Al-Baraki M.
S., Elgorashy S. A., and Taha I. E., In vitro and in
vivo Evaluation of Sustained Release Hydralazine
Hydrochloride Tablets Prepared by Thermal Granulation
Technique, Aust. J. Basic Appl. Sci 2009, 3(3), 28662875.
8. Breitenbach J., Melt extrusion: from process to drug

delivery technology, Eur. J. Pharm. Biopharm. 2002,54,
107 117.
21. Ochoa L., Igartua M., Rosa M., Hernandez,

Gascon A.R. and Pedraz J.L., Preparation of sustained
release hydrophilic matrices by melt granulation in a highshear mixer, J. Pharm. Pharmaceut. Sci. 2005, 8(2),
132-140.
9. Walker G.M., Andrews G., and Jones D., Effect of process

parameters on the melt granulation of pharmaceutical
powders, Powder Technol. 2006, 165,161166.
22. Royce A., Suryawanshi J., Shah U., and Vishnupad K.,
Alternative Granulation Technique: Melt Granulation
Drug Dev. Ind. Pharm. 1996, 22(9&10), 917-924.
10. Haider S.S., Monnujan N.and Shahriya S.M., Sustained

release preparation of metoclopramide hydrochloride
based on fatty matrix, Indian Drugs. 2002, 39 (2), 7380.
11. Heng W.S. and Wong T.W., Melt processes for oral solid
dosage forms, Pharm. Tech. 2003, 1-6.
12. Evrard B.and Delattre L., In vitro evaluation of lipid
matrices for the development of a sustainedrelease
sulfamethazine bolus for lambs, Drug Dev. Ind. Pharm.
1996, 22 (2), 111-118.
13. Kowalski J., Kalb O., Joshi Y.M. and Abu T.M., Application
of melt granulation technology to enhance stability of
moisture sensitive immediate-release drug product, Int.
J. Pharm. 2009, 381, 5661.
14. Tayel S.A., Soliman I.I. and Louis D., Formulation
of ketotifen fumarate fast-melt granulation sublingual
tablet, AAPS Pharm. Sci. Tech. 2010, 11(2),
679-685.
15. Kumar R., Patil S., Patil M.B., Patil S.R. and Paschapur
M.S., Design and in vitro evaluation of oral floating matrix
tablets of aceclofenac, Int. J. ChemTech Res. 2009,
1(4), 815-825.
16. Eliasen H., Kristensen H.G. and Schafer T., Growth
mechanism in melt agglomeration with low viscosity
binder, Int. J. Pharm. 1999, 186, 149-159.
23. Vilhelmsen T., Schfer T., Agglomerate formation

and growth mechanisms during melt agglomeration
in a rotary processor, Int. J. Pharm. 2005, 304,152
164.
24. Passerini N., Perissutti B., Monoghini M., Voinovich D.,
Albertini B., Cavallari C. and Rodriguez L., Characterization
of carbamazepineGelucire 50/13 microparticles prepared
by a spray congealing process using ultrasounds, J.
Pharm. Sci. 2002, 91(3), 699-707.
25. Maejima T., Osawa T., Nakjima K. and Kobayashi M.,
Application of tumbling melt granulation method
for preparing controlled release beads coated with
hydrogenated castor oil, Chem. Pharm. Bull. 1997,
45(5), 904-910.
26. Kidokoro M., Sasaki K., Haramiishi Y. and Matahira
N., Effect of crystallization behavior of polyethylene
glycol 6000 on the properties of granule prepared by
fluidized hot melt granulation (FHMG), Chem. Pharm.
Bull. 2003, 51(5), 487493.
27. Walkera G.M., Hollanda C.R., Mohammad , Ahmada M.N.,
Duncan Q.M. Craig, Influence of process parameters
on fluidised hot-melt granulation and tablet pressing of
pharmaceutical powders, Chem. Eng. Sci. 2005,60,
3867 3877.
13
original research articles

Effectiveness of microwave drying in improving granule
characteristics in tablets
Tarkase K. N*., Tarkase M. K., Deshpande A. P., Wagh V. S. and Dokhe M. D.
(Received 30 April 2012) (Accepted 04 February 2013)
Abstract
In the present study, metformin hydrochloride is used as model drug and the granules were formed
by using tray drying and microwave technique. The granules prepared by microwave technique and
tray dryer technique are evaluated for parameters such as amount of fines, drying time, bulk density,
compressibility and angle of repose. The study indicated that the granules retained their structure in
comparison with the conventional drying process. The prepared granules were compressed into tablets
and evaluated for hardness, friability, disintegration, and dissolution. Moreover, the microwave drying
effectively improves the characteristics of granules in tablets. By adopting microwave drying technique,
tablets can be prepared in less duration of time, at least 10 times less than with tray drying procedure.
This can prove to be time, energy and cost efficient. The microwave technique rather advantagous
because the heating or drying process is by radiation rather than conduction and convection as in
conventional methods of drying and transferring heat to every molecule at same time and intensity. The
only necessary criterion for microwave drying is the polarity of the solvent to be evaporated.
Keywords: Metformin hydrochloride tablets,

Microwave drying, Tray drying.
INTRODUCTION
The aim of the present study was to standardize
the drying process for pharmaceutical granulations
by microwave technique and compare the present
release of drug obtained by microwave technique with
other drying technique. Major limitations of classical
pharmaceutics experiments are longer time, higher
cost, longer reaction time and environmental pollution
due to the use of large quantities of solvents/reagents.
Since the heating process is very short in microwave
procedure, it saves fuel/electricity and chemicals
which helps to reduce environmental pollution. The
*For correspondence
Department of Quality Assurance Technique
Padmashree Dr. Vithalrao Vikhe Patil Foundations
College Of Pharmacy, Vilad ghat
Ahmednagar. Maharashtra. 414111.
E-mail: kntarkase2007@rediffmail.com
14
microwave systems are more compact, requiring

smaller equipment space or footprint. Microwaves
generate higher power densities, enabling increased
production speeds and decreased production costs.
Synthesis of drugs, intermediates, chemicals,
activation of chromatographic adsorbents, sterilization
of glasswares and auxiliaries, drying of granules for
the preparation of tablets, enzyme inactivation of
food products, hydrolysis of proteins and peptides
and saponification of oils are a few examples of use
& of microwaves in laboratories. The wavelengths
of microwaves are in the range of about 0.01 to
1 m and lie in electromagnetic spectrum between the
infrared and radio waves. The energy varies from 0.5
to 30 Ghz. In microwave spectroscopy, the source is
monochromatic, at a well defined single wavelength
which can be rapidly varied.
METHODS AND MATERIALS
Materials:- Metformin hydrochloride IP was
received as gift sample to PDVVPFs College of
pharmacy, Ahmednagar. The polymers hydroxy
propyl methyl cellulose (K100 M) 100 and ethyl

cellulose (18 centipoise) were purchased from Merck,
India. The other excipients, namely microcrystalline
cellulose, colloidal silicon dioxide (Aerosil) and
magnesium stearate, were purchased from
Qualigens, Mumbai.
The instruments used were from PDVVPFs College of pharmacy and with following specifications.
Instrument
UV Spectrometer
Friability
Apparatus
Dissolution
apparatus
Compression
Machine
Tablet
Disintegration
Micrometer screw
gauge
Make
Jasco-V-630-double beam
spectrometer
Rolex friability test apparatus
Electrolab-TDT-08L Dissolution
tester (USP type II)
Labpress 8 station, single
rotary(0.9 mm punch)
Veego-Digital-tab
disintegration test apparatus
Alex instruments
(K100 M) & ethyl cellulose (18 Centipoises) were

used as release rate controlling polymers.
Procedure for Preparation of Granules by Tray
Drying (Td)1,2,3
Granules were prepared using wet granulation
technique .The required quantities of drug and other
excipients (Table IV) were weighed and passed
through ASTM standard sieve no: 85 to get uniform
particle size. The powders were then mixed to get
uniform blend. The granulating medium (distilled
water) was added to the powder blend and mixed
well until a smooth dough was obtained. The wet
granules were passed through sieve no.16, and
dried at 500C for 20 minutes in a tray dryer for a
batch. The dried granules were passed through
sieve no: 22.
Microwave Granulation Procedure2,3,4
The required quantities of drug and other
excipients (Table IV) were weighed and passed
through standard sieve no: 85, to get uniform particle
size. The powders were then mixed to get a uniform
blend. The granulating medium (distilled water) was
added to the powder blend and mixed well until
smooth dough was obtained. The wet granules were
passed through sieve no: 16 and dried at 840 w in
microwave for different time intervals. After every 15
seconds, the granules were observed for dryness
and if not dried, the drying process was continued
until the granules were completely dried. After
complete drying, the dried granules were passed
through sieve no: 22.
Digital weighing
balance
Shimadzu
Melting point
apparatus
Veego, Model - VMP-D, India
Tablet hardness
tester
Monsanto tablet hardness

tester, Campbell Electronics
India
Tapped density
apparatus
PSAW Inc. India
Vibrator Stirrer
Remi India
Hot Air Oven
Universal Laboratories, India
Evaluation of Granules1,3,4
Microwave
Veego, India
IR Moisture
Balance
IR moisture balance, Jeseng

laboratories.
The granules using both tray dryer and microwave

procedure were evaluated for percentage of fines,
bulk density, compressibility and flow properties
using angle of repose and moisture content
determinations.
Formulation of Granules1,2,3
Granules were prepared using metformin
hydrochloride as model drug with starch as binder as
well as disintegrant, Aerosil as glidant and magnesium
sterate as lubricant. Hydroxy propyl methyl cellulose
Percentage of Fines
The granules were passed through standard
sieve no: 16 & 22.The material retained on sieve
15
no:22 was collected separately and weighed. From

this, the percentage of fines was calculated.
Moisture Content Determinations
Moisture content (loss on drying) of granules
before and after drying was determined. In both
methods the weight of granules before starting drying
process and after drying process was calculated and
the percentage loss weight was determined .The total
moisture content allowed in the final dried granules
was 1 %, which was calculated with the help of IR
moisture balance. The granules from microwave
muffle were estimated for LOD every 15 seconds to
assure the moisture content.
Bulk Density
A given quantity of sample was transferred to a
measuring cylinder and was tapped mechanically,
using a tapping device till a constant volume was
obtained, which is referred to as bulk volume. The
bulk density was calculated by
Bulk density =mass of sample/bulk volume
Compressibility
The compressibility index of the granules was
determined by using loose and tapped bulk densities
of granules, according to the equation below;
Carrs consolidation index= [(Tapped bulk
density-loose bulk density) x100]/Tapped bulk
density
Flow Properties
A funnel was fixed at a particular height h cm on
a burette stand and graph paper was placed below
the funnel table. The sample whose angle of repose
is to be determined was poured into the funnel by
closing the bottom of the funnel. The bottom was
opened and sample was allowed to fall onto the
paper. The height of the formed pile was measured
and the circumference of the pile was drawn with
the pencil on the graph sheet. The radius of the pile
was noted as r cm and the angle of repose was
calculated as follows:
16
tan=h/r or = tan-1(h/r)
where h=height of the pile, r=radius of pile and
=angle of repose
Preparation of Tablets4,5,6
The granules were mixed with glidant and lubricant
and compressed using an 8-station rotary tablet
machine with 10mm standard punches. The batch size
was 500 tablets. Two batches of tablets were prepared,
corresponding to tray drying granulation procedure
and other batch corresponding to microwave drying
at 840 watt. The prepared tablets were evaluated for
weight variation, hardness, friability, drug content, and
disintegration time and invitro dissolution profile.
In vitro drug release study2,3,4
Drug release studies were carried out using USP
(II) dissolution apparatus following paddle method.
Freshly prepared buffer of pH 5.8 (900mL) was
placed in the dissolution flask and allowed to attain
a temperature of 371oC.The tablet was placed at
the bottom of the dissolution flask. The paddle was
rotated at 50 rpm for 720 minutes. One ml of the
sample was withdrawn at different time intervals at 15,
30, 45, 60 minutes and afterwards at hourly intervals.
After each withdrawal, the medium was replaced
by equal amount of fresh buffer. The samples were
diluted to 10 ml with dissolution medium and used
for measurement of absorbance 233nm, in jasco
UV-visible spectrophotometer.
Percentage release of drug = Absorbance of sample
content of standard Dilution factor/ Absorbance
of standard label claim.
RESULTS & DISCUSSION
Evaluation of Granules
One batch of corresponding granules of tray dried
wet granulation and other corresponding batch of
microwave dried were prepared and evaluated for
percentage of fines, bulk density, compressibility and
flow properties using angle of repose. The granule
drying time was found to be very less in case of
Table I: Properties of Metformin HCl granules using tray dried and microwave methods
Physical properties
Amount of fines(%)
Bulk density(g/cc)
Compressibility
Angle of repose(q)
Drying time(min)
Loss on drying (%)
Microwave dried granules at 840 w

10.61
0.980.006
16.25 0.005
16.00 0.5
3.44 0.52
2.5-3.45
Tray dried granules

12.10
0.960.5
10.12 0.003
14.86 0.42
36.2 0.31
3.0-4.5
All the values are represented as mean s.d; n=3

Table II: Properties of Metformin HCl tablets prepared using tray dried and microwave dried methods
Evaluation parameters
Average weight (mg)
Hardness(kg/cm2)
Friability (%)
Drug content(mg)
Disintegration(sec)
Microwave dried tablets at 840 w

770 0.5
8.30.02
0.109
5010.024
55.020.1
Tray dried tablets

7500.4
5.560.04
0.124
5000.046
44.660.4
All the values are represented as mean s.d; n=3

Table III: Cumulative release of drug from two batches of tablets prepared by microwave
and tray drying methods
Time (h)
2.00
4.00
6.00
8.00
10.00
12.00
Cumulative drug release from

Microwave dried tablets at 840 w (%)*
30.15
34.81
49.75
65.93
77.54
99.87
Cumulative drug release from

Tray dried tablets (%)*
29.41
31.85
44.59
59.70
74.11
95.54
*Average of three determinations

Table IV : Composition of tablet formulations
Ingredients (per tablet) mg
Metformin hydrochloride
Hydroxy propyl methyl cellulose (K100 M)
Ethyl cellulose (18 centipoise)
Microcrystalline cellulose
Colloidal silicon dioxide (Aerosil)
Magnesium stearate
Formulations
500
100
100
75
0.006
0.012
17
% drug release
Fig. 1: In vitro dissolution profiles of two batches of

tablets prepared by microwave and tray dried methods
microwave drying. The tray drying method took 30

minutes for complete drying of granules whereas the
microwave method took a maximum of 3 minutes
at 840 watt. The results of evaluations of granules
shown in Table I.
Evaluation of Tablets7,8
The tablets were evaluated for weight variation,
hardness, friability, drug content, disintegration and
in vitro dissolution. The results of evaluations of tablets
shown in Table II.
Dissolution test9
From the results, it was found that the tablets
prepared by tray dried granulation and those prepared
by microwave granulation at intensity of 840 watt
exhibited good release profiles.
They achieved 98-99.5 release in 720 minutes
time. From the results, it can be concluded that the
batch which were dried at an intensity of 840 watt
was ideal batch, and the results were comparable with
that of tray dried tablets. Hence, higher intensities can
be used for drying of granules in normal cases. The
results of in vitro dissolution studies of two batches
of tablets were shown in Table III and Fig. 1.
CONCLUSION
Metformin hydrochloride is a highly water soluble
drug. Its poor inherent compressibility coupled with
high dose (500mg) poses a significance challenge
18
for developing an extended release dosage form.

For developing extended release matrix tablet with
desirable drug release profile, cost effectiveness and
broader regulatory acceptance combination of HPMC
(K100M) and EC (18 CPS) was choosen as release
controlling polymers. Moreover the microwave drying
effectively improves the characteristics of granules
in tablets. It can be stated that the tablet granulation
can be dried successfully using a microwave oven.
By adopting microwave drying technique, tablets
can be prepared in less duration of time, at least 10
times less than tray drying procedure. This can save
time, energy and cut down the cost of conducting
practical classes. Also, use of such technique can
reduce environmental pollution.
The microwave technique causes its advantages
because the heating or drying process is by
radiation process than conduction and convection
in conventional methods of drying. Further more the
advantages are attributed to its directly transferring
heat to every molecule at same time and at same
intensity, also since less energy is wasted hence less
energy is required, more over less energy causes
less increase in temperature of matter hence thermal
shielding is offered to the matter.
The only necessary criterion for microwave
drying is the polarity of solvent to be evaporated.
All the flow properties and dissolution parameters
of both techniques are within acceptable limits,
however, the microwave technique provides
comparatively better results, and also the time
and energy consumption is lowered to a significant
level.
Acknowledgements
We wish to acknowledge our institute PDVVPFs
College of pharmacy, Ahmednagar, for availing their
analytical, pharmaceutical facilities and the API
Metformin for this project .
References
1.
Pederson AV. Erosion-based drug delivery. Manuf Chem.

2006;11:16.
2.
3.
4.
5.
Washington N, Wilson C. G. Erosion-based delivery.

Drug Deliv Technol. 2006; 6(9): 71-74.
Andrews G. P., Laverty T.P., Jones D.S. Mucoadhesive
polymeric platforms for controlled drug delivery. Eur J
Pharm Biopharm. 2009;71(3):505-518.
Sharma S. V, Sharma GVSR, Suresh B; A ecofriendly
technology, Ind. J. Pharm. sci, 2002; jul - aug; 64 (4):
337-344.
Walter J Moore; Physical chemistry, 5th Edition, 1999,
Orient Longman limited, 761.
6. www.industrialmicrowave.com/faqs.htm
7. Leon Lachman, Liberman H. A., Kanj L. J; Theory and
practice of industrial pharmacy, 3rd Edition, Varghese
publishing -House, 1987; 293-345.
8. Lieberman H. A., Leon Lachman, Schwartz B. J;
Pharmaceutical dosage forms: Tablets, Vol. 2, 2nd
Edition, 1989; Replika- Press, 245-335.
9. Martin A, Bustamanate P, Chun A. H. C, Physical
Pharmacy, 4th Edition, Gopsons papers, 2003;
423-490.
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DEVELOPMENT AND VALIDATION OF UV SPECTROPHOTOMETRIC METHOD

FOR ESTIMATION OF PROCHLORPERAZINE MALEATE AND PYRIDOXINE
HYDROCHLORIDE IN TABLET DOSAGE FORM BY UV USING MULTICOMPONENT MODE OF ANALYSIS
Bhagwat G. B.*, Wate S. P. and Mundhey A. S.
(Received 05 July 2012) (Accepted 04 February 2013)
Abstract
Prochlorperazine maleate and pyridoxine hydrochloride in combination are available as tablet dosage
forms in the ratio of 1:5. A simple, reproducible and efficient method for the simultaneous determination
of prochlorperazine maleate and pyridoxine hydrochloride in tablet dosage form has been developed. The
developed method is based on the simultaneous estimation by UV Spectroscopy, using multi-component
mode of analysis. In this method 0.3M HCl was used as solvent. Wavelengths selected for estimation
of prochlorperazine maleate and pyridoxine hydrochloride in multi-component mode of analysis method
were 254.5 nm and 290.5 nm respectively. Both drugs obey Beer-Lamberts law in concentration range of
1-5 g/mL (prochlorperazine maleate) and 5-25 g/mL (pyridoxine hydrochloride). The results of analysis
have been validated statistically and by recovery studies.
Keywords: Prochlorperazine Maleate, Pyridoxine

Hydrochloride, Multi-component mode of analysis,
Standard addition, Validation.
INTRODUCTION
Prochlorperazine maleate (PCM), 2-Chloro10-[3-(4-methylpiperazin-1-yl)propyl] 10H
phenothiazine bis[hydrogen(Z)-butenedioate]
(Fig.1), is an antiemetic and antipsychotic drug1
while pyridoxine hydrochloride (PDH), 5-hydroxy6-methyl-3,4-pyridine dimethanol hydrochloride
(Fig. 2), is a nutritional component2. Both the drugs
are official in IP, BP and USP. Literature survey
revealed that HPLC3-5 methods have been reported
for pyridoxine hydrochloride with other drug. A recent
research about emergency department treatment of
migraine headache indicated that prochlorperazine
was statistically superior to other optional drugs
for pain relief in migraine patients6. Some methods
*For correspondence
Department of Pharmaceutical Chemistry
Kasturi Shikshan Sansthas College of Pharmacy
Shikrapur, Pune 412 208 (M. S.)
E-mail: rajbhagwat07@gmail.com
20
have been reported such as LC-MS for detection of

both drugs along with other drugs7-10. The present
developed method is economic, rapid and precise
for simultaneous estimation of these two drugs in
combined dosage form. This prompted us to develop
simple, rapid, accurate, economical and sensitive
multi-component mode of analysis method for their
determination in combination.
MATERIAL AND METHODS
Shimadzu 1700 UV/Visible spectrophotometer
with matched cuvettes was used for experimental
work. The chemicals used were of analytical grade.
Commercially available tablets of prochlorperazine
maleate and pyridoxine hydrochloride in combination
were procured from local market. Standard
prochlorperazine maleate and pyridoxine
hydrochloride were received as gift sample from
FDA, Mumbai.
METHOD
Selection of solvent
A 0.3M HCl solution was selected as the suitable
solvent for estimation of PCM and PDH.
Table I: Results for Estimation of Pcm and Pdh in Laboratory Mixture

Sr.No
1
2
Amount of pure drug taken

PCM (1-5 g/mL)
PDH (5-25 g/mL)
% of drug estimated*
100.02
100.01
SD
0.09628
0.12194
RSD
0.00927
0.01487
SE
0.00742
0.0119
PCM is prochlorperazine maleate, PDH is pyridoxine hydrochloride, SD is standard deviation, RSD is relative
standard deviation, SE is the standard error of the mean and *Results are mean of five replicates.
Table Ii: Results of Estimation of Pcm and Pdh in Marketed Formulation
Sr.No
1
2
Amount of pure drug taken

PCM (1-5 g/mL)
PDH (5-25 g/mL)
% of drug estimated*
100.52
100.83
SD
0.62235
0.80661
RSD
0.38732
0.65062
SE
0.30986
0.5205
PCM is prochlorperazine maleate, PDH is a pyridoxine hydrochloride, SD is standard deviation, RSD is relative
standard deviation, SE is the standard error of the mean and *Results are mean of five replicates.
Table Iii: Results of Recovery Study
Sr.No
1
2
3
Amount of pure drug Added

(mg)
PCM
PDH
5
25
5
25
5
25
Amount of pure drug recovered

(mg)
PCM
PDH
5.000
25.001
5.0904
25.452
4.9819
24.910
% Recovery
PCM
100.00
101.33
99.98
PDH
100.02
101.46
99.99
PCM is prochlorperazine maleate and PDH is a pyridoxine hydrochloride.

Table Iv: Results of Specificity Study
Sr.No
1
2
3
4
Sample
PCM
100.61
98.78
99.89
101.01
Normal
Alkali
Acid
oxide
% Label claim
PDH
100.93
99.00
99.68
101.21
PCM is prochlorperazine maleate and PDH is a pyridoxine hydrochloride.

Table V: Results of Ruggedness Study
Condition
Intraday
Interday
Different
analyst
% label claim*
PCM
PDH
100.01
100.03
100.18
100.13
100.19
100.12
PCM
0.0251
0.1212
0.1569
SD
PDH
0.0360
0.0981
0.1474
PCM
0.00063
0.0147
0.0246
RSD
PDH
0.0023
0.0096
0.0120
PCM is prochlorperazine maleate, PDH is pyridoxine hydrochloride, SD is standard deviation, RSD is relative
standard deviation and *Results are mean of five replicates.
21
Preparation of standard stock solutions

Accurately weighed quantities, 25 mg each of
PCM and PDH were transferred to two separate
100 mL volumetric flasks. Both drugs were dissolved
in about 50 mL of 0.3M HCl and then volume was
made upto the mark with the same solvent. This gave
standard stock solutions having concentration 250g/
mL of PCM and PDH respectively.
Selection of scanning range and sampling
wavelengths
. HCI
Fig. 2: Structure of pyridoxine hydrochloride
The standard stock solutions of PCM and

PDH were diluted with 0.3 M HCl individually
to get concentration of 10 g/mL of each drug.
The solutions were scanned in U.V range of
200-400 nm. The sampling wavelengths 254.5 and
290.5 nm was selected on trial and error basis and this
is shown in Fig. 3, 4. The concentrations of individual
drugs (i.e. PCM and PDH) in respective five mixed
standard solutions were fed to the multicomponent
mode of instrument and all five mixed standards
were scanned in range of 400-200 nm. The overlain
spectra of PCM and PDH and mixture are shown
in Fig.5.
Study of Beer-Lamberts law at selected
wavelengths
Fig. 3: UV Spectrum of PCM

PCM is a prochlorperazine maleate, 254.5 nm is a max of
PCM.
The basic necessity for the application of

proposed method is that all the selected sampling
wavelengths of the mixed standard solutions must
follow the Beer- Lamberts law in the concentration
between 1-5g/mL for PCM and 5-25 g/mL for
PDH respectively.
2
Fig. 1: Structure of prochlorperazine maleate
22
Fig. 4: UV Spectrum of PDH

PDH is a pyridoxine hydrochloride, 290.5 nm is a max of
PDH
the range of mixed standards. The results of this are

shown in Table I.
Peak - 2
Peak - 1
Fig. 5: Overlain spectra of PCM and PDH in mixed

standard solution
Prochlorperazine maleate (PCM, Peak 1) at wavelength
of 254.5nm in concentration of 1-5 g/mL and Pyridoxine
hydrochloride (PDH, Peak 2) at wavelength of 290.5nm in
concentration of 5-25 g/mL.
Fig. 6: The plot of linearity and range study

for PCM and PDH.
Prochlorperazine maleate (PCM) its R2 value is 0.999,
Pyridoxine hydrochloride (PDH) its R2 value is 0.999, ABS
is absorbance, CONC is concentration of PCM and PDH
respectively.
Analysis of laboratory mixture by proposed

method
Standard solution: Mixed standard solutions of 1
to 5g/mL were prepared.
Sample solution: Accurately weighed quantities, 5
mg of PCM and 25 mg of PDH were dissolved in 100
mL of 0.3M HCl. The above solution was appropriately
diluted with 0.3M HCl to obtain final concentration in
Analysis of marketed formulation by proposed

method
Marketed tablets Emidoxyn Forte (Shreya
Life Science Pvt Ltd., Mumbai) were used for the
simultaneous estimation of PCM and PDH. Twenty
tablets were accurately weighed. Average weight of
tablet was calculated. A quantity of tablet powder
equivalent to 5 mg of PCM was transferred to 100
mL volumetric flask and dissolved in 0.3M HCl and
volume was made to 100 mL, filtered and dilutions
were made as shown in Table II.
Method Validation
Accuracy
Accuracy of an analytical method is the
closeness of test results obtained by that method
to the true value. It was ascertained on the basis
of recovery studies performed by standard addition
method.
The preanalysed tablet powder equivalent to
about 5 mg PCM was taken in 100 mL volumetric
flask and accurately weighed quantities of 5 mg of
PCM and 25 mg of PDH were added to it followed by
addition of 50 mL 0.3M HCl. The mixture was shaken
for 15 min and volume was adjusted to the mark with
0.3M HCl. The solution was filtered through Whatman
filter no. 41. An aliquot portion of the resultant solution
was appropriately diluted with 0.3M HCl to get final
concentration within the range of mixed standard.
Results of recovery studies and statistical data are
shown in Table III.
Precision
Precision of analytical method is the degree
of agreement among individual results when the
method is applied repeatedly to multiple readings of
homogenous sample. It is expressed as the SD or
RSD of series of measurements. It was ascertained
by replicate estimation of the drugs by proposed
method.
23
Specificity
Accurately weighed quantities of tablet powder
equivalent to 5 mg of PCM were taken in different
50.0 mL volumetric flask and were stored for 24 hr
under following conditions.
1. At room temperature (normal)
2. At 500C after addition of 1.0 mL of 0.1N NaOH
(alkali)
3. At 500C after addition of 1.0 mL of 0.1N HCl
(acid)
4. At 500C after addition of 1.0 mL of 3% H2O2
(oxide)
The samples were diluted upto the mark with
0.3M HCl and filtered through grade-1 filter paper.
Aliquot of the filtrate was diluted with 0.3M HCl to get
10 g/mL concentration of PCM. The solution was
analyzed as per the procedure described for analysis
of laboratory mixture. The results of specificity studies
are shown in Table IV.
Ruggedness
Test for ruggedness was carried out by repeating
the procedure under different conditions
A. Days (Intraday and Interday)
B. Different analysts
Results of ruggedness study are shown in Table V.
Linearity and range
Accurately weighed quantities of tablet powder
equivalent to 80, 90, 100, 110 and 120% of label claim
were taken and dilutions were done appropriately
to obtain a concentration in the range of 80-120%
of the test concentration and absorbance values
were recorded at 254.5 nm and 290.5 nm. PCM and
PDH were found to be linear in 80-120% of the test
concentration. The plot of linearity and range for PCM
and PDH is shown in Fig. 6.
analytical method for simultaneous estimation of

PCM and PDH in their combined dosage form.
In this method drugs obey Beers law in the
concentration range 1-5 g/mL for PCM and 5-25
g/mL respectively.
The result of % estimation of drugs is shown in
Table II. The method was validated as per the ICH
and USP guidelines 11, 12, 13. The results of recovery
study were found to be within the prescribed limit of
98-102%, proving the accuracy and showing that the
method is free from interference from excipients. The
results are shown in Table III. For precision, replicate
estimations of both PCM and PDH in the same batch
of tablet were done by the proposed method, which
yielded quite concurrent results, indicating reliability
of the method. The values of SD or RSD are within
the prescribed limit of 2%, showing high precision of
method, as shown in Table II.
In the specificity study, sample was exposed to
different stress condition like acid, alkali, peroxide
and heat. The results for specificity study are shown
in Table IV. For ruggedness, proposed method was
repeated under different conditions like at different
time, on different days and by different analysts.
The results shown in Table V, prove that the method
is reproducible. During the linearity study it was
observed that absorbance values of PCM and PDH
in marketed formulation were linear in the range of
80% to 120% of test concentration with R2 close to 1
for this method of analysis. Plot of linearity and range
is shown in Fig.6.
From the study of validation parameters namely
accuracy, precision (SD and RSD), ruggedness
(interday, intraday and different analyst), specificity,
linearity and range, it was observed that the method
is specific, accurate, precise, reproducible and
rugged.
RESULTS AND DISCUSSION
CONCLUSION
An attempt has been made to develop a fast,

sensitive, precise, reproducible and economical
The present study comprises a UV spectroscopic,

multi-component mode of analysis for the simultaneous
24
determination of PCM and PDH in tablet dosage

form. From the study of validation parameters, it
was observed that the method is specific, accurate,
precise, reproducible and rugged. The proposed
method could be applied for routine analysis in quality
control laboratories.
ACKNOWLEDGEMENTS
The authors wish to thank Principal of Sharad
Pawar College of Pharmacy, Rashtrasant Tukadoji
Maharaj Nagpur University for providing necessary
facilities. They also thank Mr. Kamlesh Shende, FDA
Department, Mumbai for providing the authentic
sample of drugs.
REFERENCES
1. Budhavari S., The Merck index: An Encyclopedia of
Chemicals, Drugs and Biologicals. 13th ed. Whitehouse
Station (NJ): Merck Research Lab, Division of Merck;
2001.7850.
2. Budhavari S., The Merck index: An Encyclopedia of
Chemicals, Drugs and Biologicals. 13th ed. Whitehouse
Station (NJ): Merck Research Lab, Division of Merck;
2001.8072.
3. Poongothai S, Ilavarasan R and Karrunakaran CM:
Simultaneous and accurate determination of vitamins B1,
B6, B12, and alpha-lipolic acid in multivitamin capsule by
Reverse-phase high performance liquid chromatographic
method, Int J Pharm Pharm Sci. 2010,2(4),133-139.
4. Khor Swan-Choo and Tee E-Siong: Development of a
HPLC method for simultaneous determination of several
B-vitamins and ascorbic acid, Mal J Nutr. 1996,2,
49-65.
5. RadaAmidzic, JasminaBrboric and OliveraUdina :RPHPLC Determination of vitamins B1, B3, B6, folic acid
and B12 in multivitamin tablets, J. Serb. Chem. Soc.
2005, 70 (10), 12291235.
6. David Borsook, The Migraine Brain, David Borsook, Arne
May, Peter J. Goadsby, Richard Hargreaves, Oxford
University Press, New York 1st Edition.
7. Misao N., Katsuhiko M., Tsukioka T., Yamashita H.,
Inagaki N., Sugiyama T. and Itoh Y.: In vitro and in
vivocharacteristics of prochlorperazineoral disintegrating
film, ij Pharma.2009, 368. (2), 98102.
8. CaihongMou,Ganju N., Sridhar K. S. andAwtarKrishan:
Simultaneous quantitation of plasma doxorubicin and
prochlorperazine content by high-performance liquid
chromatography, J. Chromatography B. 1997,703,
217 224.
9. Adnan El-Yazigi, Fida Abdel and WahabBarmaAfrane:
Stability study and content uniformity of prochlorperazine
in pharmaceutical preparations by liquid chromatography,
J. Chromatography A.1995, 690. (1), 7176.
10. M a r c i n L e s z e k M a r s z a l l , A n n a L e b i e d z i s k a ,
WojciechCzarnowski and PiotrSzefer: High-performance
liquid chromatography method for the simultaneous
determination of thiaminehydrochloride, pyridoxine
hydrochloride and cyanocobalamin in pharmaceutical
formulations using coulometric electrochemical and
ultraviolet detection, J. Chromatography A. 2005, 1094,
(2), 9198.
11. ICH Q2B; Guidelines on validation of analytical procedure;
Definitions and terminology, Federal Register, 1995, 60,
11260.
12. ICH Q2B; Guidelines on validation of analytical procedure;
Methodololgy, Federal Register, 1996, 60, 27464.
13. The United States Pharmacopoeia 24/ National Formulary
19, The United States Pharmacopoeial Convection,
Rockville, 2000, 2151.
Available DVD of IP 2010

DVD of IP 2010 is available from the office of the Secretary-cum-Scientific Director,
I.P.Commission, Sector-23, Raj Nagar, Ghaziabad 201002, (U.P.), @ Rs.25,000/-per
DVD.For more details please visit the website: www.ipc.gov.in; E.mail Id: ipclab@vsnl.net
or contact to The Publication Division, I.P.Commission, Tel.Nos.: 0120-2783392, 2783400,
Extn.309, 308 & 112.
25
A Simple and Rapid HPLC Method for Estimation of Dimethicone

from Formulations
Jadhav J. J.*, Mungekar S., Velada J. V., Doshi H. A. , Gajbe V. and Raunak K.
Abstract
A simple, sensitive, precise and specific normal phase high performance liquid chromatography (HPLC)
method was developed and validated for the determination of dimethicone from tablet dosage forms.
It was found that the excipients used in the tablet dosage form did not interfere in the quantification of
dimethicone. The HPLC separation was carried out by normal phase chromatography on Princeton
Sphere Cyano, 250 x 4.6mm, 5 with a mobile phase composed of hexane : ethanol : ethyl acetate
(80:20:0.2) in isocratic mode at a flow rate of 0.5mL/min. Dimethicone was quantified using a refractive
index detector. The calibration curve for dimethicone was linear from 1.75 to 3.25 mg/mL. The inter-day
and intra-day precisions were found to be within limits. The proposed method has adequate sensitivity,
reproducibility and specificity for the determination of dimethicone from tablet dosage forms.
Keywords: Dimethicone, HPLC, Refractometer,

Isocratic, Validation.
Simethicone is a mixture of liquid dimethicone
containing finely divided silicon dioxide to enhance
the defoaming properties of the silicone. Simethicone
is used for the relief of flatulence and abdominal
discomfort due to excess gastrointestinal gas in
disorders such as dyspepsia and gastro-oesophageal
reflux disease. It lowers surface tension and,
when given orally, causes bubbles of gas in the
gastrointestinal tract to coalesce, thus aiding their
dispersion1. It contains not less than 90.5% and not
more than 99% of polydimethylsiloxane (Dimethicone)
and not less than 4% and not more than 7% of silicon
dioxide2 (Fig. 1).
The limited analytical methods for dimethicone
are based on Fourier transform infrared spectroscopy
(FT-IR) and reversed phase high performance liquid
chromatography (RP-HPLC) using evaporative
*For correspondence
Indeus Life Sciences Pvt. Ltd
SU Motors Complex
Balrajeshwar Road, Opp. Model Town
Mulund (W), Mumbai 400080
E-mail: Jaideep.jadhav@indeus.in
26
light scattering detection (ELSD)3,4,5. The active

ingredient has an official monograph in the United
States Pharmacopeia (USP)6 and the European
Pharmacopoeia (EP)2 in which the method is based
on quantitative infrared (IR) spectroscopy.
We report a simple and rapid HPLC method for
quantification of dimethicone using a cyano column
and a refractive index detector.
MATERIALS AND METHODS
The chromatographic separation was performed
on a Waters (Philadelphia, USA) HPLC equipped with
a refractive index detector (model no. 2414). Waters
Empower 2 software was used for data capture and
analysis.
Hexane, ethanol and ethyl acetate were procured
from Merck (Mumbai, India) and were of HPLC grade.
Dimethicone working standard was procured from
Riocare India Pvt. Ltd. (Navi Mumbai, India).
Dimethicone content from tablets was estimated
by two different methods. The infrared method is
reported in the USP while the HPLC method was
developed in-house. The details of the methods are
as follows:
y = 69252x 15545
r=0.999
Fig. 1: Simethicone = Dimethicone + Silicon dioxide

0.45
Abs
0.4
Fig. 4: Linearity plot for dimethicone obtained by the

new HPLC method
Quantification using infrared spectroscopy
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0.05

1320 1305 1290 1275 1200 1245 1230 1215 1200 1185
Fig. 2: Infra-red overlay spectrum of dimethicone

standard and sample solution
Sufficient tablets were crushed to a fine composite

and a sample equivalent to 50 mg of dimethicone
was transferred to a 200 mL volumetric flask. To this
25 mL of toluene was added and the contents were
shaken on a mechanical shaker for 10 minutes. To
this 50 mL of dilute hydrochloric acid was added and
was shaken again for 20 minutes, after which it was
left to settle in a separating funnel for 2 hours. The
toluene layer was separated and was treated with
0.5 g of sodium sulphate. The toluene layer was
then analyzed on an infrared spectrophotometer in
a 0.5 mm NaCl cell. A scan was run between 1180
and 1330 cm-1. The absorbance was determined at
1261.45 cm-1.
The standard solution was prepared in the same
way, using dimethicone. A blank was prepared
by treating toluene with sodium sulphate. The
infrared spectra of standard and sample were
recorded (Fig. 2).
Quantification using HPLC method
Fig. 3: Representative overlay chromatogram of the

diluent, dimethicone standard and sample solution
Sufficient tablets were crushed to a fine composite

and sample equivalent to 500 mg of dimeticone was
transferred to a 200 mL volumetric flask. To this, 150
mL of a mixture of hexane and ethanol (4:1) was
added. The contents were sonicated for 10 minutes
with intermittent shaking. The flask was allowed to cool
to room temperature and the contents were diluted to
200 ml using the same diluent. After thorough mixing,
27
the dispersion was filtered through a 0.45 PTFE

filter. The first 2 mL of the filtrate was discarded and
20l from the remaining part was injected onto a
HPLC. The chromatographic conditions are denoted
in Table I.
Table I: Chromatographic conditions for the
analysis of dimethicone
Column: Princeton Sphere Cyano
(250 x 4.6 mm), 5
Mobile phase: hexane : ethanol : ethyl acetate
(1600 : 400 : 4)
Column temperature: 35C
Table III: Method validation parameters for the

HPLC method
Parameter
Linearity
Correlation coefficient (r)
Slope
Y-intercept (%)
Range / Value
1.75 3.25 mg/mL
0.999
692527
-0.2
Accuracy (%) (n=12)

101.0%
RSD (%) for mean accuracy 1.06 %
(%) (n=12)
Method precision, mean 101.0%
recovery (%) (n=6)
RSD (%)
0.56
Auto-sampler temperature:10C
RI Detector Sensitivity: 32
Injection volume: 20 l
Intermediate precision
101.2
RSD (%) for intermediate 1.25
precision (n=12)
Flow rate: 0.5 mL/min

Run time: 20 min
RI Detector Temperature: 40C
Filter recovery (%)

Table II: Results of the analysis of dimethicone
content from simethicone tablets using infrared
spectroscopy and HPLC methods
Sample
1
2
3
4
5
6
7
8
9
10
11
12
Mean
SD
RSD %
N
28
IR
94.9
95.2
95.2
94.9
96.9
94.9
96.4
95.5
95.6
96.8
97.4
96.9
95.9
0.93
0.97
12
HPLC
94.6
94.1
94.1
94.3
94.5
95.0
95.0
94.9
95.0
95.9
96.0
96.0
95.0
0.70
0.73
12
Nylon filter (0.45m)
99.02%
PVDF Filter
99.37%
PTFE Filter
101.35%
Stability in analytical solution The standard and

sample were found
to be stable for 72
hours
Precision
System precision: RSD (%)
(n=6)
Intermediate precision:
Cumulative RSD (%) (n=12)
Total
95.4
0.93
0.98
24
1.45%
1.25%
Twelve samples were analyzed by infrared

spectroscopy and other twelve by HPLC.
Subsequently, the methods were assessed for
interchangeability. Chromatograms of the blank,
standard and sample were found to be free from
interference (Fig. 3).

The quantification of dimethicone content from
tablets by the two different methods yielded the results
summarized in Table II.
The newly developed HPLC method was validated
as per the requirements of the current ICH guidelines
and the results of the validation are mentioned in
Table III. The response was found to be linear from
1.75 3.25 mg/mL (Fig. 4).
No interference was observed from common
excipients generally used in tablets.
Samples were analyzed using the already
reported pharmacopoeial infrared method and using
the newly developed and validated HPLC method.
The results from the two different methods were
compared to assess interchangeability. The WHO
guideline8 on transfer of analytical methods was
used to determine the criteria for interchangeability
between the methods. Two criteria were evaluated,
viz., the variability in terms of the relative standard
deviation (RSD) and the difference between the
means.
The methods can be considered interchangeable
if the RSDs calculated are less than 2%. The RSDs
(%) were found to be 0.97%, 0.73 % and 0.98
% for the IR method, the HPLC method and the
complete data set (i.e. the IR and HPLC results
combined), respectively. Thus the first criterion
was fulfilled, as the RSDs were calculated to be
less than 2 %.
The methods could be considered interchangeable
if the difference between means of both the methods
was smaller than 2 % at a 95 % confidence level.
To compare the means, an independent two sample
t-test was applied. The 95% confidence interval of
the difference was found to be 0.24% - 1.63%. Thus
second criterion was also fulfilled, as the difference
between means of both the methods, although being
significant (p=0.011), was smaller than 2% at 95%
confidence level.
By meeting both criteria, it can be concluded that

interchangeability of the IR method and the HPLC
method was confirmed.
CONCLUSION
While pharmacopoeial method are based on
quantitative IR spectroscopy, new rapid and simple
HPLC method for the estimation of dimethicone
from tablets was developed and validated. This
method may be used to estimate dimethicone
from simethicone and from other dosage forms
as both IR and HPLC methods were found to be
interchangeable.
ACKNOWLEDGEMENTS
The authors wish to thank Onne Backers of
Disphar International BV for his expert help with the
statistical analysis of data and Rasielle Gonzales for
review of the manuscript.
REFERENCES
1. Monograph on Simethicone, Martindale The Extra
Pharmacopoeia, www.medicinescomplete.com, 2012.
2. Monograph on Simethicone, The European Pharmacopoeia
Volume 7.2, 2012, pp. 3711.
3. Torrado G, Garca-Arieta A, de los Ros F, Menndez JC,
Torrado S.: Quantitative determination of Dimethicone in
commercial tablets and capsules by Fourier transform
infrared spectroscopy and antifoaming activity test. J.
Pharm. Biomed. Anal. 1999, 19(3-4), 285-292.
4. Moore D.E., Liu T. X., Miao W.G., Edwards A, Elliss
R.: A RP-LC method with evaporative light scattering
detection for the assay of Simethicone in pharmaceutical
formulations. J. Pharm. Biomed. Anal. 2002, 30(2),
273-278.
5. Mojsiewicz-Piekowska K. Size exclusion chromatography
with evaporative light scattering detection as a method
for speciation analysis of polydimethylsiloxanes. III.
Identification and determination of dimethicone and
simethicone in pharmaceutical formulations. J. Pharm.
Biomed. Anal. 2012, 58, 200-207.
6. Monograph for Simethicone Tablets, The United States
Pharmacopeia, Volume 35, 2012, 4639
7. ICH guideline Q2B: Validation of Analytical Procedures:
Methodology, IFPMA Geneva
8. WHO Technical Report Series, No. 961, 2011, Annex
7, WHO Guidelines on Transfer of Technology in
Pharmaceutical Manufacturing, pp: 19.
29
Simultaneous Estimation of Montelukast Sodium and Desloratadine

in Bulk and in Tablet Formulation by UV-Spectophotometry
Jain R. R., Patil P. O.* and Bari S. B.
Abstract
Novel, simple, sensitive and rapid spectrophotometric method has been developed for simultaneous
estimation of montelukast sodium (MONTE) and desloratadine (DES). The method involved solving
simultaneous equations based on measurement of absorbance at two wavelengths 285.6 nm and 245
nm, the max values of MONTE and DES, respectively. Beers law was obeyed in the concentration
range of 4-24 g/mL and 2 12 g/mL for MONTE and DES, respectively. The method was validated for
accuracy, precision and recovery studies. The developed method was precise, reproducible, selective,
specific and accurate for quantitative estimation of MONTE and DES, in combination. The wide linearity
range, sensitivity, accuracy, and simple procedure demonstrated the method to be appropriate for
routine analysis and quality control assays of their tablets. The method was validated according to the
ICH guidelines.
Keywords: Montelukast sodium, Desloratadine,

Simultaneous equation method, Spectrophotometry.
INTRODUCTION
Montelukast sodium [1-((((1R)-1-(3-((1E)2-(7-chloro-2-quinolinyl)ethenyl)phenyl)-3-(2-(1hydroxy-1-methylethyl)phenyl)propyl)thio)methyl)
cyclopropaneacetic acid], monosodium salt, is a
white colored powder, freely soluble in ethanol, and
methanol 1. Molecular weight of montelukast sodium is
608.2 and molecular formula is C35H35ClNO3S.Na2.
Montelukast (sodium salt) is potent, selective
CysLT1 receptor antagonist. It is indicated for the
prophylaxis and chronic treatment of asthma in adults
and pediatric patients. The drug is commercially
available in various forms of once-daily oral dosage
formulations including oral granules. In oral dosage
form, each packet contains montelukast sodium
equivalent to 10 mg of montelukast 3.
*For correspondence
H. R. Patel Institute of Pharmaceutical
Education and Research,
Shirpur, Dist: Dhule (M.S.) 425405
E-mail: rxpatilpravin@yahoo.co.in
30
Desloratadine is an H1 receptor antihistaminic

drug, developed in 2005. It is a metabolite of
loratadine, a second generation antihistaminic drug.
Desloratadine is a second generation tricyclic anti
histaminic drug, which has a selective and peripheral
H1 antagonist action. It is an active form of a prodrug,
loratadine. Chemically, it is 8-chloro-6, 11- di hydro11(4-piperdinylidene)- 5H (5,6 cycloheptyal (1,2-b)
pyridine. It is a white to off white powder with molecular
weight of 310.84 4. The chemical structures of both
the drugs are as shown in Fig. 1 & 2.
Montelukast is official in Indian Pharmacopoeia 1.
Literature survey reveals few UV-spectrophotometric 3-5 and RP-HPLC 5-9 methods for the estimation of MONTE in combination with other drugs in bulk
and in pharmaceutical dosage forms. Similarly, several
methods, viz., UV-spectrophotometric 10, RPHPLC11-12,
LC-MS/MS13-14 UPLC 15 are reported in the literature
for determination of DES in bulk and in pharmaceutical formulations. Patel et al., have reported derivative
spectroscopic method for simultaneous estimation of
montelukast sodium and desloratadine in bulk and
combined dosage form 16.
In the present work, an endeavor has been made
to estimate both these drugs simultaneously by a
simple UV-spectrophotometric method (simultaneous

equation method). These methods were validated
according to the ICH guidelines 17-19.
MATERIAL AND METHODS
Chemicals and Reagents
The bulk drugs, montelukast sodium and
desloratadine, were obtained from Ranbaxy Lab. Ltd,
Gurgaon, India, as gift samples. Methanol (HPLC
Grade) was purchased from Merck (India) Ltd., Worli,
Mumbai, India, and was selected as common solvent
for studying spectral characteristics of drugs. The
Mondeslor tablets, containing 10 mg of MONTE and
5 mg of DES were purchased from Indian market.
Instrumentation
mL for MONTE and 2 - 12 g/mL for DES. The

absorbances of these standard solutions were
measured at 285.6 nm and 245.0 nm and calibration
curves were plotted. Two simultaneous equations
(in two variables C1 and C2) were formed using E
(1%, 1cm) values (Table II).
A1 = 336.875 CMONTE + 570.0 CDES
A2 = 333.750 CMONTE + 137.5 CDES
(2)
where, CMONTE and CDES are the concentrations

of MONTE and DES, respectively, in g/100 mL in a
sample solution and A1 and A2 are the absorbances
of the mixture at the selected wavelengths, 285.6 nm
and 245.0 nm, respectively.
A UV-Visible spectrophotometer (Shimadzu1700, UV Probe 2.21 software) with spectral

bandwidth of 1 nm was employed for all spectroscopic
measurements, using a pair of 1.0 cm matched quartz
cells over the range of 200 400 nm.
Table I: Linearity studies

Parameter
MONTE
DES
Linearity
4 - 24
2 12
-1
[g mL ]
Linearity
Y = 0.038X +
Y = 0.058 X+
Equation
0.068
0.004
4
Molar
2.048 10
1.767 104
Absorptivity
Mean
101.080.00467 101.320.01137
Recovery (%)
Precision
(% RSD)
Inter day
1.244
1.583
Intra day
1.309
1.410
Specificity
Specific
Specific
Robustness
Robust
Robust
Selection of a solvent
Methanol was selected as common solvent for
studying spectral characteristics of drugs.
Preparation of stock solutions of standards
Stock solutions of MONTE and DES were
separately prepared by dissolving 10 mg of each in
50 mL methanol and volume was made up to 100 mL
with the same solvent to obtain a final concentrations
as100 g/mL.
Simultaneous equation method
From the stock solutions of 100 g/mL of both the
drugs, working standard solutions were prepared by
appropriate dilution and scanned in the UV-region.
Linearity was found in the concentration range of
4-24 g/mL and 2 12 g/mL for MONTE & DES,
respectively (Table I). From the overlain spectra
(Fig. 3) two wavelengths, 285.6 (max of MONTE)
and 245.0 (max of DES), were selected for
developing equations. Standard solutions were
prepared in the concentration range of 4-24 g/
(1)
Table II: E (1%, 1cm) values of MONTE and DES

Sr. No
(E 1%, 1cm) at
(E 1%, 1cm) at
285.6 nm
245.0 nm
1
MONTE
DES
MONTE
DES
Mean 336.875
137.5
333.75
570.0
SD 0.001414 0.001789 0.001789 0.004535
%RSD 0.2623
1.6411
0.3349 0.9967
a
Average of six estimations

31
By applying the Cramers rule 20 to equations 1

and 2, the concentrations CMONTE and CDES, can be
obtained as follows:
C1 = (A2 x 137.5) - (A1 x 570) / - (146125.28)
(3)
C2 = (A1 x 333.75) - (A2 x336.875) / - (146125.28) (4)
Assay of tablet formulation

Twenty tablets containing MONTE and DES were
weighed and the mean weight of a tablet was found out.
The tablets were crushed and an accurately weighed
tablet powder, equivalent to 10 mg of MONTE that
contains 5 mg DES, was transferred into a 100 mL
volumetric flask, containing 50 mL of methanol. The
volume was made up to the mark with methanol and
the solution filtered through 0.45 m Whatman filter
paper. An appropriate volume of solution was further
diluted with methanol to obtain concentration of 16
g/mL of MONTE and 8 g/mL of DES. Absorbance
of a sample solution was recorded at 285.6 nm
and 245.0 nm and the concentrations of two drugs
in the sample were determined using equations
3 and 4. The analysis procedure was repeated six
times with tablet formulation. The results of analysis
are mentioned in Table III.
RSD of the assay results, expressed as a percentage

of the label claim, was used to evaluate the method
precision. The obtained RSD values were found to
be 1.16 to 1.54% and 1.13 to 1.73% for MONTE and
DES, respectively. The inter-day precision was also
determined by assaying the tablets in triplicate,
per day for consecutive 3 days, which were found
to be 1.10 to 1.48% and 1.15 to 1.93% for MONTE
and DES, respectively. The results are shown in
Table I. The results are revealed good precision of
the developed method.
Sensitivity
Sensitivity of the method was determined
individually for each drug by calculating the LOD
and LOQ (Table VII).
The accuracy of the proposed analytical method

was determined by recovery experiments. The
recovery studies were carried out at three different
concentration levels (80, 100 and 120%). A known
amount of drug was added to the pre-analysed tablet
formulation at three different concentration levels,
i.e. 80 %, 100 % and 120 %, and percent recovery
was calculated. The results of recovery studies were
satisfactory and are presented in Table IV.
Montelukast and desloratadine followed linearity

in the concentration range of 4 - 24 g/mL and 2
12 g/mL, respectively and the results are shown
in Table I, (Fig. 4,5). Marketed brand of tablets was
analyzed and the amount of MONTE and DES,
determined by the proposed method, was found
to be 100.82 % for MONTE and 101.07%, for DES
respectively. The % recoveries ranged from 99.33 to
102.37 for DES and 100.80 to 101.30% for MONTE,
respectively. Precision was calculated as inter and
intraday variations (% RSD is less than 2) for both
drugs and as repeatability (% RSD is less than 2),
for both the drugs, and are presented in Tables V,
VI. The LOD and LOQ of MONTE and DES were
found to be sufficiently low (Table VII). The method
is sufficiently sensitive to permit determination of
MONTE and DES upto 2 g mL-1 and 4 g mL-1
respectively.
Precision
CONCLUSION
Precision of an analytical procedure expresses

the closeness of agreement between a series of
measurements obtained from multiple samplings of
the same homogenous samples under the prescribed
conditions. The precision of the method was verified
as intra-day, inter-day and repeatability studies. The
The results of our study indicate that the

proposed UV spectroscopic method is simple,
rapid, precise and accurate. The developed UV
spectroscopic method was found suitable for
determination of MONTE and DES as bulk drugs
and also for the marketed solid dosage formulation
Percent recovery studies
32
Table III: Results of simultaneous estimation of marketed tablets, Mondeslor

Component Label Claim (mg) Amount Taken (g/mL) Amount Found (g/mL) Amount found (%)
MONTE
16
16
16.10
100.62
16
16.08
100.52
16
15.96
99.78
16
16.06
100.36
16
16.04
100.27
DES
16
Mean
SD
% RSD
8
8
8
8
8
8
Mean
SD
%RSD
16.03
16.05
0.049
0.30
7.96
7.93
8.03
8.01
7.99
7.98
7.98
0.0044
0.56
100.19
100.29
0.30
0.30
99.50
99.15
100.38
100.12
99.88
99.75
99.80
0.44
0.44
Table IV : Results for recovery studies

Sr.
No.
Initial Amount
(g/mL)
% Recovery
Amount Added
Concentration of
[n = 3]
Excess Drug Added Drug Obtained
[g/mL]
to Analyte [g/mL]
MONTE
DES
MONTE DES
MONTE
DES
MONTE
DES
1.
16
12.8
6.4
12.9
6.5
101.3
2.
16
16
16.1
8.1
16
19.2
9.6
19.3
9.5
% RSD
MONTE
DES
102.25
0.70
1.49
101.1
102.37
0.17
1.42
100.8
99.33
0.38
0.82
Table V : Results from precision studies

Sr.No
Drug
Amount Taken
(g/mL)
Intraday n =3
Amount Found (g/mL)
MONTE
DES
12
16
20
4
8
12
12.23
16.21
20.21
4.09
8.09
12.20
Interday n = 3
%RSD Amount Found (g/mL) %RSD
1.09
1.47
1.15
1.54
1.67
1.15
12.07
15.97
19.97
4.09
7.86
12.19
1.53
1.15
1.23
1.07
1.13
1.36
33
Table VI: Results of Repeatability Study

Sr. No
1
2
a
Drug
[g/mL]
MONTE
DES
Amount found
in [g/mL]
16.18
8.12
%RSD
1.220
1.616
Average of six estimations

Table VII: Sensitivity
Parameter
LOD [g/mL]
LOQ [g/mL]
MONTE
0.088
0.291
DES
0.129
0.249
Fig. 3: Overlain spectra of MONTE and DES
Fig. 1: Montelukast sodium
Fig. 4: Calibration curve of montelukast sodium
Fig. 2: Desloratadine
34
Fig. 5: Calibration curve of desloratadine

without any interference from the excipients.

Statistical analysis proves that the method is
repeatable and selective for the analysis of MONTE
and DES. It can therefore be concluded that use
of the method can save time and money and it can
be used in small laboratories with accuracy and
a wide linear range.
9.
10.
ACKNOWLEDGEMENTS
The authors are thankful to H.R. Patel Institute
of Pharmaceutical Education and Research, Shirpur
(M.S.), India, for providing facilities to carry out this
research work and also thankful to the Ranbaxy
laboratories, Gurgaon, for providing gift samples of
montelukast sodium and desloratadine.
11.
12.
REFERENCES
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& Family Welfare, published by Indian Pharmacopoeial
convention, Ghaziabad,Vol. II, 2010 p.1704 -1706.
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and Biologicals, 14th edn., Merck Research Laboratories,
Whitehouse Station, New Jersey, USA, 2006 p. 514,
1117.
3. Garg L. K., Kumar B. R., Sait S. S., Krishnamurthy
T.: Determination of montelukast sodium in oral
granules dosage forms by a simple and accurate UV
spectrophotometric methods, Int J of pharm sci Rev
and Res. 2011, 7(2): 69-72.
4. Satish B., Sudarshan R.: Spectroscopic method for
determination of desloratadine in bulk and its tablet
dosage forms, Int J Pharm and Ind Res. 2011, 01(2):
131-134.
5. Radhakrishna T., Narasaraju A., Ramakrishna M.,
and Satyanarayana A.: Simultaneous determination
of montelukast and loratadine by HPLC and derivative
spectrophotometric methods, J Pharm Biomed Anal.
2003, 31(2): 359-368.
6. Choudhari V., Kale A., Abnawe S., Kuchekar B., Gawli
V., Patil N.: Simultaneous determination of montelukast
sodium and levocetirizine dihydrochloride in pharmaceutical
preparations by ratio derivative spectroscopy, Int J of
Pharm Tech Res. 2010, 2 (1): 4-9.
7. Ibrahim A. A.: Development of a stability-indicating HPLC
method for the determination of montelukast in tablets and
human plasma and its applications to pharmacokinetic
and stability studies, Saudi Pharmaceutical Journal.
2004, 12(4): 136-143.
8. Singh R. M., Saini P. K., Mathur S. C., Singh G. N., and
Lal B.: Development and validation of a RP-HPLC method
for estimation of montelukast sodium in bulk and in tablet
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Kitchen C. J., Wang A. Q., Musson D. G., Yang A. Y., Fisher
A. L.: A semi-automated 96-well protein precipitation
method for the determination of montelukast in human
plasma using high performance liquid chromatography
/uorescence detection, J Pharm Biomed Anal. 2003,
31: 647-654.
Caglar, S., Oztun A. A.: sensitive spectrophotometric
method for the determination of desloratadine in tablets,
J AOAC Int. 2007, 90(2): 372-5.
Razib B. M., Ullah A., Azad M. A., Sultana R., Yasmin
H., Hasnat A.: Validation and application of modified
RP-HPLC method for the quantification of desloratadine
in pharmaceutical dosage forms, Dhaka Univ J Pharm
Sci. 2006, 5(1-2): 1-4.
El-Sherbiny D. T., El-Enany N., Belal F. F., Hansen
S. H.: Simultaneous determination of loratadine and
desloratadine in pharmaceutical preparations using liquid
chromatography with a microemulsion as eluent, J Pharm
Biomed Anal. 2007, 43: 1236-42.
Xu H., Lie X., Chen W., Chu N.: Simultaneous
determination of desloratadine and its active metabolite
3-hydroxydesloratadine in human plasma by
LC/
MS/MS and its application to pharmacokinetics and
bioequivalence, J Pharm Biomed Anal. 2007, 45: 65966.
Jun Wen., Zhanying Hong., Yiwen Wu., Hua Wei.,
Guorong Fan., Yutian Wu.: Simultaneous determination
of rupatadine and its metabolite desloratadine in human
plasma by a sensitive LCMS/MS method, application to
the pharmacokinetic study in healthy chinese volunteers,
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Rao D. D., Satyanarayana N. V., Reddy A. M., Sait S. S.,
Chakole D., Mukkanti K. A.: validated stability-indicating
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Patel S. V., Patel G. F., Pipaliya S. G.: derivative
spectroscopic method for simultaneous estimation of
montelukast sodium and desloratadine in bulk and
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Beckett A. H., Stenlake J. B.: Practical Pharmaceutical
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and Distributors; 2002, p.272-280.
35
ANTI ATHEROSCLEROTIC EFFECT OF ETHANOLIC STEM EXTRACT OF CISSUS

QUADRANGULARIS LINN
Soumya V.S., and Dominic S.*
(Received 29 June 2012) (Accepted 23 January 2013)
Abstract
Atherosclerosis and the subsequent complications cause many deaths world wide. Though many
medications like statins and surgical procedures are available to tackle this problem, none of them is
hazard free. In the present study the ethanolic extract of the plant Cissus quadrangularis, traditionally
used for many ailments including elevated blood cholesterol is evaluated scientifically on rat model
of hypercholesterolemia for its antiatherosclerotic effect. The study done in atherogenic diet induced
hypercholesterolemic rats, in comparison with the standard drug Atorvastatin revealed the dose
dependent effect of the extract to increase the antioxidant levels like SOD and catalase and to decrease
the damage due to lipid peroxidation. Moreover, the plant exhibited significant anticoagulant and
membrane stabilizing effect apart from decreasing LDL and VLDL and increasing HDL level, all relevant
in the prevention of complications due to atherosclerosis. Histopathological examination of liver and
coronary artery of the treated rats also suggested the protective effect of the extract in atherosclerosis.
Keywords: Atherosclerosis , Cissus quadrangularis,

Antioxidants, Atherogenic diet.
INTRODUCTION
Atherosclerosis is a progressive disorder
characterized by the formation of lesions called
atherosclerotic plaques in the walls of large and
medium sized arteries including coronary, carotid,
cerebral arteries, aorta and its branches. It is
characterized by endothelial dysfunction, vascular
inflammation and the build up of lipids, cholesterol,
calcium and cellular debris within the intima of
the vessel wall. This build up results in vascular
remodeling, obstruction, abnormalities of blood flow
and diminished oxygen supply to target organs1.
A complex interaction exists among the critical
cellular elements of atherosclerotic lesion. Elevated
serum levels of cholesterol overwhelm the antioxidant
properties of the healthy endothelium and results in
abnormal endothelial metabolism of these lipid moiety.
*For correspondence
Govt. Medical College
Thiruvananthapuram- 695011, Kerala
E-mail: shinidm@yahoo.com
36
Oxidized LDL is capable of causing a wide range of

toxic effects and cell/vessel wall dysfunction that are
characteristically and consistently associated with the
development of atherosclerosis. Besides, oxidized
LDL activates inflammatory processes leading to
rupture resulting in thrombus formation, partial or
complete occlusion of blood vessel and progression
of atherosclerotic lesion.
Statins2 are the widely prescribed drugs for
preventing and treating atherosclerosis. They reduce
blood cholesterol by inhibiting HMG-CoA reductase
responsible for cholesterol formation. The main
limitation for the use of statins is hepatotoxicity apart
from the rare serious rhabdomyolysis. Many patients
still experience adverse coronary events despite statin
therapy. Many medicinal plants with minimal or no
side effects have shown their beneficial effects in
cardiovascular disorders by virtue of their antioxidant,
anti coagulant and anti-inflammatory effects3,4.
Cissus quadrangularis is a common climbing herb
in India traditionally used for many ailments5. But there
is no scientific study to prove its antiatherogenic effect
in comparison with statin therapy. So the present
work was aimed to evaluate its antiatherosclerotic
effect on rat model of cholesterol diet induced
hypercholesterolemia6 by verifying its antioxidant,

anticoagulant, anti-inflammatory and hypolipidaemic
effects along with the histopathological aspects.
Sample collection
The stem of Cissus quadrangularis collected from
Neyyardam during the month of September 2010
and authenticated by G.R. Jayakumar, Research
officer Poojappura was extracted using 95% ethanol
and this extract was suspended in 0.3% CMC for
oral administration in rats (LD 50 of this extract was
reported to be more than 5000 mg/kg)7.
Ethical clearance
The study protocol was approved by the
Institutional Animal Ethical Committee,Medical
College,Thiruvananthapuram.(IAEC No 04/17/2010/
MCT)
Atherogenic diet 8
Hypercholesterolemia in rat was induced by
gavage of 1mL/100g body weight of a cocktail
containing 1 litre peanut oil, 100g cholesterol,
30g propylthiouracil and 100g cholic acid over a period
of 14 days. The test compounds were administered
concurrently with the cocktail.
Study groups
Albino rats of either sex weighing 100-200 g
were used for the study9. The animals were divided
into five groups of six rats each and administered
the following.
Group I-The animals fed with the standard
commercial pellet diet daily (normal control).
Group II- The animals orally administered with
the atherogenic diet at 10 AM daily for 14 days days
(atherogenic control).
Group III- The animals orally administered with
Atorvastatin (30mg/kg) after the administration of the
atherogenic diet at 10 AM daily for 14 days.
Group IV- The animals orally administered with

the extract of Cissus quadrangularis (500mg/kg )
after the administration of the atherogenic diet at
10 AM daily for 14 days.
Group V- The animals orally administered with
the extract of Cissus quadrangularis (1000mg/kg )
after the administration of the atherogenic diet at 10
AM daily for 14 days .
After 14 days of drug administration animals
were sacrificed by cervical dislocation, and organs
removed.
Anti Oxidant Enzyme Assay
Different antioxidant enzyme assays were carried
in the liver homogenate.
Preparation of liver homogenate
The liver was washed in ice cold saline, 1 gm
of liver was sliced and added to four volumes of a
solution containing trihydrochloride buffer (pH 7.2)
This mixture was taken in a clean sterile centrifuge tube
and homogenized to get a 25% tissue homogenate.
Homogenate was centrifuged at 15000 rpm at 40C
for 15 minutes and the supernatant stored at 40C was
used for estimation of reactive oxygen species in terms
of lipid peroxidation, SOD and Catalase. Remaining
portion of liver was used for histopathological
examination.
Lipid Peroxidation Assay 10,11
Malondiealdehyde (MDA) is one of the several
low molecular weight end products formed via
decomposition of certain primary and secondary
lipid peroxidation products. At low pH and elevated
temperature, MDA readily participates in nucleophilic
addition reaction with 2-thiobarbituric acid (TBA)
generating a red fluorescent 1;2 MDA;TBA adduct
that absorbs strongly at 532 nm.
To 0. 2mL of 25% (w/V) liver homogenate added
0.2 mL of 8.1 % Sodium dodecyl sulphonate, 1.5
mLof 20 % acetic acid solution and adjusted to pH 3.5
with NaOH, and added 1.5 mL of 0.8% thiobarbituric
37
acid. The mixture was made up to 4 mL with distilled

water and then heated in a water bath at 95 0C for
60 minutes. After cooling under tap water added 5
ml of mixture of n-Butanol and pyridine (15 :1 V/V)
and shaken vigorously. The mixture was centrifuged
at 4000 rpm for 10 minutes, the organic layer was
separated and its absorbance was taken at 542 nm.
Pure sample of butanol pyridine mixture was taken
as blank.
MDA =
50mM phosphate buffer, the control tubes were kept

in darkness and light control tubes were kept under
an incandescent lamp. After 30 minutes of incubation,
the optical density of test, light control and control
solution were read at 560 nm.
The SOD activity of homogenate in units/mg
protein was determined using the equation
SOD = 100 - optical density of sample
Absorbance D.F 10 9
Optical density of light control/50
Molar absorptivity
The amount of soluble protein present in the
extract was determined by the standard method
proposed by Biuret et al using bovine serum albumin
as the standard.
The intensity of lipid peroxidation was expressed

as nano moles of malonaldehyde per mg of protein
using molar absorptivity 1.56x 105 M cm-1
Superoxide Dismutase Assay 12,13
Superoxide dismutase is a group of metal
containing enzymes that catalyses the dismutation of
highly reactive superoxide anion to O2 and to the less
reactive species H2O2. The assay utilizes riboflavin
as a photosensitizer and methionine as a reducing
substrate. This method employs riboflavin to generate
superoxide radicals in presence of light which react
with nitroblue tetrazolium to form a red formazan dye
which give absorbance at 560 nm.
The assay was carried out in the prepared liver
homogenate. The reaction mixture of control, light
control, blank and test solution were prepared by
adding 0.1 mL of 1. 5 M sodium carbonate,0.3 mL
of 0.13 M methionine,0.3 mL of 10mM EDTA, 0.3
mL of 0.63 mM nitroblue tetrazolium (fresh, absent
in blank) 0.3 mL of 13 M riboflavin and 0.1 mL
homogenate (absent in blank and light control). The
mixture was made upto a final volume of 3mL using
38
100
OD of sample= optical density of test- optical

density of control
Catalase Assay 14
Catalase catalyzes the conversion of hydrogen
peroxide into molecular oxygen and water. The assay
was carried out in the prepared liver homogenate.
50L of the tissue homogenate was added to a 3mL
cuvette that contained 1.95 ml of 50 mM phosphate
buffer (pH 7.0).To this 1.0 mL of 30mM hydrogen
peroxide was added and change in absorbance was
followed for 75 seconds at 240nm at 50 seconds
intervals. Pure sample of phosphate buffer was
taken as blank
C=
A 10 6
Ia
C = concentration of H2O2 decomposed

A = difference in absorbance / minute
a = Molar extinction concentration of hydrogen
peroxide (43.6 molar-1 cm-1)
I = Path length of cuvette
The catalase activity of homogenate was
expressed in terms of micromoles of hydrogen
peroxide decomposed /min/mg protein.
Anticoagulant Activity 15
Tissue thromboplastin in the presence of
calcium activates the extrinsic pathway of human
blood coagulation system. When unipack system

pack reagent, which is a ready to use calcium
thromboplastin liquid reagent, derived from rabbit
brain, is added to normal anticoagulated plasma,
the clotting mechanism is initiated forming a solid
gel clot within a specified period of time. The time
required for clot formation will be prolonged if there
is deficiency of factors in the extrinsic pathway of
coagulation.
Six New Zealand white rabbits of either sex and
of body weight between 2.1 to 2.4 kg were used. The
blood was collected from the marginal ear vein and
transferred into the anticoagulated tubes containing
0.109 M sodium citrate and centrifuged at 2000rpm
for 10 minutes to obtain plasma.
The test procedure for unipack system pack
reagent was programmed on coal LAB 6000
a. Enter the routine menu
b. Load the required number of sample plasma in
separate sample cups giving appropriate sample
numbers and created job list of PT test for the
sample plasma.
c. Loaded the required number of unipack system
pack reagent into the reagent cups
d. Measured the thromboplastin time for the
samples.
Anti-inflammatory Activity16,17
The anti-inflammatory effect of a drug can be
demonstrated through its membrane stabilization
effect by inhibiting hypotonocity induced lysis of
erythrocyte membranes.
The erythrocyte membrane is analogues to the
lysosomal membrane and its stabilization implies
that the extract can stabilize lysosomal membrane.
Stabilisation of lysosomal membrane is important in
inhibiting the inflammatory response by preventing
the release of lysosomal constituents of activated
neutrophil such as bacterial enzymes and proteases
which cause further tissue inflammation and
damage.
Blood was collected from one of the authors who

had not taken any NSAIDs or steroids for two weeks
prior to the experiment. Nine parts of the blood was
collected with 1 part of 0.109 M sodium citrate and
was centrifuged at 3000 rpm and the packed cells
were washed with isosaline and a 10% suspension
was made with isosaline. The assay mixture contained
drugs in different concentration, 1 mL of phosphate
buffer (0,15 M, pH 7.4) and 2 mL of hyposaline(0.36%)
and 0,5mL of HRBC suspension. Diclofenac was used
as the reference standard. Instead of hyposaline 2mL
distilled water was used in the control.
All the assay mixtures were incubated at
37 0 C for 30 minutes and centrifuged. The hemoglobin
content in the supernatant was estimated using
spectrophotometer at 560 nm. The percentage
hemolysis was calculated by assuming the hemolysis
produced in presence of distilled water as 100 % and
calculated as
percentage hemolysis = OD of test 100
OD of control
The percentage of HRBC membrane stabilization
was calculated using the formula
percentage protection = 100 OD of test 100
OD of control
Effect on Ldl, Vldl and Hdl18
Blood from all groups of rats was collected
by retro orbital bleeding under ether anaesthesia
(1mL ) into a clean sterile test tube and kept for about
30 minutes to clot and centrifuged at 3000 rpm for
10 minutes. Clear serum obtained as supernatant
was used for estimation of cholesterol, triglycerides
and HDL cholesterol. HDL, LDL, and VLDL were
determined as follows: To 0.2 mL of serum was
added 0.2 mL of precipitating agent to precipitate
LDL cholesterol, VLDL cholesterol and chylomicron
fractions, mixed well and kept for 30 minutes at
room temperature, centrifuged for 15 minutes at
2000rpm and separated the clear supernatant for
HDL cholesterol estimation. To 0.1 mL of supernatant
was added 1 mL of cholesterol working reagent and
the absorbance was measured at 505nm.
39
HDL cholesterol (mg/dL) = Absorbance of test 50 2

Absorbance of standard
LDL cholesterol was calculated using Friedewalds

equation as
LDL cholesterol = Total cholesterol triglycerides/5
HDL cholesterol
VLDL cholesterol is calculated as
VLDL cholesterol = triglycerides/5
Total cholesterol was determined by adding 1mL
cholesterol working reagent to 1 mL serum and
measuring the absorbance at 505 nm.
Triglyceride was determined by adding 1mL
Triglyceride working reagent to 0.01 mL serum and
measuring the absorbance at 505 nm
Histopathological Examination
Liver and coronary artery of the rats removed
were washed with normal saline and put in 10 %
formalin solution. The fixed specimen was then
trimmed, washed and dehydrated in ascending grades
of alcohol, embedded in paraffin, sliced on a rotary
microtome stained with heamatoxilin and eosin and
histopathological features were examined under a
high power.
RESULTS
Lipid Peroxidation Assay
Hypercholesterolemic animals showed a
significant increase in MDA level in their tissue
homogenate of 2.32 0.07 n mol/mg protein when
compared with normal control. ie, 1.170.04 n
mol/mg protein (p <0.001). Groups treated with
atorvastatin and extract (1000 mg/kg) showed a
significant decrease in MDA level to 1.67 0.05 and
1.510.06 n mol/mg protein when compared to group II
(p< 0.01). But the group treated with extract at the
dose of 500 mg/kg did not show any significant
decrease in MDA level (Table I).
40
Superoxide Dismutase Assay

The activities of SOD were significantly decreased
to 1.82 0.05 U/ per mol of H2O2 consumed per
mg protein per minute in diet control group when
compared with normal group ie, 2.99 0.17U
(p<0.001).The activities of SOD were significantly
increased to 2.57 0.05, 2.47 0.01 and 2.54 0.01 U
per mol of H2O2 consumed per mg protein per minute
respectively in groups treated with Atorvastatin and
extracts (1000 mg/kg and 500 mg/kg) when compared
to group II (p<0.01) (Table I)
Catalase Assay
The activity of catalase was significantly
decreased to 16.27 0.31 U/ per mol of H2O2
consumed /mg protein per minute in diet control group
when compared with normal group ie, 26.6 0.77U
( p<0.001). The activity of catalase was significantly
increased to 24.52 1.37, 20.93 0.36 and 24.4
1.39 U/ per mol of H2O2 consumed /mg protein per
minute respectively in groups treated with atorvastatin
and extracts (1000 mg/kg and 500 mg/kg) when
compared to group II (p<0.01) (Table I )
Anticoagulant Activity
The ethanolic plant extracts significantly
prolonged the prothrombin time to 26.53 1.33 and
44.08 0.57 seconds in a concentration of 300g and
600 g respectively when compared to the control
value of 9.45 0.56 but this effect was slightly lower
than that of heparin (Table II )
Anti-inflammatory Activity
The ethanolic extract at a dose of 100 and 200
g showed significant HRBC membrane stabilization
ie, 76.020.36 and 82.84 0.11% respectively when
compared with the standard drug diclofenac (Table III)
Effect on Ldl, Vldl and Hdl
On the 14th day of treatments serum LDL were
reduced to 19.544.19, 33.811.84 and14.09
1.78mg/dL for atorvastatin and 500mg/kg & 1000mg/
kg of the extract treated groups respectively. Similarly
the serum VLDLwere reduced to 19.880.61,
Table I: Lipid Peroxidation,Superoxide Dismutase & Catalase Assay

Group
I
II
III
Treatment
Standard commercial pelleted

diet + 0.3 % CMC
Atherogenic diet alone+
0.3 % CMC
Atherogenic diet + Atorvastatin
in 0.3 % CMC
Dose
MDA (n mol/mg
protein)
SOD (U /mg
protein/min)
Catalase (U mol of
H2O2 consumed/
mg protein/min)
1mL/100 gm
1.17 0.04
2.99 0.17
26.6 0.77
1mL/100 gm
2.32 0.07 **
1.82 0.05 **
16.27 0.31 **
30 mg/kg
1.67 0.05 ^
2.57 0.05 ^
24.52 1.37 ^
IV
Atherogenic diet + extract in

0.3 % CMC
500 mg/kg
2.030.03 #
2.470.01 ^
20.930.36 ^
Atherogenic diet + extract in

0.3 % CMC
1000 mg/kg
1.510.06 ^
2.540.01^
24.41.39 ^
Values are expressed as mean SEM (n=6 rats)

P values : ** <0.0001 Vs control
P values : ^ <0.01 Vs atherogenic control. # : not significant
Data were analysed by using one way ANOVA followed by Dunnet multiple comparison test and
students t test
22.870.46 and 19.84 0.45 mg/dL for atorvastatin
and 500mg/kg &1000mg/kg of the extract treated
groups. Though the reduction in VLDL withn500mg/
kg of the extract was not significant , the reduction I
LDL and VLDL was more than that of the standard
drug atorvastatin
Similarly, the increase in the level of the good
cholesterol HDL was much more for 1000 mg/kg of
the extract ie, 64.89 2.38 when compared to the
standard drug atorvastatin (Table IV).
Histopathological Examination
CORONARY ARTERY - The coronary artery wall
was of uniform thickness with no lumen bulging in the
control group (Fig. 1A). The focal proliferation and
steatosis in the vessel wall induced by atherogenic
diet (Fig. 1B) was normalized by atorvastatin
(Fig. 1C) and 1000mg/kg of the extract (Fig. 1E) but
not by 500 mg/kg (Fig. 1D).
LIVER- The control group exhibited normal
healthy hepatocytes (Fig. 2A). The observed steatosis
Table II: Effect on Prothrombin Time

Sr.
No.
1
2
3
4
5
Sample
Control
Heparin 300 g
Extract 300 g
Heparin 600 g
Extract 600 g
Prothrombin
time (sec)
9.45 0.56
52.081.48 ^
26.531.33 ^
74.782.68 ^
44.080.57 ^

P values : ^ <0.01 Vs control.
Data were analysed by using one way ANOVA
followed by Dunnet multiple comparison test and
students t test
Table Iii: Effect on Hrbc Membrane
Stabilisation
Sr.
No.
Conc
(g)
Prevention of lysis %
Extract
100
76.02 0.36
95.29 4.19
200
82.84 0.11
96.16 0.03
Diclofenac
41
Table Iv: Effect of Plant Extract on Ldl, Hdl and Vldl

Group Treatment
Dose
LDL (mg/dL)
HDL (mg/dL)
VLDL (mg/dL)
Standard commercial pelleted diet

0.3 % CMC
1mL/100 gm
20.252.53
50.240.97
17.740.65
II
Atherogenic diet alone+0.3 % CMC 1mL/100 gm
18013.03**
40.291.39**
28.892.95**
III
Atherogenic diet + Atorvastatin in

0.3 % CMC
19.544.19^
53.931.82^
19.880.61^
IV
Atherogenic diet + extract in 0.3

% CMC
mg/kg 33.811.84^
44.172.59#
22.870.46#
Atherogenic diet + extract in 0.3 % 1000 mg/kg 14.09 1.78^

CMC
64.89 2.38^
19.84 0.45^
30 mg/kg
500

P values : ** <0.0001 Vs control
P values : ^ <0.01 Vs atherogenic control. # : not significant
Data were analysed by using one way ANOVA followed by Dunnet multiple comparison test and students t test
of hepatocytes in the atherogenic diet treated
group indicates malfunction of lipid metabolism in
liver (Fig. 2B). The fatty infiltration and the other
histopathological changes in the liver induced by
the atherogenic diet were reversed by atorvastatin
(Fig. 2C) and 1000mg/kg of the extract (Fig. 2E) but
not completely by 500 mg/kg (Fig. 2D).
DISCUSSION
Similarly SOD and catalase are highly significant

in the enzymatic antioxidant defence system. They
protect the tissues from highly reactive and toxic free
radicals and play a significant role in the development
of tolerance to cellular oxidative stress. High fat
content in atherogenic diet treated group might have
caused the formation of toxic intermediates capable
of inhibiting the activity of these enzyme system. The
given extract is found to be capable of increasing
the activity of these essential antioxidant systems.
Besides, the anticoagulant and anti-inflammatory
activity of the extract are beneficial for ameliorating
the mechanisms leading to atherogenisis
High cholesterol diet induced oxidative stress

leads to free radical generation that promotes
lipid peroxidation and foam cell formation. In the
present study serum MDA level was measured
in tissue homogenate as a parameter for lipid
peroxidation. Elevated levels of MDA in atherogenic
diet treated group showed a clear manifestation of
excessive formation of free radicals and activation
of lipid peroxidation. The significant reduction in lipid
peroxidation observed with 1000mg/kg extract was
more than that with 30 mg/kg of the standard drug
Atorvastatin.
Oxidative modification of LDL appears to have

an important role in initiation and progression of
atherogenic changes in aorta. Supplementation of
cholesterol in diet rapidly results in a marked increase
in the production of cholesteryl ester rich VLDL by
the liver and intestine and a reduction in the number
and rate of cholesterol removal by the hepatic LDL
receptors. Here the significant decrease in LDL and
VLDL following concurrent administration of ethanolic
extract and atherogenic diet suggest the beneficial
modulatory influence of the extract on cholesterol
metabolism and turnover.
(The degree of magnification is same (40X)

for the same type of tissue from differently treated
groups)
42
Histopathology of rat coronary artery
Fig. 1A: Normal control
Fig. 1B: Diet control
Fig. 1C: Atorvastatin 30mg/kg
Fig. 1D: Extract 500mg/kg
Fig. 1E: Extract 1000mg/kg

43
Histopathology of rat liver
Fig. 2A: Normal control
Fig. 2B: Diet control
Fig. 2C: Atorvastatin 30mg/kg
Fig. 2D: Extract 500mg/kg
Fig. 2E: Extract 1000mg/kg

44
Thus the study revealed the antioxidant,

membrane stabilizing and anticoagulant properties
along with the beneficial effects on blood lipid level of
the ethanolic stem extract of Cissus quadrangularis in
a dose dependent manner all of which are beneficial in
the prevention and treatment of atherosclerosis. More
over, the histopathological examination of the coronary
artery and liver cells also demonstrated the protective
effect of the extract in hypercholesterolemia. There
fore this extract is worthy of further pharmacological
and phytochemical investigations which may lead to
promising new drugs.
ACKNOWLEDGEMENTS
Govt. College of Pharmaceutical Sciences
for providing the research facilities and Ranbaxy
Pharmaceuticals Pvt. Ltd for providing Atorvastatin
(gift sample).
8. Shirwaikar A., Khan S., Malini S., Antiosteoporotic

effect of ethanol extract Cissus quadrangularis. J. of
Ethnopharmacol 2003, 89 (2): 245-250.
9. Ochani P.C., Mello P.D. Antioxidant and antihyperlipidemic
activity of Hibiscus sabdariffa Linn leaves and calyces
extract in rats. India journal of Experimental Biology.
2009:47; 276-282.
10. Alagumanivasagam G., Kottai Mutthu A., Saqtheesh
kumar D. In vitro of antioxidant and lipid peroxidation effect
of methanolic extract of tuberous of roots Ipomoea digitata
( Linn) in rat fed with high fat diet. International journal
of applied Biology and pharmaceutical technology.
2010, 1 (2); 214-220
11. Shajiselvin C.D., Kottai Muthu A., Suresh K.: Evaluation
of in vitro antioxidant and lipid peroxidation effect of
various extracts of the whole plant of Borreria hispida
On rat fed with high fat diet. Indian J.Exp.Biol 2010, 3
(1) 120-124.
12. Batra S.P., Srivasta V.M.L.: Xanthine Oxidase, Superoxide
dismutase, catalase and lipid peroxidation in mastomys
nataensis effect of dipentalonema viteae infection, Indian
J.Exp.Biol 1989, 27;1067.
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Pathophysiology and the role of Novel risk factors: A
clinicobiochemical Perspective. Aniology, 2007. 58;
513-522.
14. Krishnamoorthy P.,Vaithinathan S.,Vimal rani A.,

Bhuvaneswari A. Effect of Terminalia chebula fruit extract
on lipidperoxidation and antioxidant system of testis of
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2. http;//www.Medicinenet.com/statins/article.htm.
3. Chopra N.N., Chopra I.C., Handa K.L., Kapur L.D.
Indigeinous drugs of India CSIR publication 1958;669670.
15. Camara R. B.G., Costa L.S.: Heterofucans from the brown

seeweed Canistrocarpus cervicornis with anticoagulant
and antioxidant activities. Marine Drugs 2011; 9 (1)124138.
4. Mueen Ahmed K.K., Khan M. Y.; Sivananda B.G.

Cardiovascular Diseases and role of Medicinal plants
as a reemerging health aid. Pharmacogn. Rev. 2009,3
(5),8-14
16. Shirwaikar A., Devi S., Siju E.N.: Antiinflammatory activity

of Thespesia populnea fruits by memrane stabilization.
International Journal of Pharmtech Research. OctDec 2011 3 (4) 2060-2063
5. Oben J, Kuate d, Agbor G Momo C, Talla X.: The use of

a Cissus quadrangularis Formulation in the management
of weight loss and metabolic syndrome. Lipids in health
and diseases 2006, 5; 24-27.
17. Gupta S., Kohli S and Dwivedi S. In-Vitro anti-inflammatory

activity of Sarcostemma acidum Wight. & Arn. Indian
herb by Human red blood cell membrane stabilization
method. International Journal of Pharmacy Teaching
& Practices 2011, Vol.2, Issue 4, 184-188.
6. Moghadasian Mohammed H.: Mini review.Experimental

atherosclerosis A Historical Overview . Life Sciences 70
2002; 855-865.
7. G.H. Vogel Drug discovery and Evaluation. 2
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18. Dabhi J.K., Solanki J.K., Mehta A. Antiatherosclerotic

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45
Short Notes
IMMUNOMODULATORY ACTIVITY OF METHANOLIC EXTRACTS OF SEEDS AND
BARK OF PONGAMIA GLABRA VENT. ON HUMAN NEUTROPHILS
Abstract
Methanolic extracts of seeds and bark of Pongamia glabra Vent. (250mg/kg and 500mg/kg p.o.) in the
concentration range 100,50,25,12 and 6.25 g were subjected to evaluate the phagocytic effect on human
neutrophils using the in-vitro models nitroblue tetrazolium (NBT) dye test, phagocyotosis of Candida
albicans and chemotaxis assay. The extracts of the plant in the concentration range 100,50,25,12 and
6.25 g showed significant (P <0.01) phagocytic effect on human neutrophils in the parameters studied.
Methanolic extracts of seeds and barks of Pongamia glabra Vent. exhibited immunostimulant property
in in-vitro models.
Keywords: Pongamia glabra Vent., Immunomodulatory, NBT, Candida albicans, Chemotaxis.

INTRODUCTION
In Ayurvedic system of medicine under the concept
of Rasayana, immunostimulation is considered as
an alternative to conventional chemotherapy and
prophylaxis of infection especially when the host
defense mechanism has to be activated under
conditions of impaired immune responsiveness1.
Certain agents have been shown to possess activity to
normalize or modulate pathophysiological processes
and hence are called immunomodulatory agents2.
A number of medicinal plants have been screened
systematically for their immunomodulatory activity
such as Tinospora cordifolia, Mangifera indica3.
Pongamia glabra Vent. (Syn. Pongamia pinnata).
Fam. leguminosae, commonly known as Karanja, is a
tree found all over India bearing imparipinnate leaves
and pinkish white coloured flowers. Literature survey
claims the use of bark in disease of eye, the vagina
and the skin and cures biliousness, piles, ulcers,
ascites, Vata and Kapha. The seed also claimed
to be used in keratitis, eye and skin diseases4. The
seeds of the plant reported to contain fixed oil and
traces of essential oil. Bark contains bitter alkaloid,
resin and mucilage. Ayurvedic literature reveals
the application of powdered seeds in asthenic and
46
debilitating conditions. Chakradatta, an ancient

Ayurvedic physician recommended an ointment
known as Tiktadya Ghrita, a formulation consisting
of the leaves, fruits and seeds of the plant with some
other plants to be useful as an external application
and also prescribed internally in doses of teaspoonful
with hot milk and sugar twice a day in early stages of
leprosy, unhealthy ulcerations and wounds5.
Bhavaprakash Nighantu, an Ayurvedic medicines
text claims the properties of Karanja as followsYonidoshaharta (useful in uterine diseases), Kustagna
(useful in skin diseases), Arshahara (useful in piles/
haemorrhoids), Vranahara (helps in quick wound
healing), Krumihara (relieves worm infestations),
Kaphavatahara (Kapha and Vata diseases), Mehahara
(useful in urinary tract disorders and diabetes) and
Bhedana (induces diarrhea, relieves constipation)6.
The ancient folklore claims the uses of the seeds
to treat rheumatic joints and anti-paracytic. Bark is
astringent and powdered seeds are used as febrifuge
and tonic7. Some important Ayurvedic formulations
of Karanja include- Brhanmanjisthadi kvata curna,
Mustakakaranjadi kvata curna (used internally),
Karanja taila, Karanjadya ghrta and Somaraja tailam
(used externally)8.
P. glabra (Karanja) is claimed to be used internally
as well as externally. The seed powder is used
externally as nasal therapy to relieve the phlegm
in chronic sinusitis and Kapha diseases. Internally

Karanja claims to be a valuable remedy for various
diseases as listed below. The seed powder is given in
whooping cough in children for quick relief. The bark
juice is a keen stimulant for digestive system and is
beneficial in anorexia, piles, worm infestations and
flatulence and liver diseases. In hepatosplenomegaly,
the decoction of bark works well when co-prescribed
with pippali and rock salt. Karanja also claims to be
a good blood purifier, hence used in blood disorders
as an adjunct9. The aqueous extract of seeds of
the plant has been reported of significant anti-viral
activity against Herpes simplex virus HSV cell lines
experimentally. Also, the aqueous extract of the bark
of the plant has been reported of significant sedative
and antipyretic effects in rats and antispasmodic effect
in vitro on smooth muscles10. In review of these vital
properties and pharmacological claims of the plant,
the present study was aimed at screening of seeds
and bark methanolic extracts of P.glabra Vent. for
the phagocytic effect on human neutrophils using
the in-vitro models nitroblue tetrazolium (NBT)
dye test, phagocyotosis of Candida albicans and
chemotaxis assay.
Plant Material
Pongamia glabra Vent. seeds and barks were
collected from local areas of North Karnataka and
a voucher specimen has been deposited at the
departmental herbarium. The plant was authenticated
by Dr. Srinathrao, Prof. and HOD, Dept. of Botany,
Gulbarga University, Gulbarga and Ref. No. GUG/
BOT/Herbarium/2008-09/09. The mentioned parts of
the plant were dried and pulverized to particle size (#)
40 and then were first defatted with petroleum ether
(40-600C) and extracted with methanol using Soxhlet
apparatus for 48h to obtain methanolic extracts of
seed and barks of the plant respectively. The above
procedure was followed to separate the petroleum
ether fraction as a process of Shodhana (Detoxification
process), since the petroleum ether fraction of the
seeds and the bark, especially the seed oil reported
to be toxic, which cannot be used internally. The
filtrates of the methanolic extracts were concentrated

to dryness at 400C under reduced pressure in a rota
evaporator. The yields of the methanolic extracts of
seeds and barks were 43.16gm (30.59%w/w) and
37.76gm (22.19%w/w) respectively.
Preliminary phytochemical studies
The methanolic extracts of seeds and bark were
subjected for preliminary qualitative chemical tests
and the presences of major phytoconstituents were
confirmed by TLC studies11,12.
Thin layer chromatography studies
l TLC profile for flavonoids

Solvent system: ethyl acetate: formic acid: glacial

acetic acid: water (10:1.1:1.1:2.6).
Detection: UV 365nm (blue fluorescent spots).
TLC profile for alkaloids
Solvent system: toluene: ethyl acetate:

diethylamine (7:2:1)
Detection: wagners reagent (brown colored

spots).
TLC profile for steroids
Solvent system: petroleum ether: acetone (7:3)
Detection: anisaldehyde: sulphuric acid reagent

(pink to red colored spots)
TLC profile for saponins
Solvent system: chloroform: glacial acetic acid:

methanol: water (6.4:3.2:1.2:0.8).
Detection: anisaldehyde: sulphuric acid reagent

(pink colored spots).
In-vitro screening Methods13,14,15

Preparation of test solutions
Stock solutions for in-vitro studies of concentrations
in the range of 100g/mL, 50g/mL, 25g/mL, 12.50g/
mL and 6.25g/mL were prepared by dissolving the
methanolic extracts of seeds and bark of Pongamia
glabra Vent. in 0.5 mL dimethyl sulphoxide (DMSO)
and with phosphate buffer salt solution (PBS solution
prepared by dissolving 2.38 g of disodium hydrogen
47
phosphate 0.19g of potassium dihydrogen phosphate

and 8.0g of sodium chloride in sufficient water to
produce 1000mL).
Nitroblue tetrazolium (NBT) dye test
0.3% solution of NBT was prepared in 0.34%
sucrose solution. One part of this was added to one
part of PBS (phosphate buffer solution) and was used
fresh. At the same time a suspension of leucocytes
(5x106/mL) was prepared in 0.5mL PBS.
The stock solutions of the methanolic extracts of
seeds and bark of P. glabra Vent. in concentrations
of 100g/mL, 50g/mL, 25g/mL, 12.50g/mL and
6.25g/mL were added individually with 0.2mL freshly
prepared 0.15% NBT solution and suspension of
leucocytes.
In another test tube containing 0.1mL of endotoxin
activated plasma added with the above solutions
except test extracts and was used as positive control
(standard).
A normal control was also maintained in another
test tube with only suspension of leucocytes and
NBT solution. All the test tubes containing above
solutions were incubated separately at 370C for 20 min,
centrifuged gently at 400g for 3-4 min. The supernatant
was discarded and a drop of PBS was added and
gently resuspended the cells in small volumes of fluid
at the bottom of the test tube. A film was made by
allowing a drop of this fluid to dry on a microscope
slide, fixed gently by heating and counterstained with
dilute carbol-fuchsin, washed, dried and mounted
using a 100X oil immersion objective, 200 neutrophils
were counted and the percentage of NBT positive
cells containing blue deposits was determined.
Phagocytosis of killed candida albicans
Preparation of heat killed Candida albicans
suspension
The Candida albicans was grown on Saborauds
2% dextrose broth for 48 hours at 370C to obtain
organisms in the yeast phase only. The cultures spun
48
at 1500g for 10min and the deposit washed twice with

PBS and filtered twice through sterile gauze. 5x106
cell/mL Candida albicans resuspended in Hanks
(buffer) solution. They were killed by heating at 1000C
for 30min. The sterility of the heated batch checked
and were cooled and stored at 200C.
0.25mL of predetermined concentrations i.e.,
100g/mL, 50g/mL, 25g/mL, 12.50g/mL and
6.25g/mL of the methanolic extracts of seeds and
bark of Pongamia glabra Vent. were taken in separate
test tube and were individually added with 0.25ml
Hanks solution, 0.25mL heat killed Candida albicans
and 0.25mL neutrophil suspension.
In another test tube 0.25mL of pooled serum
was taken to which the above solutions except the
test extracts were added and was maintained as
positive control.
At the same time a normal control was also
maintained in another test tube, which contains 0.25mL
of neutrophil suspension, 0.25mL of Hanks solution
and 0.25mL of heat killed Candida albicans.
All the above test tubes were slightly shook for
proper mixing and incubated at 370C for 30 min,
centrifuged at 200g for 5 min and the supernatant
removed with a pasteur pipette leaving a droplet into
which the sediment was resuspended. Smears were
made, dried in air and stained with May-Grunwald
Giemsa stain. About 200 neutrophils were examined,
the number of ingested Candida albicans associated
with each cell was counted and the mean particle
number associated with each cell i.e., neutrophil
was calculated.
Neutrophil locomotion and Chemotaxis
Chemotaxis experiment can be carried out using
a special apparatus arrangement called chemotaxis
chamber, which consists of an upper chamber - in
which the neutrophil cells suspension is placed,
which is separated by a micropore filter from a
lower chamber - in which the chemotactic factor i.e.
extract is placed. It is a self constructed apparatus
in which the lower chamber, a 5mL beaker is placed

in a sandwich box with holes bored in its lid and the
upper chamber is a Sawnoff tuberculin syringe with
the filter glued to its lower end.
Neutrophil cells were prepared in Hanks
(buffer) solution at about 106 cells/mL. The lower
compartments of the chemotaxis chambers with

appropriate chemotactic agent pre-adjusted to a pH
of 7.2.
Chamber 1 : Hanks solution (Normal control)
Chamber 2 : Casein 1mg/mL (positive control)
Table I: Thin Layer Chromatography results of Pongamia glabra Vent. seeds

and bark methanolic extracts
Sr.
no.
1
2
Extracts
Seed
Ext.
Bark
Ext.
Flavonoids
No. of
Rf value
Spots
0.59,
2
0.63
0.62,
2
0.71
Alkaloids
No. of
Rf
Spots
value
0.89,
2
0.80
0.82,
2
0.53
Steroids
No. of
Rf Value
spots
0.61,
2
0.76
0.64,0.72,
4
0.78, 0.88
Saponins
No. of
Rf
spots
value
0.58,
2
0.65
0.72,
2
0.87
Table II: Nitroblue tetrazolium (nbt) qualitative test for methanolic extracts of Pongamia glabra Vent.
seeds and bark on human neutrophils
Normal Control Endotoxin Activated Plasma
(Positive Control) Conc.
1mg/mL
180.0
Conc.
(g/mL)
Methanolic Ext. of
seeds of Pongamia
glabra
Mean Percentage of Reduced Neutrophils

100
85
50
79
860.0
25
67
12.50
43
6.25
40
MeanSEM
62.80ns9.178
Methanolic
Ext. of bark of
Pongamia glabra
76
74
56
32
30
53.60**9.867
Table III: Phagocytosis of killed Candida albicans for methanolic extracts of Pongamia glabra Vent.
Normal Control
20.0
Pooled Serum
(Positive control)
50.0
Conc.
(g/mL)
Methanolic Ext. of seeds Methanolic Ext. of bark

of Pongamia glabra
of Pongamia glabra
Mean Particle Number

100
5
50
5
25
4
12.50
3
6.25
3
MeanSEM
4.00ns0.447
4
4
3
3
3
3.40**0.245
49
Table IV: Neutrophil locomotion and chemotaxis for methanolic extracts of Pongamia glabra Vent.
Methanolic Ext. of bark
Conc.
Methanolic Ext. of
Normal Control
Casein
of Pongamia glabra
(g/mL)
seeds of Pongamia
(Positive Control)
glabra
Conc. 1mg/mL
Mean Number of Neutrophils per field
100
170
150
50
160
150
150.0
2000.0
25
120
130
12.50
100
110
6.25
90
90
MeanSEM
128.00***15.94
126.00***11.66
Significant difference from positive control by One Way ANOVA followed by Dunnets t test (n=4),
Test drug treated groups were compared with positive control group.
** P <0.01 Significant, ns- non-significant
*** P <0.001 Highly Significant
Chamber 3 : Pre-determined concentrations
of extracts (100g/mL, 50g/mL,
25g/mL, 12.50g/mL and 6.25g/
mL in separate chambers).
The upper compartments filled with the neutrophil
cells suspension ensuring that the fluid level in
the upper chamber was the same as in the lower
to avoid gradient disturbance.The filters allowed
for wetting from the top before putting them in the
lower compartments, when the contents of upper
compartments were placed in the lower compartments.
The concentration of chemotactic factor throughout
the filter was zero when it was not placed in the
lower compartment and as soon as the filter was
placed in chemotactic solution, the gradient began
to form the bottom of the filter. The arrangement
was not disturbed until the end of the experiment. It
was incubated for 3 hours at 370C in air. After about
45min the upper compartments were removed and
inverted them to empty fluid out of them. The fluid
of the upper compartments was fixed by immersing
the filters in 70% ethanol or methanol. After few
minutes in alcohol, the glue melted and the fluid
became loose, this was picked off gently with dental
packing forceps. It was stained with haematoxylin and
xylol respectively, mounted under microscope using
50
mountant like glycerin and covered with a coverslip.

The cell migration measured microscopically by
counting the number of cells, which have reached the
lower surface of the filter after a given time interval.
The count of lower surface was taken, as the count
of cells on the lower surface is directly proportional
to the number of cells placed on top of the filter at
the start of the experiment.
Statistical Analysis
Data were expressed as mean SEM and
differences between the groups were statistically
determined by analysis of variance followed by
Dunnets test.
RESULTS
The preliminary qualitative chemical tests of
both the methanolic extracts of seeds and bark were
indicated equally the presence of carbohydrates,
glycosides, proteins, steroids, saponins, flavonoids,
alkaloids and tannins. The results TLC studies are
showed in Table I.
Nitroblue tetrazolium (NBT) dye test
The results indicate that the methanolic extracts
of seeds and bark of Pongamia glabra Vent. have
stimulated the neutrophils for phagocytosis of NBT dye

to the extent of 62.80% 9.178 and 53.60% 9.867
respectively at the concentration range of 100g/mL,
50g/mL, 25g/mL, 12.50g/mL and 6.25g/mL. The
methanolic extract of bark showed significant (P<0.01)
activity compared to the positive control i.e. endotoxin
activated plasma (86%). However the methanolic
extract of seeds showed higher percentage of
phagocytosis, but it was statistically nonsignificant.
The activity of both the extracts was comparative at
the higher concentrations (100g/mL, 50g/mL and
25g/mL) and lower at the concentrations 12.50g/
mL and 6.25g/mL. (Table II)
Phagocytosis of killed candida albicans
Phagocytosis of killed candida albicans was
determined as the mean particle numbers (MPN) of
candida albicans phagocytosed. The MPN for the
methanolic extracts of seeds and bark of Pongamia
glabra Vent. were found to be 4.000 0.447 and 3.400
0.245 respectively. The methanolic extract of bark
exhibited significant (P<0.01) activity compared to
the positive control i.e. pooled serum. However the
MPN for the methanolic extract of seeds was high
but it was statistically nonsignificant. (Table III)
Neutrophil locomotion and chemotaxis
The chemotactic activity of the methanolic
extracts of seeds and bark of Pongamia glabra
Vent. was determined as the mean number of
neutrophils attracted per field. The average count
for the methanolic extracts of seeds and bark was
found to be 128.0015.94 and 126.00 11.66
respectively. The activity of both the extracts were
highly significant (P<0.001) compared to the positive
control i.e. casein (200). The chemotactic activities
of both extracts were concentration dependent i.e.
higher at concentrations 100g/mL, 50g/mL and
25g/mL and lower at concentrations 12.50g/mL
and 6.25g/mL. (Table IV)
DISCUSSION
Immune activation is an effective as well as
protective approach against emerging infectious
diseases16. Immunomodulatory activity of methanolic

extract of seeds and bark of Pongamia glabra Vent.
was explored by evaluating their phagocytic effects
on human neutrophils.
Nitroblue tetrazolium (NBT) assay is a qualitative
test, in which stimulated neutrophils absorb nitroblue
tetrazolium stain. Formazone granules can be
seen in stimulated (opsonized) neutrophils. Mean
percentage of reduced neutrophils was observed
with extracts against positive control i.e., endotoxin
activated plasma.
Phogacytosis of killed candida albicans assay is a
method of measuring phagocytosis rely on the uptake
of particles by phagocytes over a brief period. In this
the number of particles phagocytosed by neutrophil
cells was counted under the microscope.
Neutrophil locomotion and chemotaxis is
a process of chemoattraction i.e., attraction of
neutrophils towards certain chemicals. These
chemical substances are called chemoattractants.
This is a micropore filter method, in which the extracts
are tested to contain chemoattractants and the mean
number of neutrophils attracted per field was noted
against positive control - casein.
From the study of these parameters the process
of immunomodulation (immunostimulation) of the
methanolic extract groups of seeds and barks of
Pongamia glabra Vent. at various concentration
range can be assessed by observing stimulation
(Opsonization) of neutrophils (NBT assay), particle
ingestion (phagocytosis assay) and chemoattraction
of neutrophils (chemotaxis).
The phytochemical investigations of the methanolic
extracts of seeds and barks revealed the presence
of steroids, saponins, tannins, flavonoids, alkaloids,
proteins and carbohydrates. Saponins are either
triterpenoid or steroidal glycosides proven as important
phytoconstituent with different pharmacological
activities such as antiallergic, cytotoxic, antitumour,
antiviral, immunomodulating, antihepatotoxic,
and antifungal activities. Recently three diosgenyl
51
saponins isolated from Paris polyphylla were reported

for immunostimulating activity17. Tannins are also
known to possess immunostimulating activites. The
well known ayurvedic formulation, Triphala contains
Terminalia chebula, Terminalia belenica and Emblica
officialis, which are rich in tannins reported for
immunostimulating activity18. Hence the collective
presence of steroids, saponins, tannins and flavonoids
in the methanolic extracts would be attributed for
immunostimulating activity. It is concluded from the
studies of in-vitro models that the methanolic extracts
of seeds and bark of Pongamia glabra Vent. possess
immunostimulating property.
ACKNOWLEDGEMENT
Authors are thankful to the authorities of HKE
Society and MTR Institute of Pharmaceutical sciences,
Gulbarga, Karnataka India, for providing necessary
facilities to carry out the study.
REFERENCES
1. Charak Samhita (Trans.): Shree Gulab Kunvera Ayurvedic
Society, Jamnagar, India.1949, 249-250.
2. Wagner H.: Immunomodulatory agents, Proceedings of
the Alfred Benzon Symposium. 1983, 20, 559.
3. Makare N., Bodhankar S., Rangari V.: Immunomodulatory
activity of alcoholic extracts of Mangifera indica Linn. in
mice, J Ethnopharmacol. 2001, 78, 133-137.
4. Kirtikar K.R. and Basu B.D..: Indian Medicinal Plants 2nd
ed. Vol. 1,Oriental Enterprises; Dehradun. 1984, 830832.
5. Nadkarni K.M..: Indian Materia Medica Vol 1,Popular
Prakashan Ltd., Mumbai. 1996, 10011004.
6. Bhavaprakash Nighantu, Guduchyadi Varga : Shree
Gulab Kunvera Ayurvedic Society, Jamnagar, India.1949,
119-120.
Dept. of Pharmacognosy and Phytochemistry

HKESs MTR Institute of Pharmaceutical Sciences
Gulbarga 585105, Karnataka
E-mail: ssheroor@gmail.com
7. Hartwell J.L.: Plants used against cancer, A Survey,

Lloydia. 1967-1971, 30-34.
8. The Ayurvedic Pharmacopoeia of India Part-I, 1st ed.
Vol.II, Government of India, Ministery of Health and
Family welfare, Dept. of Indian System of Medicine and
Homeopathy, New Delhi, 1999, 79-80.
9. www.herbalcureindia.com
10. Khare C.P.: Indian Medicinal Plants, an illustrated
dictionary, Springer-Verlag/ Heidelberg. 2007,
511-512.
11. Khandelwal K.R.: Practical Pharmocognosy Techniques
and Experiments 10th Ed. Nirali Prakashan, Pune.
2003,149 158.
12. Hildebert Wagner and Sabine Bladt: Plant Drug Analysis-A
Thin layer Chomoatography Atlas 2nd ed., Springerverlag, Berlin Heidelberg, New York. 2001, 1-3, 195-197,
206-305.
13. Wilkinson P.C.: Neutrophils leucocyte function test,
chapter 13 in Techniques in clinical immunology edited
by Thomson R.A. 2nd ed., Blackwell scientific publication,
oxford London. 1981, 279-290.
14. Reevas W.G.: Laboratory investigation of immunological
Diseases, 6th series, Allergy and clinical immunology,
Med. Internat.1994, 2, 238-244.
15. Chaning R. and Rodger P.: Basic and clinical immunology,
edited by stites DP. Terr AT. 8th ed. ,Lange International,
New Jersey. 1994, 151.
16. Hackett C.J.: Allergy, Clin. Immunol. 2003, 112, 686694.
17. Xiu-feng Z., Yan C., Jiajun H., Ya-Zhou Z., Zhou N.:
Immunostimulating properties of diosgenyl saponins
isolated from Paris polyphylla, Bioorganic and Med.
Chem. Letters. 2007.
18. Srikumar R., Parthasarathy N.J., Sheeladevi R.:
Immunomodulatory activity of Triphala on Neutrophil
Functions, Biol. Pharm. Bull. 2005, 28(8),
1398 1403.
Heroor S.S.*, Beknal A.V. and Mahurkar N.K.
*For correspondence
(Received 22 November 2012) (Accepted 04 February 2013)
52
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Vol.

INDIAN DRUGS 50(03) march 2013

INDIAN DRUGS 50(03) march 2013

original research articles

Indian Drug Manufacturers' Association

INDIAN DRUGS 50(03) march 2013

Mr. J. L. Sipahimalani, B. Pharm. Hons. (London), MRCS, FRPharmS

Dr. S. G. Deshpande, M.Sc. (Tech.), Ph.D.

Editorial Advisory Board

Prof. K. G. Akamanchi, Ph.D. (Tech.)

Prof. Y. K. Agrawal, Ph.D., F.I.C., F.R.M.S.

Dr. Evans Coutinho, Ph.D. (Tech.)

Prof. H. L. Bhalla, Ph.D.

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.

Dr. K. N. Ganesh, Ph.D.

Dr. C. L. Kaul, Ph.D.

Dr. (Mrs.) Gopa Ghosh, Ph.D.

Dr. S. P. S. Khanuja, Ph.D.

Dr. Parthajyoti Gogoi, Ph.D.

Prof. J. K. Lalla, Ph.D.

Dr. Nirmala D. Grampurohit, Ph.D.

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Dr. (Mrs.) S. S. Mahajan, M.Sc. (Tech.), Ph.D.

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Dr. M. K. Raina, Ph.D.

Dr. Sanjay Singh, M.Pharm, Ph. D.

Dr. A. V. Rama Rao, Ph.D. (Tech.), D.Sc.

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.

INDIAN DRUGS 50(03) march 2013

Fluidised hot melt granulation

Industrial application of extrusion processes dates

Fluidised hot melt granulation (FHMG) has

& drying phases eliminated, thus less time

l There are no requirements on compressibility

of active ingredients, entire procedure simple,

swellable and water insoluble nature .

l Clinically advantaged dosage forms, such as drug

abuse and dose dumping deterrent technology.

HME process among granules of different size

be applied to heat-sensitive materials owing to

melting or softening of the binder occurs during

temperatures and can contribute to instability

Applications in the pharmaceutical industry

2. Controlling or modifying the release of the

Table I : Hydrophilic Meltable Binders in the

Hydrophilic Meltable Typical Melting