Indian Drugs March PDF 09 Final(1)

INDIAN DRUGS 50(03) mARch 2013 1

Vol.50 Issue No.03 march 2013

2 INDIAN DRUGS 50(03) mARch 2013


Vol. 50

No. 03

march 2013

IndIan drug Manufacturers' assocIatIon102-B, 'A-Wing', Poonam Chambers, Dr. A.B. Road, Worli, Mumbai - 400 018Tel : 022-2494 4624 / 2497 4308 Fax: 022-2495 0723E-mail: [email protected] / [email protected], Website: www.idma-assn.org

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


editorial BoardProf. K. G. Akamanchi, Ph.D. (Tech.)

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

Prof. Padma Devarajan, m.Pharm., Ph.D. (Tech.)

Dr. Prashant m. Dikshit, Ph.D.

Prof. A. K. Gadad, m.Pharm., Ph.D.

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

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

Dr. Parthajyoti Gogoi, Ph.D.

Dr. Nirmala D. Grampurohit, Ph.D.

Dr. (mrs.) S. S. mahajan, m.Sc. (Tech.), Ph.D.

Prof. Bhushan Patwardhan, Ph.D.

Dr. Ashwinikumar Raut, m.D.

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

Prof. Saranjit Singh, m.Pharm., Ph.D.

Prof. N. Udupa, m.Pharm., Ph.D.

Dr. K. Valliappan, m.Pharm., Ph.D.

Dr. A. A. Natu, Ph.D.

Dr. N. G. N. Swamy, Ph.D.

Founder editor*Dr. A. Patani, D.Sc. (Germany)

editorial committeeeditorDr. Gopakumar G. Nair, Ph.D.

associate editorsmr. J. l. Sipahimalani, B. Pharm. hons. (london), mRcS, FRPharmS Dr. Nagaraj Rao, D.Sc. (Germany) Dr. George Patani, Ph.D.

consulting editorDr. S. G. Deshpande, m.Sc. (Tech.), Ph.D.

editorial BoardProf. K. G. Akamanchi, Ph.D. (Tech.)

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

Prof. Padma Devarajan, m.Pharm., Ph.D. (Tech.)

Dr. Prashant m. Dikshit, Ph.D.

Prof. A. K. Gadad, m.Pharm., Ph.D.

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

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

Dr. Parthajyoti Gogoi, Ph.D.

Dr. Nirmala D. Grampurohit, Ph.D.

Dr. (mrs.) S. S. mahajan, m.Sc. (Tech.), Ph.D.

Prof. Bhushan Patwardhan, Ph.D.

Dr. Ashwinikumar Raut, m.D.

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

Prof. Saranjit Singh, m.Pharm., Ph.D.

Prof. N. Udupa, m.Pharm., Ph.D.

Dr. K. Valliappan, m.Pharm., Ph.D.

Dr. A. A. Natu, Ph.D.

Dr. N. G. N. Swamy, Ph.D.

editorial advisory BoardProf. Y. K. Agrawal, Ph.D., F.I.c., F.R.m.S.

Prof. h. l. Bhalla, Ph.D.

Dr. B. N. Dhawan, m.D.

Prof. S. S. handa, Ph.D.

Dr. c. I. Jolly, Ph.D.

Dr. c. l. Kaul, Ph.D.

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

Prof. J. K. lalla, Ph.D.

Dr. D. B. Anantha Narayana, Ph.D.

Dr. Nitya Anand, Ph.D.

Dr. harish Padh, Ph.D.

Dr. m. K. Raina, Ph.D.

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

Dr. G. N. Singh, m.Pharm., Ph.D.

Prof. R.T. Sane, Ph.D.

Prof. m. N. Saraf, m.Pharm., Ph.D.

Dr. P. D. Sethi, Ph.D.

Dr. Ashok Vaidya, m.D., Ph.D., F.A.I.m.

Dr. J. S. Yadav, Ph.D., FNA

Founder editor*Dr. A. Patani, D.Sc. (Germany)

editorial committeeeditorDr. Gopakumar G. Nair, Ph.D.

associate editorsmr. J. l. Sipahimalani, B. Pharm. hons. (london), mRcS, FRPharmS Dr. Nagaraj Rao, D.Sc. (Germany) Dr. George Patani, Ph.D.

consulting editorDr. S. G. Deshpande, m.Sc. (Tech.), Ph.D.


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.

*For correspondence department of Pharmaceutics modern college of Pharmacy, sector no.21 Yamuna nagar , nigdi, Pune – 411044 e-mail: [email protected]

introduction

Industrial application of extrusion processes dates back to 1930’s. 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

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. Schaefer’s 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.

advantageslNeither 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.


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 nature1, 3.

lclinically 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.

lReduced number of unit operations.

l Production of a wide range of performance dosage forms1-4,10,11.

disadvantages l Thermal process (drug/polymer stability).

lRequires 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.

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


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. no.

type examples

1 citrate esters triethyl citrate, tributyl citrate, acetyl triethyl citrate, acetyl tributyl citrate

2 Fatty acid esters

butyl stearate, glycerol monostearate, stearyl alcohol

3 Sebacate esters

Dibutyl sebacate

4 Phthalate esters

diethyl phthalate, dibutyl phthalate, dioctyl phosphate

5 Glycol derivatives

Polyethylene glycol, propylene glycol

6 Others triacetin, mineral oil, castor oil7 Vitamin E

TPGSD-α-tocopheryl polyethylene 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

table i : hydrophilic meltable Binders in the melt granulation technique10,17

sr. no.

hydrophilic meltable Binders

typical melting range(oc)

1 Gelucire 50/13 44-502 Poloxamer 188 50.93 Polyethylene glycol 2000 42-534 Polyethylene glycol 3000 48-635 Polyethylene glycol 6000 49-636 Polyethylene glycol 8000 54-637 Polyethylene glycol 10000 57-648 Polyethylene glycol 20000 53-669 Stearate 6000 Wl 1644 46-58

table ii : hydrophobic meltable Binders in the melt granulation technique4,16

sr. no.

hydrophobic meltable Binders

typical melting range(oc)

1 Beeswax 56-602 carnuba wax 75-833 cetyl palmitate 47-504 Glycerin monostearate 47-635 Paraffin wax 47-656 Stearic acid 46-697 Glyceryl behenate 67-758 Glyceryl palmitostearate 48-579 Glyceryl stearate 54-6310 hydrogenated castor oil 62-8611 microcrystalline wax 58-7212 Stearyl alcohol 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


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.

• 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

conditions18. The examples of drug substances which has been processed by melt granulation technique are given in Table IV.

table iv : examples of drug substances processed by melt granulation technique

sr. no.

drug tm(0c) references

1 Diclofenac sodium

284 Bhavsar D.B. et al., 2010; lyons. J.G., 2006; Sato et al., 1997

2 Theophyllin 255 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

3 Nifedipine 175 Forster et al., 2001a, 2001c; Nakamichi et al., 2002

4 carbamazepine 192 Perissutti et al., 2002

effect of Process variables on granulation1. 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, low-shear 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 80°c. A lower-melting-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


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

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. Spray-congealed particles (10–3000 µm 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 non-meltable 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. high-

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


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

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.

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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, 56–61.

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.

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, 909–926.

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), 2866-2875.

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 high-shear mixer,” J. Pharm. Pharmaceut. sci. 2005, 8(2), 132-140.

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.

23. Vilhelmsen T., Schæfer 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 carbamazepine–Gelucire 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.

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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.


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.

*For correspondence department of quality assurance technique Padmashree dr. vithalrao vikhe Patil Foundation’s college of Pharmacy, vilad ghat ahmednagar. maharashtra. 414111. e-mail: [email protected]

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

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 PDVVPF’s 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 PDVVPF’s col-lege of Pharmacy and with following specifications.

instrument makeUV Spectrometer Jasco-V-630-double beam

spectrometerFriability Apparatus

Rolex friability test apparatus

Dissolution apparatus

Electrolab-TDT-08l Dissolution tester (USP type II)

compression machine

labpress 8 station, single rotary(0.9 mm punch)

Tablet Disintegration

Veego-Digital-tab disintegration test apparatus

micrometer screw gauge

Alex Instruments

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 Indiahot Air Oven Universal laboratories, Indiamicrowave Veego, IndiaIR moisture Balance

IR moisture balance, Jeseng laboratories.

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

(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.

evaluation of granules1,3,4

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.

Percentage of Fines

The granules were passed through standard sieve no: 16 & 22.The material retained on sieve


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:

tanө=h/r or ө= tan-1(h/r)

where h=height of the pile, r=radius of pile and ө=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 37±1oc.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 microwave dried granules at 840 w tray dried granulesAmount of fines(%) 10.61 12.10Bulk density(g/cc) 0.98±0.006 0.96±0.5

compressibility 16.25± 0.005 10.12 ± 0.003Angle of repose(q) 16.00± 0.5 14.86 ±0.42Drying time(min) 3.44± 0.52 36.2± 0.31

loss on drying (%) 2.5-3.45 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 microwave dried tablets at 840 w tray dried tablets Average weight (mg) 770± 0.5 750±0.4

hardness(kg/cm2) 8.3±0.02 5.56±0.04Friability (%) 0.109 0.124

Drug content(mg) 501±0.024 500±0.046Disintegration(sec) 55.02±0.1 44.66±0.4

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) cumulative drug release from microwave dried tablets at 840 w (%)*

cumulative drug release from tray dried tablets (%)*

2.00 30.15 29.41 4.00 34.81 31.85 6.00 49.75 44.59 8.00 65.93 59.70

10.00 77.54 74.11 12.00 99.87 95.54

*Average of three determinations

table iv : composition of tablet formulations

ingredients (per tablet) mg Formulationsmetformin hydrochloride 500

hydroxy propyl methyl cellulose (K100 m) 100 Ethyl cellulose (18 centipoise) 100

microcrystalline cellulose 75 colloidal silicon dioxide (Aerosil) 0.006

magnesium stearate 0.012


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

Fig. 1: In vitrodissolutionprofilesoftwobatchesoftablets prepared by microwave and tray dried methods

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 PDVVPF’s college of pharmacy, Ahmednagar, for availing their analytical, pharmaceutical facilities and the API metformin for this project .

reFerences1. Pederson AV. Erosion-based drug delivery. manuf chem.

2006;11:1–6.

% d

rug

rele

ase


2. Washington N, Wilson c. G. Erosion-based delivery. drug deliv technol. 2006; 6(9): 71-74.

3. 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.

4. Sharma S. V, Sharma GVSR, Suresh B; A ecofriendly technology, ind. J. Pharm. sci, 2002; Jul - Aug; 64 (4): 337-344.

5. Walter J moore; Physical chemistry, 5th Edition, 1999, Orient longman limited, 761.

6. www.industrialmicrowave.com/faqs.htm7. 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 multi-comPonent mode oF analYsisBhagwat 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-lambert’s law in concentration range of 1-5 µg/ml (prochlorperazine maleate) and 5-25 µg/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-chloro-10-[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-hydroxy-6-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 sanstha’s college of Pharmacy shikrapur, Pune 412 208 (m. s.) e-mail: [email protected]

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 amount of pure drug taken % of drug estimated* sd rsd se1 Pcm (1-5 µg/ml) 100.02 0.09628 0.00927 0.007422 PDh (5-25 µg/ml) 100.01 0.12194 0.01487 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 amount of pure drug taken % of drug estimated* sd rsd se1 Pcm (1-5 µg/ml) 100.52 0.62235 0.38732 0.309862 PDh (5-25 µg/ml) 100.83 0.80661 0.65062 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 amount of pure drug added (mg)

amount of pure drug recovered (mg)

% recovery

Pcm Pdh Pcm Pdh Pcm Pdh1 5 25 5.000 25.001 100.00 100.022 5 25 5.0904 25.452 101.33 101.463 5 25 4.9819 24.910 99.98 99.99

PCM is prochlorperazine maleate and PDH is a pyridoxine hydrochloride.

table v: results of ruggedness study

condition % label claim* sd rsdPcm Pdh Pcm Pdh Pcm Pdh

Intraday 100.01 100.03 0.0251 0.0360 0.00063 0.0023 Interday 100.18 100.13 0.1212 0.0981 0.0147 0.0096Different 100.19 100.12 0.1569 0.1474 0.0246 0.0120 analyst

PCM is prochlorperazine maleate, PDH is pyridoxine hydrochloride, SD is standard deviation, RSD is relative standard deviation and *Results are mean of five replicates.

table iv:ResultsofSpecificityStudy

sr.no sample % label claimPcm Pdh

1 Normal 100.61 100.932 Alkali 98.78 99.003 Acid 99.89 99.684 oxide 101.01 101.21

PCM is prochlorperazine maleate and PDH is a pyridoxine hydrochloride.


Fig. 1: structure of prochlorperazine maleate

Fig. 2: structure of pyridoxine hydrochloride

Fig. 3: uv spectrum of PcmPcm is a prochlorperazine maleate, 254.5 nm is a λmax of

Pcm.

Fig. 4: uv spectrum of PdhPDh is a pyridoxine hydrochloride, 290.5 nm is a λmax of

PDh

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 250µg/ml of Pcm and PDh respectively.

selection of scanning range and sampling wavelengths

The standard stock solutions of Pcm and PDh were diluted with 0.3 m hcl individually to get concentration of 10 µg/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-lambert’s law at selected wavelengths

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- lambert’s law in the concentration between 1-5µg/ml for Pcm and 5-25 µg/ml for PDh respectively.

. hcI

2


the range of mixed standards. The results of this are shown in Table I.

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.

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.

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 µg/ml and Pyridoxine hydrochloride (PDh, Peak 2) at wavelength of 290.5nm in

concentration of 5-25 µg/ml.

analysis of laboratory mixture by proposed method

Standard solution: mixed standard solutions of 1 to 5µg/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

Peak - 1

Peak - 2


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 µg/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.

results and discussion

An attempt has been made to develop a fast, sensitive, precise, reproducible and economical

analytical method for simultaneous estimation of Pcm and PDh in their combined dosage form. In this method drugs obey Beer’s law in the concentration range 1-5 µg/ml for Pcm and 5-25 µg/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.

conclusion

The present study comprises a UV spectroscopic, multi-component mode of analysis for the simultaneous


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.

reFerencesBudhavari S., The merck 1. Index: An Encyclopedia of chemicals, Drugs and Biologicals. 13th ed. Whitehouse Station (NJ): merck Research lab, Division of merck; 2001.7850.Budhavari S., The merck 2. Index: An Encyclopedia of chemicals, Drugs and Biologicals. 13th ed. Whitehouse Station (NJ): merck Research lab, Division of merck; 2001.8072.Poongothai S, Ilavarasan R and Karrunakaran cm: 3. 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.Khor Swan-choo and Tee E-Siong: Development of a 4. hPlc method for simultaneous determination of several B-vitamins and ascorbic acid, mal J nutr. 1996,2, 49-65.

RadaAmidzic, JasminaBrboric and OliveraUdina :RP-5. hPlc Determination of vitamins B1, B3, B6, folic acid and B12 in multivitamin tablets, J. serb. chem. soc. 2005, 70 (10), 1229–1235.David Borsook, The migraine Brain, David Borsook, Arne 6. may, Peter J. Goadsby, Richard hargreaves, Oxford University Press, New York 1st Edition.misao N., Katsuhiko m., Tsukioka T., Yamashita h., 7. Inagaki N., Sugiyama T. and Itoh Y.: In vitro and in vivo characteristics of prochlorperazine oral disintegrating film, iJ Pharma.2009, 368. (2), 98–102.caihongmou, Ganju N., Sridhar K. S. and AwtarKrishan: 8. Simultaneous quantitation of plasma doxorubicin and prochlorperazine content by high-performance liquid chromatography, J. chromatography B. 1997,703, 217 –224.Adnan El-Yazigi, Fida Abdel and WahabBarmaAfrane: 9. Stability study and content uniformity of prochlorperazine in pharmaceutical preparations by liquid chromatography, J. chromatography a.1995, 690. (1), 71–76.Marc inLeszekMarszal l , Anna Lebiedz ińska, 10. 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), 91–98.Ich Q2B; Guidelines on validation of analytical procedure; 11. Definitions and terminology, Federal Register, 1995, 60, 11260.Ich Q2B; Guidelines on validation of analytical procedure; 12. methodololgy, Federal Register, 1996, 60, 27464.The United States Pharmacopoeia 24/ National Formulary 13. 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: [email protected] or contact to The Publication Division, I.P.commission, Tel.Nos.: 0120-2783392, 2783400, Extn.309, 308 & 112.


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, 5µ 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: [email protected]

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:


Fig. 4: linearity plot for dimethicone obtained by the new hPlc method

Fig. 1: simethicone = dimethicone + silicon dioxide

Fig. 2: infra-red overlay spectrum of dimethicone standard and sample solution

Fig. 3: representative overlay chromatogram of the diluent, dimethicone standard and sample solution

Quantificationusinginfraredspectroscopy

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).

QuantificationusingHPLCmethod

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,

y = 69252x – 15545r=0.999

1320 1305 1290 1275 1200 1245 1230 1215 1200 1185

0.45

0.4

0.35

0.3

0.25

0.2

0.15

0.1

0.05

0

0.05

Abs


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).

table i: chromatographic conditions for the analysis of dimethicone

column: Princeton Sphere cyano (250 x 4.6 mm), 5µmobile phase: hexane : ethanol : ethyl acetate (1600 : 400 : 4)

column temperature: 35°c

Auto-sampler temperature:10°c

RI Detector Sensitivity: 32

Injection volume: 20 µl

Flow rate: 0.5 ml/min

Run time: 20 min

RI Detector Temperature: 40°c

table ii: results of the analysis of dimethicone content from simethicone tablets using infrared

spectroscopy and hPlc methods

sample ir hPlc1 94.9 94.62 95.2 94.13 95.2 94.14 94.9 94.35 96.9 94.56 94.9 95.07 96.4 95.08 95.5 94.99 95.6 95.0

10 96.8 95.911 97.4 96.012 96.9 96.0 total

mean 95.9 95.0 95.4sd 0.93 0.70 0.93

rsd % 0.97 0.73 0.98n 12 12 24

the dispersion was filtered through a 0.45µ PTFE filter. The first 2 mL of the filtrate was discarded and 20µl from the remaining part was injected onto a hPlc. The chromatographic conditions are denoted in Table I.

table iii: method validation parameters for the hPlc method

Parameter range / valuelinearity 1.75 – 3.25 mg/mlCorrelation coefficient (r) 0.999Slope 692527Y-intercept (%) -0.2

Accuracy (%) (n=12) 101.0%RSD (%) for mean accuracy (%) (n=12)

1.06 %

method precision, mean recovery (%) (n=6)

101.0%

RSD (%) 0.56

Intermediate precision 101.2RSD (%) for intermediate precision (n=12)

1.25

Filter recovery (%)

Nylon filter (0.45µm)

PVDF Filter

PTFE Filter

99.02%

99.37%

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)

1.45% 1.25%



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.

reFerences1. 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, García-Arieta A, de los Ríos F, menéndez 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-Pieńkowska 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.


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 λmax values of mONTE and DES, respectively. Beer’s law was obeyed in the concentration range of 4-24 µg/ml and 2– 12 µg/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-(1-hydroxy-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: [email protected]

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 hydro-11(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 estima-tion of mONTE in combination with other drugs in bulk and in pharmaceutical dosage forms. Similarly, several methods, viz., UV-spectrophotometric 10, RP–hPlc11-12, lc-mS/mS13-14 UPlc 15 are reported in the literature for determination of DES in bulk and in pharmaceuti-cal 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


table ii: e (1%, 1cm) values of monte and des

sr. no (e 1%, 1cm) at 285.6 nm

(e 1%, 1cm) at 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

aAverage of six estimations

table i: linearity studies

Parameter monte deslinearity [µg ml-1]

4 - 24 2 – 12

linearity Equation

Y = 0.038X + 0.068

Y = 0.058 X+ 0.004

molar Absorptivity

2.048 × 104 1.767 × 104

mean Recovery (%)

101.08±0.00467 101.32±0.01137

Precision (% RSD)Inter day 1.244 1.583Intra day 1.309 1.410

Specificity Specific Specific Robustness Robust Robust

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

A UV-Visible spectrophotometer (Shimadzu-1700, 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.

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 µg/ml.

simultaneous equation method

From the stock solutions of 100 µg/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 µg/ml and 2 − 12 µg/ml for mONTE & DES, respectively (Table I). From the overlain spectra (Fig. 3) two wavelengths, 285.6 (λmax of mONTE) and 245.0 (λmax of DES), were selected for developing equations. Standard solutions were prepared in the concentration range of 4-24 µg/

ml for mONTE and 2 - 12 µg/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 ………… (1)

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.


By applying the cramer’s 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 µm Whatman filter paper. An appropriate volume of solution was further diluted with methanol to obtain concentration of 16 µg/ml of mONTE and 8 µg/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.

Percent recovery studies

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.

Precision

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

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).


montelukast and desloratadine followed linearity in the concentration range of 4 - 24 µg/ml and 2 – 12 µg/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 µg ml-1 and 4 µg ml-1 respectively.

conclusion

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


table iii: results of simultaneous estimation of marketed tablets, mondeslor

component label claim (mg) amount taken (µg/ml) amount Found (µg/ml) amount found (%)mONTE 16 16 16.10 100.62

16 16.08 100.5216 15.96 99.7816 16.06 100.3616 16.04 100.2716 16.03 100.19

mean 16.05 100.29SD 0.049 0.30

% RSD 0.30 0.30DES 8 8 7.96 99.50

8 7.93 99.158 8.03 100.388 8.01 100.128 7.99 99.888 7.98 99.75

mean 7.98 99.80SD 0.0044 0.44

%RSD 0.56 0.44

table iv : results for recovery studies

sr. no.

initial amount (µg/ml)

concentration of excess drug added to analyte [µg/ml]

amount added drug obtained

[µg/ml]

% recovery [n = 3]

% rsd

monte des monte des monte des monte des monte des

1. 16 8 12.8 6.4 12.9 6.5 101.3 102.25 0.70 1.49

2. 16 8 16 8 16.1 8.1 101.1 102.37 0.17 1.42

3 16 8 19.2 9.6 19.3 9.5 100.8 99.33 0.38 0.82

table v : results from precision studies

sr.no drug amount taken (µg/ml)

intraday n =3 interday n = 3amount Found (µg/ml) %rsd amount Found (µg/ml) %rsd

1 mONTE 12 12.23 1.09 12.07 1.5316 16.21 1.47 15.97 1.1520 20.21 1.15 19.97 1.23

2 DES 4 4.09 1.54 4.09 1.078 8.09 1.67 7.86 1.13

12 12.20 1.15 12.19 1.36


table vi: results of repeatability study

sr. no drug [µg/ml]

amount found in [µg/ml]

%rsd

1 mONTE 16.18 1.2202 DES 8.12 1.616

aAverage of six estimations

table vii: sensitivity

Parameter monte deslOD [µg/ml] 0.088 0.129lOQ [µg/ml] 0.291 0.249

Fig. 5: calibration curve of desloratadine

Fig. 1: montelukast sodium

Fig. 2: desloratadine

Fig. 3: overlain spectra of monte and des

Fig. 4: calibration curve of montelukast sodium


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.

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.

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& Family Welfare, published by Indian Pharmacopoeial convention, Ghaziabad,Vol. II, 2010 p.1704 -1706.

2. The merck Index – An Encyclopedia of chemicals, Drugs and Biologicals, 14th edn., merck Research laboratories, Whitehouse Station, New Jersey, USA, 2006 p. 514, 1117.

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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

dosage form, indian J Pharm sci. 2010, 72 (2): 231-235.

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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.

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

*For correspondence govt. medical college thiruvananthapuram- 695011, Kerala e-mail: [email protected]


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


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 = Absorbance × D.F × 10 9

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 µM riboflavin and 0.1 mL homogenate (absent in blank and light control). The mixture was made upto a final volume of 3mL using

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 × 100

Optical density of light control/50

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. 50µl 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

I × a

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-inflammatoryActivity16,17

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.


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, 1.17±0.04 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 1.51±0.06 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).

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 300µg and 600 µg 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-inflammatoryActivity

The ethanolic extract at a dose of 100 and 200 µg showed significant HRBC membrane stabilization ie, 76.02±0.36 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 19.54±4.19, 33.81±1.84 and14.09 ±1.78mg/dl for atorvastatin and 500mg/kg & 1000mg/kg of the extract treated groups respectively. Similarly the serum VlDlwere reduced to 19.88±0.61,


table i: lipid Peroxidation,superoxide dismutase & catalase assay

group treatment dose mda (n mol/mg protein)

sod (u /mg protein/min)

catalase (u mol of h2o2 consumed/mg protein/min)

I Standard commercial pelleted diet + 0.3 % cmc

1ml/100 gm 1.17± 0.04 2.99± 0.17 26.6± 0.77

II Atherogenic diet alone+ 0.3 % cmc

1ml/100 gm 2.32 ±0.07 ** 1.82 ±0.05 ** 16.27 ±0.31 **

III Atherogenic diet + Atorvastatin in 0.3 % cmc

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 2.03±0.03 # 2.47±0.01 ^ 20.93±0.36 ^

V Atherogenic diet + extract in 0.3 % cmc

1000 mg/kg 1.51±0.06 ^ 2.54±0.01^ 24.4±1.39 ^

Values are expressed as mean ± SEM (n=6 rats)P values : ** <0.0001 Vs controlP values : ^ <0.01 Vs atherogenic control. # : not significant

Data were analysed by using one way ANOVA followed by Dunnet multiple comparison test and student’s t test

table ii: effect on Prothrombin time

sr. no.

sample Prothrombin time (sec)

1 control 9.45 ± 0.562 heparin 300 µg 52.08±1.48 ^3 Extract 300 µg 26.53±1.33 ^4 heparin 600 µg 74.78±2.68 ^5 Extract 600 µg 44.08±0.57 ^

Values are expressed as mean ± SEM (n=6 rats)P values : ^ <0.01 Vs control. Data were analysed by using one way ANOVA followed by Dunnet multiple comparison test and student’s t test

table iii: effect on hrBc membrane stabilisation

sr. no.

conc (µg)

Prevention of lysis %

extract diclofenac 1 100 76.02 ±0.36 95.29 ± 4.19

2 200 82.84 ±0.11 96.16 ± 0.03

22.87±0.46 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 iv: effect of Plant extract on ldl, hdl and vldl

group treatment dose ldl (mg/dl) hdl (mg/dl) vldl (mg/dl)

I Standard commercial pelleted diet 0.3 % cmc

1ml/100 gm 20.25±2.53 50.24±0.97 17.74±0.65

II Atherogenic diet alone+0.3 % cmc 1ml/100 gm 180±13.03** 40.29±1.39** 28.89±2.95**III Atherogenic diet + Atorvastatin in

0.3 % cmc 30 mg/kg 19.54±4.19^ 53.93±1.82^ 19.88±0.61^

IV Atherogenic diet + extract in 0.3 % cmc

500 mg/kg 33.81±1.84^ 44.17±2.59# 22.87±0.46#

V Atherogenic diet + extract in 0.3 % cmc

1000 mg/kg 14.09 ±1.78^ 64.89 ±2.38^ 19.84 ±0.45^

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 student’s 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).

(The degree of magnification is same (40X) for the same type of tissue from differently treated groups)

discussion

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.

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

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.


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


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


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).

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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 µg 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 µg 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., Immunomodu-latory, 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

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 follows- Yonidoshaharta (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 studiesl 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).

l TLC profile for alkaloids

Solvent system: toluene: ethyl acetate: diethylamine (7:2:1)

Detection: wagner’s reagent (brown colored spots).

l TLC profile for steroids

Solvent system: petroleum ether: acetone (7:3)

Detection: anisaldehyde: sulphuric acid reagent (pink to red colored spots)

l 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 100µg/ml, 50µg/ml, 25µg/ml, 12.50µg/ml and 6.25µg/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


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 100µg/ml, 50µg/ml, 25µg/ml, 12.50µg/ml and 6.25µg/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 Saboraud’s 2% dextrose broth for 48 hours at 370c to obtain organisms in the yeast phase only. The cultures spun

at 1500g for 10min and the deposit washed twice with PBS and filtered twice through sterile gauze. 5x106 cell/ml Candida albicans resuspended in hank’s (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., 100µg/ml, 50µg/ml, 25µg/ml, 12.50µg/ml and 6.25µg/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 hank’s 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 hank’s 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


table i: thin layer chromatography results of Pongamia glabra vent. seeds and bark methanolic extracts

sr. no.

extractsFlavonoids alkaloids steroids saponins

no. of spots

rf valueno. of spots

rf value

no. of spots

rf valueno. of spots

rf value

1Seed Ext.

20.59, 0.63

20.89, 0.80

2 0.61, 0.76

2 0.58, 0.65

2Bark Ext.

20.62, 0.71

20.82, 0.53

40.64,0.72, 0.78, 0.88

2 0.72, 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

conc. (µg/ml)

methanolic ext. of seeds of Pongamia

glabra

methanolic ext. of bark of

Pongamia glabramean Percentage of reduced neutrophils

18±0.0 86±0.0

100 85 7650 79 7425 67 56

12.50 43 326.25 40 30

mean±SEm 62.80ns±9.178 53.60**±9.867

table iii: Phagocytosis of killed Candida albicans for methanolic extracts of Pongamia glabra vent. seeds and bark on human neutrophils

normal control Pooled serum (Positive control)

conc. (µg/ml)

methanolic ext. of seeds of Pongamia glabra

methanolic ext. of bark of Pongamia glabra

mean Particle number2±0.0 5±0.0 100 5 4

50 5 425 4 3

12.50 3 36.25 3 3

mean±SEm 4.00ns±0.447 3.40**±0.245

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 : hank’s solution (Normal control)

chamber 2 : casein 1mg/ml (positive control)


chamber 3 : Pre-determined concentrations of extracts (100µg/ml, 50µg/ml, 25µg/ml, 12.50µg/ml and 6.25µg/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

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 Dunnet’s 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

table iv: neutrophil locomotion and chemotaxis for methanolic extracts of Pongamia glabra vent. seeds and bark on human neutrophils

normal control casein (Positive control)

conc. 1mg/ml

conc. (µg/ml)

methanolic ext. of seeds of Pongamia

glabra

methanolic ext. of bark of Pongamia glabra

MeanNumberofNeutrophilsperfield

15±0.0 200±0.0

100 170 15050 160 15025 120 130

12.50 100 1106.25 90 90

mean±SEm 128.00***±15.94 126.00***±11.66

Significant difference from positive control by One Way ANOVA followed by Dunnet’s ‘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


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 100µg/ml, 50µg/ml, 25µg/ml, 12.50µg/ml and 6.25µg/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 (100µg/ml, 50µg/ml and 25µg/ml) and lower at the concentrations 12.50µg/ml and 6.25µg/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 128.00±15.94 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 100µg/ml, 50µg/ml and 25µg/ml and lower at concentrations 12.50µg/ml and 6.25µg/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


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.

reFerences1. 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, 830-832.

5. Nadkarni K.m..: Indian materia medica Vol 1,Popular Prakashan ltd., mumbai. 1996, 1001–1004.

6. Bhavaprakash Nighantu, Guduchyadi Varga : Shree Gulab Kunvera Ayurvedic Society, Jamnagar, India.1949, 119-120.

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., Springer-verlag, 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, 686-694.

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.

dept. of Pharmacognosy and Phytochemistry heroor s.s.*, Beknal a.v. and mahurkar n.K. hKes’s mtr institute of Pharmaceutical sciences gulbarga 585105, Karnataka e-mail: [email protected] *For correspondence (Received 22 November 2012) (Accepted 04 February 2013)


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