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Education & Research Vol. 44(4), Oct - Dec, 2010

CONTENTS


Education & Research Vol. 44(4), Oct - Dec, 2010

Formulation and Evaluation of Diclofenac Sodium Gels Using Sodium Carboxymethyl Hydroxypropyl Guar and Hydroxypropyl MethylcelluloseSwamy N.G.N., Mazhar Pasha and Zaheer Abbas ..................................................................................... 310 -314

Mucoadhesive films of Losartan Potassium for Buccal delivery: Design and CharacterizationMarina Koland, R.N. Charyulu and Prabhakara Prabhu .......................................................................... 315 - 323

Formulation and Evaluation of Topical Liposomal Gel for FluconazoleB. V. Mitkari, S. A. Korde, K. R. Mahadik and C. R. Kokare ...................................................................... 324 - 333

Development of Fast Dissolving Tablets of Glibenclamide Using Crospovidone and its Kneading MixtureJeevana Jyothi. B and Suneela. G .............................................................................................................. 334 - 340

Stability Indicating HPTLC method for Estimation in the Bulk Drug and Tablet Dosage Form

Effect of Different Acids on the Formation of E and Z Isomers of Doxepin G.K. Rao, A.R. Ramesha, Amit Kumar Jain and B.V. Adavi Rao.................................................................. 345 - 349

Application of Factorial Design in Optimization of Synthetic Reactions: a Novel ApproachPal Tanushree, Somani Rakesh R. and Kadam Vilasrao J. ......................................................................... 350 - 357

Microwave Assisted Synthesis and Biological Evaluation of Pyrazole Derivatives of BenzimidazolesR. Kalirajan, Leela Rathore, S. Jubie, B. Gowramma, S. Gomathy, S. Sankar and K. Elango ................. 358 - 362

Anti-inflammatory and Antioxidant Activities of Methanolic extract of Buchanania Lanzan KernelWarokar A. S., Ghante M. H., Duragkar N. J. and Bhusari K. P. ............................................................... 363 - 368

Phytochemical investigation and evaluation of anthelmintic activity of extract from leaves of Eupatorium odoratum linn. Debidani Mishra, Deb Kumar Sarkar, Bhabani Shankar Nayak, Prasant Kumar Rout, P. Ellaiah and S. Ramakrishna ..................................................................................................................... 369 - 374

Determination of In-vitro Sun Protection Factor (SPF) of Murraya Koenigii L. (Rutaceae) Essential oil Formulation

Rekha B Patil, Shantanu Kale, Devanshi M Badiyani and A.V. Yadav ....................................................... 375 - 379

In vitro and In Vivo evaluation of Stimuli Sensitive Hydrogel for ophthalmic drug deliveryVinod Singh, S.S. Busheetti S Appala Raju , Rizwan Ahmad, Mamta Singh .............................................. 380 - 385,

Total Quality Management in Pedagogy (TQM_P): An UpdateRam Chakkaand G.T. Kulkarni .................................................................................................................. 386 - 390

Trandolapril

Vikas, Rao J.R, Sathiyanarayanan L and Yadav S.S ..................................................................................... 341 - 344

Abstract

In this investigation, Diclofenac sodium gels were formulated employing Sodium carboxymethyl hydroxypropyl guar

and Hydroxypropyl methylcellulose as gelling agents. Hydroxypropyl methylcellulose (K4M) was employed at 5 %

w/w strength whereas, Sodium carboxymethyl hydroxypropyl guar formed a gel at 2.5 % w/w strength. Gels were

subjected for various evaluation tests such as pH measurement, assay, stability study, rheological evaluation, and in-

vitro release studies across hairless albino rat skin. Gels formulated using Sodium carboxymethyl hydroxypropyl

guar displayed a pH value of 7.48 whereas, Hydroxypropyl methylcellulose gels revealed a pH value of 7.26. Stability

studies revealed good physical stability and assay values did not show much variation from the initial drug content in 0 0both the cases with formulations stored at 25 C, 60% RH and 40 C, 70% RH for six months. Hydroxypropyl

methylcellulose at 5% w/w strength revealed shear-thinning property whereas, Sodium carboxymethyl

hydroxypropyl guar at 2.5 % w/w strength revealed both pseudoplastic and thixotropic property. The rheological data

were fitted into Martin and co-workers equation to obtain a linear relationship and from the linear curve fittings, the

'N'- values; the possible flow indices for pseudoplasticity were arrived at. A 'N' value of 4.65 was obtained for Sodium

carboxymethyl hydroxypropyl guar gels in contrast to a 'N' value of 1.52 in case of Hydroxypropyl methylcellulose

gels. When subjected to In-vitro release studies across hairless albino rat skin, Sodium carboxymethyl hydroxypropyl

guar based gels revealed a % cumulative drug release of 25.66 in contrast to a % cumulative drug release of 20.80 in

case of Hydroxypropyl methylcellulose based gels at the end of 6 hours. From the above observations, Sodium

carboxymethyl hydroxypropyl guar seems to be a promising pharmaceutical adjuvant in the formulation of

Diclofenac sodium gels.

Keywords: Diclofenac sodium gels, Sodium carboxymethyl hydroxypropyl guar, Hydroxypropyl methylcellulose,

Rheological evaluation, Pseudoplasticity index

INTRODUCTION1Gels consist of liquids gelled by means of suitable

gelling agents. Gels comprise of homogenous

preparations intended to be applied to the skin or certain

mucous membranes; Gels may contain auxiliary

substances such as antimicrobial preservatives, 2antioxidant and stabilizers. The active ingredients in gel

based formulations are better percutaneously absorbed

than cream or ointment bases. A gel based formulation

can hold/contain more percentage of ethyl alcohol than

ointment and creams. A number of polymers are used to

provide the structural network for gel system. The

polymers are used in the concentration range of 0.5 to

15%. In the present study, two polymers have been used

as gelling agents namely Sodium carboxy methyl 3hydroxy Propyl guar (NaCMHPG) and hydroxy Propyl

4methyl cellulose (HPMC) .5Diclofenac Sodium , a non steroidal antiinflammatory

agent is frequently prescribed for the long term treatment

of rheumatoid arthritis, osteoarthritis and ankylosing 6spondylitis. The drug undergoes substantial first pass

effect and only 50% of drug is available systemically.

Further, the drug is known to induce ulceration and

bleeding of the intestinal wall. To avoid the adverse

effect, alternate routes of administration have been tried 7, 8by investigators . Delivery of Diclofenac sodium via

5skin offers the potential advantage of bypassing hepato-Indian Journal of Pharmaceutical Education and ResearchReceived on 6/4/2010; Modified on 23/7/2010Accepted on 28/8/2010 © APTI All rights reserved

Formulation and Evaluation of Diclofenac Sodium Gels Using

Sodium Carboxymethyl Hydroxypropyl Guar and Hydroxypropyl

Methylcellulose

Swamy N.G.N.*, Mazhar Pasha and Zaheer AbbasDepartment of Pharmaceutics, Government college of Pharmacy, Bangalore – 560 027

*Author for Correspondence: [email protected]

310

Indian J.Pharm. Educ. Res. 44(4), Oct - Dec, 2010

gastrointestinal first pass metabolism associated with

oral administration. The drug is prescribed in a dose of

75 to 150 mg daily in divided doses by oral route. The

dosing frequency can be reduced if patients are instructed

to use topical products along with the conventional

tablets.

Materials and method:

Materials: Diclofenac sodium was obtained as a gift

sample from Bangalore Pharmaceutical and Research

Laboratories. NaCMHPG used in the formulation was

prepared in our laboratory The polymer revealed a DS of 9 10value of 1.5 and sodium content of 10.44%w/w.HPMC

was obtained from Zydus cadila pharmaceuticals limited

Bangalore; Propylene glycol, sodium methyl paraben

and sodium Propyl paraben were obtained from Nice

chemicals.

Method

A quantity of HPMC (gel-I)/NaCMHPG (gel-II) as

specified in table-1 were dispersed separately in about 40

ml of deionised water. In case of HPMC dispersion, it was 11warmed to form a gel . Diclofenac sodium was dispersed

in 40 ml of deionised water. To the drug solution,

propylene glycol and preservatives (sodium salts of

methyl paraben and Propyl paraben) were added and

stirred until the solutioning was effected. The drug,

humectant and preservatives solution was added to the

polymer solution in small increments with constant

stirring using a propeller mixer. Stirring speed was

adjusted to minimize the air entrapment in the gel and

deionised water was added to adjust the gel weight to 100

grams. The agitation was continued until a uniform gel

resulted. The prepared gels were filled in empty

collapsible tubes and sealed by crimping the ends and

preserved in a cool and dry place until further use.

EVALUATION OF DICLOFENAC SODIUM GELS

The Diclofenac sodium gels were subjected for extensive

rheological evaluation, drug content estimation.pH

measurement, stability study and drug release study

across hairless albino rat skin.

Rheological studies: Brookfield synchrolectric 12 13 viscometer , analog model was used for the studies.

First, the spindle was dipped into the gel till the notch on

the spindle touched the gel surface.100 g each of gel I and

gel II was used in the study. The spindle no.7 was selected

based on the viscosity of the (gel for) both the

formulations. This spindle was rotated at 0.5 rpm, and

dial reading was recorded until 2 consecutive similar

readings were obtained. Similarly dial readings were

recorded at 1.0, 2.5, 5.0, 10.0, 20.0, and 50.0 and up to

100 rpm. As soon the sample was sheared at the highest

rate, another set of dial readings were recorded by

reducing the spindle rotation in the decreasing order to

the pool the data on the down curve. Rheograms were

constructed by plotting the dial readings on the X-axis

and rpm values along the Y-axis. Rheological data were

pooled for (i) polymer dispersion in preservative solution

(ii) dispersion of polymer and drug in preservative

solution (iii) dispersion of polymer and humectant in

preservative solution (iv) all together.

Drug content estimation: An accurately weighed 1 gm

quantity of the gel was transferred into a 250ml stoppered

volumetric flask and shaken vigorously with 2x25 ml

quantity of methanol to extract the drug. The contents

were filtered into a 50 ml volumetric flask and volume

was made up to the mark with methanol. From the above

solution, 0.5 ml was pipetted in to a 25 ml volumetric

flask and volume was made up to 25 ml with methanol.

Finally, the UV absorbance of the resulting solution was

measured at 280 nm against the blank solution of

methanol.14Diclofenac sodium obeys Lambert's beers law in the

concentration range of 2 to 16 µg/ml. A calibration curve

was constructed which revealed a slope value of 0.0421

and intercept value of 0.0025. These values were used in

finding the drug content in the formulation after

extracting the drug in suitable dilutions and recording the

absorbance at 280 nm.15,16pH measurement: The pH measurement was carried

out by using a calibrated digital type pH meter by dipping

the glass electrode and the reference electrode

completely into the gel system so as to cover the

electrodes.17In-vitro release studies: The hairless albino rat skin

obtained from the discards of the animal sacrifice at the

pharmacology department in the college was used. The 18skin was soaked in 0.32 N ammonium hydroxide

solution for 30 to 35 minutes to remove subcutaneous fat

and hair. The skin was rinsed well with saline followed by

distilled water.19Franz diffusion cell was used for permeability study: 1 g


311


of the gel was uniformly spread over the rat skin

membrane and tied over the donor compartment. The

skin was placed with stratum corneum facing the donor

compartment and the dermis facing the receptor

compartment containg 100ml distilled water. At hourly

intervals, 5ml of sample was withdrawn from the receptor

and replaced with fresh 5ml distilled water.The 5 ml

withdrawn sample was made up to 25 ml with distilled

and the absorbance was recorded at 280nm. The receptor

medium was magnetically stirred for uniform

distribution and was maintained at a temperature of 37°C

± 0.2°C .

Stability study

Stability studies for Diclofenac sodium gels were carried 20out as per ICH guidelines .

0 0The gel samples were stored at 25 C, 60% RH and 40 C,

70% RH, in stability chambers for a period of 6 months,

samples were drawn at regular interval for stability

analysis. At the end of 6 months assay was carried out to

find out if there is any interaction between the drug and

other ingredients of the formulation upon storage.

RESULTS AND DISCUSSION

1%w/w Diclofenac sodium gels were formulated using

two different polymers namely NaCMHPG and HPMC at

2.5% w/w and 5% w/w respectively. A 5% w/w

dispersion of HPMC containing 10% w/w humectant and

0.2% w/w of preservatives in water revealed a viscosity

of 1, 44,000 cps units when sheared at 5 rpm using

spindle-7 in Brookfield synchrolectric viscometer RVT

make, analog model in contrast to a value of 1,52,000 cps

units for a 2.5% w/w NaCMHPG dispersion of similar

composition when sheared under similar conditions.

From the rheological investigations, it is observed that

while HPMC gels have revealed only shear thinning

property, NaCMHPG gels have revealed both thixotropic

as well as pseudoplastic behavior. The rheological

behavior for the gel samples is depicted in fig 1. Further,

based on martin and co-worker’s equation, the 21rheological data are transposed to a linear plot by

construction of a log- log graph to obtain the value for

'N'. Such plots were constructed to know the influence of

drug, humectant and the preservative on variations in 'N'

value in the system. The NaCMHPG gels by virtue if its

higher intrinsic viscosity and improved interaction co-

efficient have revealed a higher value of 'N' compared to

HPMC gels. The log-log plot for NaCMHPG and HPMC

gels is shown in fig 2 .The N values for HPMC based gels

(gel-1) varied between 1.49 to 1.52, whereas, for

NaCMHPG based gels (gel-2), the value varied between

4.52 - 4.65. This is indicative of the fact that, the

contributions by the components towards the N values

are insignificant.

Gels prepared using NaCMHPG had a pH value 7.48

whereas HPMC based gels revealed a pH of 7.46. The

gels which have pH value in the range of 5.5 to 7.5 are

most ideal, as they near the pH of the skin and do not

cause irritation.

In-vitro release study for the drug across hairless albino

rat skin with NaCMHPG based gels revealed a % CDR of

25.66 while, HPMC based gels revealed a % CDR of 2220.80 at the end of 6 hrs .The drug release pattern is

depicted in fig 3. The release of drug from both the 23formulations followed zero-order kinetics . Stability

studies for gels revealed good physical stability, color and

consistency for the formulations. The drug content

remained the same as was seen in the gel formulations

before being subjected for the stability study.

CONCLUSION

A 1% w/w gel of Diclofenac sodium in a 5% w/w

dispersion of HPMC gel base revealed shear thinning

qualities only whereas, in a gel base of 2.5% w/w

NaCMHPG, both pseudoplastic as well as thixotropic

properties were observed. The NaCMHPG based

Diclofenac sodium gels with a higher 'N' value are

expected to have better ease of application. Further, better

speardability is expected in case of NaCMHPG based

gels. Since NaCMHPG in half the strength has displayed

better performance in respect to the drug release , in

contrast to HPMC based gels, it can be concluded that

NaCMHPG is a better gelling agent in the formulation of

Diclofenac gels.

ACKNOWLEDGEMENT

The authors wish to thank

(I) Messrs Juggat Pharma Pvt. Ltd, Kumbalagodu,

Bangalore for sponsoring gift sample of propylene oxide

(II) Bangalore Pharmaceutical Research Laboratories for

sponsoring gift sample of Diclofenac sodium.

(III)The Principal, Govt. College of Pharmacy for

permitting to avail the research facilities in the college.

312


Ingredients Gel-I Gel-II

Diclofenac sodium 1.0 gm 1.0 gm

HPMC 5.0 gm -

NaCMHPG - 2.5 gm

Propylene glycol 10 gm 10 gm

Methyl paraben 0.18 gm 0.18 gm

Propyl paraben 0.02 gm 0.02 gm

Purified water QS 100 gm 100 gm

Table-1: Composition of gels

Fig 1: Rheogram of HPMC gels (5% w/w) and

NaCMHPG gels (2.5% w/w) containing Drug (1% w/w),

Humectant (10% w/w) and preservative (0.2% w/w) .

Fig 2: Log Rheogram of HPMC gels (5% w/w) and

NaCMHPG gels (2.5% w/w) containing Drug (1% w/w),

Humectant (10% w/w) and preservative (0.2% w/w) )

Fig 3: Comparative in-vitro release studies of Diclofenac

gels formulated using NaCMHPG and HPMC

% d

rug

reta

ined

in

the

gel

Comparative Release Rate Profile for Diclofenac Sodium from Gel I and Gel II Prepared using NaCMHPG and HPMC

313


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ointment (cream) in topical application. JPn

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guar gum. Indian Drugs, 2001; 63(1): 74-78.

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NJ,USA :Merck and Co. Inc., 2006: P 522

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permeation enhancers of flubiprofen from gel

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– 204.

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from poloxamer gels in a membraneless diffusion

cell. J. Pharm Sci., 1991; 80(3): 280 – 283.

17. Swamy N G N, Dharmarajan T S and Paranjothi K L

K., Study of hydroxypropyl guar derivative for its

gelling property and its use in the formulation of

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transdermal therapeutic systems, Indian Drugs ,

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H. Development and in vitro evaluation of

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K., Study of film forming properties of hydroxy

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314

Abstract

Buccal delivery is considered to be an important alternative to the peroral route for the systemic administration of

drugs. Losartan potassium is an angiotensin II receptor antagonist with an oral bioavailability of only 33% due to

extensive first pass metabolism. Mucoadhesive buccal films of losartan potassium were prepared using

hydroxypropyl methyl cellulose (HPMC) and retardant polymers ethyl cellulose (EC) or eudragit RS 100. Thermal

analysis by DSC of formulations show no interaction between drug and polymers. Ex vivo permeation studies of

losartan potassium solution through porcine buccal mucosa showed 90.2 % absorption at the end of 2 hours. The

films were subjected to physical investigations such as uniformity of thickness, weight, drug content, folding

endurance, tensile strength, elongation at break, surface pH and mucoadhesive strength. Films were flexible and

those formulated from EC were smooth whereas those prepared from Eudragit were slightly rough in texture. The

mucoadhesive force, swelling index, tensile strength and percentage elongation at break was higher for those

formulations containing higher percentage of HPMC. In vitro drug release studies reveal that all films exhibited

sustained release in the range of 90.10 to 97.40 % for a period of 6 hours. The data was subjected to kinetic analysis

which indicated non fickian diffusion for all formulations except E2. Ex vivo permeation studies through porcine

buccal mucosa indicate that films containing higher percentage of the mucoadhesive polymer HPMC showed slower

permeation of the drug for 6-7 hours.

Keywords: Losartan, eudragit, ethyl cellulose, buccal mucosa


INTRODUCTION

Buccal drug delivery has lately become an important

route of drug administration. The rich vascularization of

the oral mucosa and its permeability to many drugs

makes this route an attractive alternative to the oral and

parenteral routes for systemic drug delivery. Absorption

of therapeutic agents from the oral mucosa overcomes

premature drug degradation due to enzyme activity and

pH of the gastrointestinal tract, avoids active drug loss

due to first-pass hepatic metabolism and therapeutic

plasma concentration of the drug can be rapidly 1achieved . The buccal mucosa permits a prolonged

retention of a dosage form especially with the use of

mucoadhesive polymers without much interference in

activities such as speech or mastication unlike the sub-2lingual route . Various bioadhesive mucosal dosage

forms have been developed which include tablets, gels,

patches and films all of which make use of polymers such

as carbopols, hydroxy propyl methyl cellulose etc. that

prolong the residence time of the dosage form. A few

examples of such formulated dosage forms by various

investigators are shown in Table 1. Mucoadhesive buccal

films or patches are preferred in terms of flexibility,

comfort, patient compliance and better adhesion of the 8system to the oral mucosa . The antihypertensive,

Losartan potassium is an angiotensin II receptor (type

AT ) antagonist, orally active and undergoes substantial 1

first-pass metabolism by cytochrome P450 enzymes. The

terminal half-life of losartan is about 2 h. The drug is

orally administered as 25 mg tablets once or twice daily

with total daily doses ranging from 25 to 100 mg.

Following oral administration, losartan is well absorbed

(based on absorption of radiolabeled losartan) and

undergoes substantial first-pass metabolism; the

systemic bioavailability of losartan is approximately 933% . In view of these facts, this drug can be considered

Indian Journal of Pharmaceutical Education and ResearchReceived on 29/9/2009; Modified on 21/1/2010Accepted on 23/7/2010 © APTI All rights reserved

Mucoadhesive films of Losartan Potassium for Buccal delivery:

Design and Characterization

Marina Koland*, R.N. Charyulu and Prabhakara PrabhuNitte Gulabi Shetty Memorial Institute of Pharmaceutical Sciences, Derelakatte, Mangalore,

Karnataka-574160


315

as a suitable candidate for buccal delivery. In this study,

an attempt is made to investigate the feasibility of

mucoadhesive buccal films as a medium for the sustained

delivery of losartan potassium with better bioavailability.

MATERIALS AND METHODS

Losartan Potassium was generously gifted to us by Sun

Pharmaceutical Industries Ltd. Vapi, Gujarat. Ethyl

cellulose and HPMC were procured from Merck India

Ltd., Mumbai. Organic solvents used were of analytical

grade and other chemicals of Laboratory grade.

Preparation of Polymeric films of Ethyl cellulose (EC)

and Hydroxypropylmethyl cellulose (HPMC):

Films were prepared by the solvent casting method

using EC and HPMC in the ratios of 1:0.5, 1:1 and 0.5: 1.

Higher levels of EC gave films which could not be

removed from the Petri dish. Propylene glycol was used

as the plasticizer. Losartan potassium was dissolved in 10

ml of ethanol. EC was then added to this solution and

stirred till dissolved. To this solution, 8 ml of

dichloromethane was added, followed by the HPMC. The

mixture was constantly stirred on a magnetic stirrer until

the polymers had completely gone into solution and a

clear gel was obtained. Propylene glycol was mixed and

the volume was adjusted to 20 ml with alcohol. The

vessel was closed and kept aside for a few hours until all

the entrapped air had escaped. The solution was then cast

into a glass Petri dish of 9 cm diameter and allowed to dry

overnight at room temperature. The films were removed

carefully and circular patches of 15mm diameter were

punched out so that each patch contained 10 mg of the

drug. The samples were packed in aluminum foil and

stored in a glass container maintained at room

temperature and 58% relative humidity. This condition

maintained the integrity and elasticity of the patches.

Preparation of Polymeric films of Eudragit RSPO

and HPMC:

Films were prepared as above but using Eudragit RSPO

in the place of EC. The composition of various films is

shown in Table 2.

Evaluation of prepared buccal films:

Uniformity of weight:

The individual weight each of 10 samples of each

formulation was determined. The average weight was

calculated.

Thickness:

The thickness of each of 10 patches of each type of

formulation was measured using a micrometer screw

gauge and the average was determined as shown in Table

3. Surface pH:

The surface pH of the films was determined in order to

investigate the possible side effects due to change in pH

in vivo, since an acidic or alkaline pH may cause irritation

to the buccal mucosa. The film to be tested was placed in a

Petri dish and was moistened with 0.5 ml of distilled

water and kept for 1 h. The pH was noted after bringing

the electrode of the pH meter in contact with the surface

of the formulation and allowing equilibrating for 1.0 10min . The average of three determinations for each

formulation is shown in Table 3.

Thermal Analysis:

The ethyl cellulose and eudragit films were subjected to

thermal analysis by Differential Scanning Calorimetry

(DSC) to confirm the absence of any interactions

between drug and excipients.

Folding Endurance:

The folding endurance was determined by repeatedly

folding one patch at the same place till it broke or folded

up to 300 times which is considered satisfactory to reveal

good film properties. The number of times the film could

be folded at the same place without breaking gives the 11value of the folding endurance .

Uniformity of drug content:

This parameter was determined by dissolving one patch

of 15 mm diameter designed to contain 10 mg of losartan

potassium by homogenization in a mixture of 5 ml ethyl

alcohol and 2ml of dichloromethane for 5 h with

occasional shaking and diluted to 50 ml with distilled

water. After filtration to remove insoluble residue, 1 ml of

the filtrate was diluted to 10 ml with simulated saliva of

pH 6.8. The absorbance was measured at 250 nm using an

UV spectrophotometer. The experiments were carried out

in triplicate for the films of all formulations and average

values were recorded as shown in Table1.

Measurement of Swelling Index:

The studies for Swelling Index of the films were

conducted in simulated salivary fluid of pH 6.8. The film 2 sample (surface area: 1.75 cm ) was weighed and placed

in a preweighed stainless steel wire sieve of

approximately 800 µm mesh. The mesh containing the


316

film sample was then submerged into 15 ml of the

simulated salivary medium contained in a porcelain dish.

At definite time intervals, the stainless steel mesh was

removed, excess moisture removed by carefully wiping

with absorbent tissue and reweighed. Increase in weight

of the film was determined at each time interval until a

constant weight was observed. The degree of swelling

was calculated using the formula:

S.I = (w -w )/w t 0 0

Where S.I is the Swelling Index, w is the weight of film at t

8time t and w is the weight of the film at time 0 .0

Measurement of bioadhesive strength:

Satisfactory bioadhesion is essential for successful

application of bioadhesive drug delivery systems in order

to increase the residence time at the site of application and

hence to provide the prolonged release of the drug. The

tensile strength required to detach the bioadhesive patch

from the mucosal surface was applied as a measure of the

bioadhesive performance. Several techniques have been

reported in literature for measurement of bioadhesive

strength. In the present work a specially fabricated

assembly based on published literature was used. Porcine

cheek pouch was used as the model surface for

bioadhesion testing. After the cheek pouch was excised

and trimmed evenly, it was then washed in simulated

salivary fluid and then used immediately.

Fabrication of the test assembly: The working of a

double beam physical balance formed the basis of the

bioadhesion test assembly. The left pan was removed and

hung with a stainless steel chain. A Teflon block with 1.5

in height and 1.5 in diameter was hung with the stainless

steel chain to balance the weight of the other pan. The

height of the total set up was adjusted to accommodate a

glass container or beaker below it leaving a head space of

about 0.5 cm in between. Another Teflon block of 2 in

height and 1.5 in diameter was kept inside the glass

vessel, which was then positioned below the top hung

Teflon block. Suitable weights were added (15.0 g) on the 11right pan to balance the beam of the balance .

Method: The porcine cheek membrane was attached

with the mucosal side upward onto the lower Teflon block

which was then placed in the glass vessel. Sufficient

simulated salivary fluid was filled into the beaker so that

the surface of the fluid just touches the mucosal surface to

keep it moist. The beaker was positioned below the upper


Teflon block. The film under test was fixed to the surface

of the upper block with glue. The 15.0 g weight on the

right pan was removed and this lowers the upper Teflon

block with film, so that it is in contact with mucosal

surface. A load of 20.0 g was placed as initial pressure on

the upper block for 3 min. Slowly weights were added

onto the right pan starting from 500 mg at 30 s time

intervals. The total weight at which detachment of the

film from the mucosal surface takes place is noted and the

bioadhesion force was calculated per unit area of the

patch as follows:

F = (W x g)/Aw

2Where F is the bioadhesion force (kg/m/s ), W is the w

mass applied (g), g is the acceleration due to gravity 2 2(cm/s ) and A is the surface area of the patch (cm ). The

results are tabulated in Table 3 for all formulations.

Tensile Strength Measurement:

This mechanical property was evaluated using Instron

universal testing instrument (Model 1121, Instron Ltd.,

Japan, NITK, Suratkal) with a 5-kilogram load cell. Film

strips in special dimension and free from air bubbles or

physical imperfections were held between two clamps

positioned at a distance of 3 cm. During measurement,

the strips were pulled by the top clamp at a rate of 100

mm/m; the force and elongation were measured when the

film broke. Results from film samples, which broke at

and not between the clamps, were not included in the

calculations. Measurements were run in triplicate for

each film.

Two mechanical properties, namely, tensile strength and

% elongation were computed for the evaluation of the

film. Tensile strength is the maximum stress applied to a

point at which the film specimen breaks and can be

computed from the applied load at rupture as a mean of

three measurements and cross sectional area of fractured 8film as described from the following equation .

Tensile strength =Force at break (N)

2Initial cross sectional area of the sample (mm )

Percent elongation can be obtained from the following

equation

%Elongation at break= Increase in length

Original length× 100

Values for tensile strength and percentage elongation for

all formulations are shown in Table 3.

317


Ex vivo mucoadhesion time:

The ex vivo mucoadhesive time was performed by

application of the film on freshly cut porcine buccal

mucosa. The porcine tissues were fixed on the internal

side of a beaker with cyano acrylate glue. The film was

wetted with 50 µl of simulated saliva fluid and was pasted

to the porcine buccal tissue by applying a light force with

a finger tip for 20 s. The beaker was filled with 200 ml 0 simulated saliva fluid and kept at 37 C. After 2 minutes,

a 50 rpm stirring rate was applied to simulate the buccal

cavity environment and during the test, the time taken for

the film to completely erode or detach from the mucosa 5was observed as the ex vivo mucoadhesion time .

In vitro drug release studies:

In vitro release studies were carried out by a slight

modification of the method suggested by Ilango et al and

Perioli L., et al. A buccal film was attached to the wall of

the dissolution vessel such as a 250 ml beaker, midway

from the bottom with instant adhesive or cyanoacrylate

glue. After 2 min, the vessel was filled with 200 ml of

simulated saliva of pH 6.8 and placed on a magnetic

stirrer. The temperature of the dissolution medium was 5, 12maintained at 37 ± 0.5º C and stirred at 50 rpm .

Samples of 3ml were withdrawn at predetermined time

intervals and replaced with fresh medium. The samples

were filtered through 0.45µm Whatman filter paper and

after appropriate dilutions with simulated saliva were

assayed spectrophotometrically at 250 nm. Four film

samples of each formulation were subjected to drug

release studies in this manner and the average cumulative

percentage drug released was determined.

Ex Vivo Drug permeation studies:

Permeation studies were carried using the modified Franz

diffusion cell of internal diameter of 2.5 cm. porcine oral

mucosa was used as the model membrane. The buccal

pouch of the freshly sacrificed pig was procured from the

local slaughter house. The buccal mucosa was excised

and trimmed evenly from the sides and then washed in

isotonic phosphate buffer of pH 6.6 and used

immediately. The membrane was stabilized before

mounting in order to remove the soluble components.

The mucosa was mounted between the donor and

receptor compartments. The receptor compartment was

filled with 200 ml of isotonic phophate buffer of pH 7.4

which was maintained at 37 ± 0.2º C and the

hydrodynamics were maintained by stirring with a 13, 14magnetic bead at 50 rpm .

Permeation of losartan potassium from aqueous

solution:

Before the film formulations are actually subjected to ex

vivo buccal permeation studies, it was considered

necessary to determine whether losartan potassium was

able to penetrate the buccal mucosa at all and what would

be the extent of permeation. For this study, the drug in the

most available form, i.e. an aqueous solution (10 mg in

5ml of simulated saliva) was placed in the donor

compartment. At predetermined time intervals, a 1ml

sample was withdrawn and analysed using an UV

spectrophotometer at λ of 250 nm. The experiments max

were performed in triplicate and the profile is shown in

Figure 3.

Permeation of losartan potassium from formulated

films:

Since the percentage of drug permeated from solution is

appreciable. It can be concluded that losartan potassium

does have sufficient buccal permeability and is therefore

suitable for further study from film formulations. The

patch was placed in intimate contact with the mucosal

surface of the membrane that was previously moistened

with a few drops of simulated saliva. The donor

compartment was filled with 1 ml of simulated saliva of

pH 6.8. The fluid in the receptor compartment was

replaced with fresh medium and the experiment was

conducted as described above.


Preparation of film formulations:

All the film formulations containing EC or Eudragit

RSPO as the retardant polymer and HPMC as the

mucoadhesive polymer with propylene glycol as

plasticizer were readily prepared by solvent casting. A

solvent mixture of ethanol and dichloromethane was

required to keep both polymers in solution. Films with

higher percentage of EC or the Eudragit could not be

prepared since they could not be removed easily from the

Petri dish in which they were cast and tended to fragment.

Evaluation of Prepared Films:

From the results of the tests for physical characterization

conducted, it is observed that the weight and thickness of

all film samples was uniform within each formulation.

Films formulated from EC were smooth whereas those

318

prepared from Eudragit were slightly rough in texture. All

films were translucent and flexible. All film formulations

exhibited good folding endurance exceeding 300,

indicating that they are tough and flexible.

Surface pH

An acidic or alkaline pH of administered dosage forms

can irritate the buccal mucosa. The measured surface pH

was found to be close to neutral in all the formulations

which means that they have less potential to irritate the

buccal mucosa and therefore they should be fairly

comfortable.

Thermal Analysis

Thermal analysis of ethyl cellulose and Eudragit film

formulations containing losartan potassium do not reveal

any additional peak for the drug as seen in Figures 1 and

2.

indicates that the drug did not interact with

excipients used in the films.

Drug content

All the film formulations of losartan potassium

containing ethyl cellulose and eudragit polymers show

uniform drug content as seen in Table 3.

Swelling Index

The measurement of Swelling Index indicates that

maximum swelling takes place in the formulations

containing higher proportions of HPMC namely E3 and

E6 and the least in those containing higher proportions of

Eudragit RSPO and ethyl cellulose which are water

insoluble and less hydrophilic and therefore subject to

lesser swelling upon hydration. It was also observed that

films containing the hydrophilic polymers disintegrated

very fast. The presence of the hydrophilic polymer,

HPMC seems to increase the surface wettability and

swelling of the films.

Measurement of bioadhesive strength

The bioadhesive force measured was found to be higher

for those film formulations containing higher proportions

of the mucoadhesive polymer, HPMC as in the case of E3

and E6. Moreover HPMC hydrates fast achieving

maximum swelling at shorter periods which could

promote interpenetration of the polymer chain with the

tissue.

The DSC thermograms of the films showed sharp

distinct endothermic peaks for losartan potassium and the

polymers. This corresponds to the peaks of individual

drug and polymer without exhibiting any modification

which

Tensile strength measurement

The tensile testing gives an indication of the strength and

elasticity of the film reflected by the parameters, Tensile

strength (TS) and Elongation at break (E/B). A weak and

soft polymer is characterized by a low TS and E/B; a hard

and brittle polymer shows a moderate TS and low E/B; a

soft and tough polymer is characterized by a moderate TS

and high E/B whereas a hard and tough polymer shows a 8high TS and E/B . It is observed from the results of the test

that as the percentage of the mucoadhesive polymer,

HPMC in the formulations increased, the tensile strength

and percentage elongation at break also increased.

Proportions of EC or Eudragit RSPO higher than that

used in these films make them more brittle and weak

Mucoadhesion time

The films composed of larger amounts of the

mucoadhesive polymer, HPMC showed the greatest

mucoadhesion time of nearly 6 h indicating their

suitability for use in buccal drug delivery. Comparatively

shorter mucoadhesion time was observed with films

containing higher amounts of the retardant polymers .

In vitro drug release studies

In vitro drug release studies in simulated saliva show

more than 90 % release of losartan potassium from all

film formulations, i.e., E1, E2, E3, E4, E5 and E6 after 6

hours with E3 showing a maximum percentage drug

release of 97 %. This could be attributed to the higher rate

and extent of swelling of the larger proportion of the

hydrophilic polymer, HPMC. The higher percentage of

the retardant polymers EC and Eudragit RS 100 in E1 and

E4 was responsible for the comparatively slower drug

release from them.

Kinetic analysis of in vitro release data:

In order to determine the release mechanism that

provides the best description to the pattern of drug

release, the in vitro release data were fitted to zero-order,

first-order, Hixson Crowell equation and Higuchi matrix

model. The release data were also kinetically analyzed 15using the Korsmeyer–Peppas model . The release

exponent (n) describing the mechanism of drug release

from the matrices was calculated by regression analysis 15using the following equation .

Where Mt/M is the fraction of drug released (using ∞

nMt/ M = Kt∞


319


showed nearly 90% permeation in less than 2 h,

indicating the suitability of formulating losartan

potassium for buccal delivery. Ex vivo permeation studies

of losartan potassium from the film formulations indicate

slow and sustained permeation of the drug for 6-7 h as

seen in Figure 6. The rank order of drug permeation from

films was found to be E4>E1>E5>E2>E3>E6.

CONCLUSION

This investigation shows that it is possible to formulate

mucoadhesive films of losartan potassium with the

intention of obtaining better therapeutic efficiency by

controlling drug release thereby improving patient

compliance and increasing bioavailability with

decreased dosing and fewer side effects. The use of

retardant polymers succeeded in delaying drug release,

however higher percentage of these tended to decrease

the mucoadhesive properties. Ex vivo permeation studies

through porcine buccal mucosa revealed the possibility

of permeation through human oral mucosa also.

ACKNOWLWDGEMENTS

The authors would like thank the Nitte University,

Mangalore and the National Institute of Technology,

Karnataka for providing the necessary facilities for

carrying out this investigation.

values of M/M within the range 0.10–0.60) at time t and ∞

K is a constant incorporating the structural and geometric

characteristics of the release device. A value of n=0. 5

indicates case I (Fickian) diffusion, 0.5<n<1 indicates

anomalous (non-Fickian) diffusion, and n=1 indicates

case II transport (Zero order release), n>1 indicates Super

case II transport. From the mathematical treatment of the

in vitro release data of losartan potassium from buccal 2films, the values of K, n and R (coefficient of

determination) has been obtained as presented in Table 5.

The values of n were obtained by the linear regression of

log (Mt/M ) vs. log t and were between 0.5 to 1 indicating ∞

non fickian diffusion or anomalous transport for

formulations E1, E2, E5 and E6. In the case of E3 and E4,

values of n close to 0.5 were obtained indicating possible

fickian diffusion as the release mechanism. The best fit 2with the highest correlation r and determination R

coefficients was shown by Korsmeyer and Peppas model

closely followed by the Higuchi matrix model and then

the first order equation and the Hixson-Crowell equation.

All the formulations follow Peppas model, except E2

whose drug release conforms to matrix model.

Ex vivo drug permeation studies:

Drug permeation studies through porcine buccal mucosa

conducted on the aqueous solution of the pure drug

S.No Mucoadhesive dosage form Drug Polymers used 31. Tablet Propranolol Hydrochloride Sodium CMC,

Carbopol 934 42 Tablet Nicotine HPMC K 4M, Sodium-

Alginate, Carbopol 934 53 Film Ibuprofen PVP, Sodium CMC

64 Film Glibenclamide Chitosan 7 5 Gel Triamcinolone acetonide Carbopol 934,

Poloxamer 407

Table 1. Drugs and use of polymers in mucoadhesive dosage forms reported in literature.

Ingredients in g. E1 E2 E3 E4 E5 E6

Losartan potassium 0.36 0.36 0.36 0.36 0.36 0.36

Ethyl cellulose 0.5 0.5 0.25 --- --- ---

Eudragit RSPO --- --- --- 0.5 0.5 0.25

Hydroxypropylmethyl cellulose 0.25 0.5 0.75 0.25 0.5 0.75

Propylene glycol 1.0 1.0 1.0 1.0 1.0 1.0

Table 2. Composition of drug loaded films

320


Formula Content uniformity Thickness Folding Mucoadhesion Surface pHCode (mg)* (mm) Endurance* Time (min)

E1 10.12 ± 0.43 0.51 ± 0.05 >300 185 6.5 ± 0.14

E2 9.78 ± 1.02 0.68 ± 0.02 >300 280 6.53 ± 0.15

E3 9.86 ± 0.35 0.50 ± 0.04 >300 365 6.60 ± 0.21

E4 10.52 ± 0.75 0.52 ± 0.03 >300 130 6.73 ± 0.13

E5 9.63 ± 0.63 0.70 ± 0.05 >300 300 6.48 ± 0.15

E6 9.18 ± 1.21 0.53 ± 0.06 >300 350 6.70 ± 0.15

Table 3. Physical characteristics of the formulated drug loaded films

All observations represent the mean ± S.D (Standard Deviation) and n=10 for thickness and n=3 for others.

Formula Code Tensile strength* % Elongation* Bioadhesive Force 2 2(Kg/mm ) ( Kg/m/s )*

E1 0.196 ± 0.02 13.3 ± 2.32 17.818 ± 1.02

E2 0.360 ± 0.03 33.3 ± 1.66 18.931± 1.22

E3 0.475 ± 0.01 36.66 ± 4.20 19.488 ± 0.63

E4 0.240 ± 0.02 20.0 ± 4.80 17.261 ± 0.46

E5 0.346 ± 0.01 36.66 ± 2.15 20.045 ± 2.34

E6 0.518 ± 0.02 40.0 ± 3.57 23.386 ± 1.42

Table 4. Mechanical properties of drug loaded films

* Values represented as mean ± SD ( Standard Deviation) and n=3

2 R 0.8516 0.9196 0.816 0.838 0.8737 0.8552

k 0.073 0.0671 0.079 0.0747 0.0683 0.0697 2R 0.8853 0.9369 0.8898 0.874 0.8909 0.8757

k 0.0882 0.0762 0.0673 0.0873 0.0796 0.0712 2R 0.9781 0.9917 0.9396 0.9791 0.9852 0.9927

k 1.1719 1.0395 1.3016 1.2002 1.0712 1.0990 2 R 0.9908 0.9901 0.9885 0.9896 0.9873 0.9951

k 1.1249 0.9508 2.2898 1.3318 0.6724 0.9427

n 0.5158 0.5109 0.4978 0.4870 0.5893 0.5316 2R 0.8746 0.9315 0.8669 0.8649 0.8854 0.8712

k 0.0745 0.0567 0.0834 0.0854 0.0836 0.0545

E1 E2 E3 E4 E5 E6Formulation Code

Zero Order

First Order

Higuchi Matrix

Peppas

Hixson Crowell

Best fit Model Peppas Matrix Peppas Peppas Peppas Peppas

Release Model

Table 5. Kinetic Analysis of in vitro drug release data

321

Fig. 1: DSC curves of film formulation E3,

ethyl cellulose, HPMC and Losartan potassium

Fig. 2: DSC curves of film E6, Eudragit RSPO,

HPMC and Losartan potassium

Fig 3: Swelling profile of different film

formulations in simulated saliva

Fig. 4: In vitro release profile of Losartan potassium in simulated saliva


322

Fig.5: Ex vivo permeation profile of losartan

potassium from aqueous solution through

porcine buccal mucosa

Fig.6: Ex vivo drug permeation profile from

film formulations through porcine buccal mucosa

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Mathiowitz E, editors, Bioadhesive Drug Delivery

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2. Swarbrick J, Boylan JC, editors. Buccal Absorption

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Technology; Vol.2. Marcel Dekker Inc., 1990:189-

208.

3. Patel VM, Prajapati BG, Patel MM. Formulation,

Evaluation, and Comparison of Bilayered and

Multilayered Mucoadhesive Buccal Devices of

Propranolol Hydrochloride. AAPS PharmSciTech.

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4. Lewis S, Subramanian G, Pandey S, Udupa N.

Design, evaluation and pharmacokinetic study of

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5. Perioli L, Ambrogi V, Angelici F, Ricci M,

Giovagnoli S, Capuccella M, Rossi C. Development

of mucoadhesive patches for buccal administration

of ibuprofen. J Control Rel 2004; 97: 269-79.

6. Manvi FV, Rajashree M, Chaitanya GY, Roopesh

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patches of glibenclamide. The Indian Pharmacist

2004; 27(3): 55-58.

7. Sang-Chul Shin, Ja-Young Kim. Enhanced

permeation of triamcinolone acetonide through the

buccal mucosa. Eur J Pharm Biopharm 2000; 50(2):

217-20

8. Peh KK, Wong CF. Polymeric fims as vehicles for

buccal delivery: Swelling, Mechanical and

Bioadhesive properties. J Pharm Pharmaceut Sci

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Pharmaceutical Press, London 1996, p. 427-428.

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Coomans SD, Michotte Y, Slop D. Development and

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323

Abstract

Liposomal carriers, well known for their potential in topical drug delivery have been choosen to help fluconazole

molecules in the skin layers. In the present work statistical study for the formulation of liposomes for topical delivery

of fluconazole using the factorial design approach was undertaken. Amount of phospholipid (PL 90H) and

cholesterol (CH) were taken at three different levels and liposomes were prepared using film hydration technique.

Gels containing liposomes (optimized batch) were prepared in Carbopol® 934 NF and were characterized for

rheology, spreadability, permeation and drug deposition in the rat skin. Results of regression analysis revealed that

vesicle size and entrapment efficiency were dependant on the cholesterol and lipid concentration. Rheological studies

of all liposomal gels prepared with 1%, 1.5%, and 2% w/w carbopol gave a clear idea of concentration of carbopol

required. Liposomal dispersion and gels were found to increase the skin permeation and deposition compared to

control and marketed gel. Liposome dispersion and gel formulation were found to be stable for 60 days.

Key Words: Factorial design; fluconazole; liposomes; gels; topical


INTRODUCTION

Topical drug delivery is an attractive route for local and 1systemic treatment . The delivery of drugs onto the skin is

recognized as an effective means of therapy for local

dermatologic diseases. Fluconazole, a synthetic

antifungal agent, is a triazole derivative. It is used in the

treatment of oropharyngeal, esophageal, or vulvovaginal

candidiasis as well as other serious systemic candidal

infections. It is also effective against superficial fungal 2infections and dermatophytoses . Fluconazole is

available commercially as tablets and injections only in

spite of its well known adverse effects including nausea,

vomiting, bloating and abdominal discomfort. Oral

fluconazole cannot be taken in conjunction with a

number of medications. In order to bypass these

disadvantages, the liposomal gel formulations have been

proposed as topical application.

Liposomes are acceptable and superior carriers and have

ability to encapsulate hydrophilic and lipophilic drugs 3,4 and protect them from degradation . It also has affinity

to keratin of horny layer of skin and can penetrate deeper

into skin and hence give better absorption. In the

formulation of topical dosage forms, attempts are being

made to utilize drug carriers that ensure adequate

localization or penetration of drug within or through the

skin in order to enhance the local and minimize the

systemic effects or to ensure adequate percutaneous 5absorption . Applied on the skin, liposomes may act as a

solublizing matrix for poorly soluble drugs, penetration

enhancer as well as local depot at the same time

diminishing the side effects of these drugs. Topical

liposome formulations could be more effective and less 6 toxic than conventional formulations . Fluconazole was

7successfully incorporated by Singh et al (1993) into

multilamellar (MLV) and large unilamellar liposomes

(LUV). Hence liposomal carriers, well known for their

potential in topical drug delivery have been chosen to 7help fluconazole molecules in the skin layers . These

vesicles are also expected to provide lipid enriched

hydrating conditions to retain the drug molecules within

the dermal layers. With this objective fluconazole loaded

liposomal systems have been prepared and their topical

performance has been compared with non liposomal

systems containing fluconazole.Indian Journal of Pharmaceutical Education and ResearchReceived on 3/11/2009; Modified on 17/5/2010Accepted on 3/7/2010 © APTI All rights reserved

Formulation and Evaluation of Topical Liposomal Gel for Fluconazolea a a a,bB. V. Mitkari , S. A. Korde , K. R. Mahadik and C. R. Kokare *

aDepartment of Pharmaceutics, Bharati Vidyapeeth Deemed University, Poona College of Pharmacy,

Pune– 411038, India.bDepartment of Pharmaceutics, STES, Sinhgad Institute of Pharmacy, Narhe, Pune-411041, India


324


Materials

Fluconazole (Glenmark Pharmaceutical Industries, Goa)

and saturated soy lecithin (PC; Phospholipon 90H) was a

generous gift from Nattermann phospholipids GmbH,

Germany. Cholesterol (CHOL), Stearic acid (SA) was

purchased from Qualigens Fine Chemicals, Mumbai,

India and Research Lab, Mumbai, India, respectively.

Marketed Flucos gel was procured from local market.

Carbopol® 934 NF (poly acrylic acid polymer) was gift

sample from Noveon, India. All other chemicals used

were of HPLC or analytical grade.

Liposome preparation

Aqueous liposomal formulations were prepared by 8,9conventional lipid film hydration method . Different

weight ratio of phospholipids: choleseterol and stearic

acid were weighed and dissolved in chloroform:

methanol mixture (2: 1 v/v) in 250 ml round bottom flask.

A thin film was formed on the inner side of round bottom

flask by evaporating organic solvent under vacuum in

rotary evaporator at 45-50 °C. Subsequently, the flask

was kept overnight under vacuum to ensure the complete

removal of residual solvent. The dry lipid film was

hydrated with 20 ml phosphate buffer solution (pH 7.4)

containing fluconazole at a temperature of 60±2 °C. The

dispersion was left undisturbed at room temperature for

2-3 h to allow complete swelling of the lipid film and

hence to obtain vesicular dispersion.

Effect of variables

To study the effect of variables on liposome performance

and characteristics, different batches were prepared using 2the 3 factorial design approach. Amount of PL 90H and

CH were selected as two independent variables. Vesicle

sizes, entrapment efficiency (EE) were selected as

dependent variables. Amount of SA (30 mg) and

fluconazole (30 mg) were kept constant. Values of all

variables and batch codes are shown in Table 1.

Size distribution

Prepared liposomal batches were monitored for their 10 morphological attributes using optical microscope .

Mean vesicle size and size distribution profile of

liposome was determined by using Malvern particle size

analyzer model SM 2000, which follows Mie's theory of

light scattering. Diluted liposome suspension was added

to the sample dispersion unit containing stirrer and stirred

at 2000 rpm in order to reduce the interparticle

aggregation, and laser obscuration range was maintained

between 10-20%. The average particle size was measured

after performing the experiment in triplicate.

Entrapment efficiency

Fluconazole associated with liposome was separated 11 from unentrapped drug using centrifugation method .

Liposomes were centrifuged at 20000 rpm for 1 h at ocontrolled temperature of 4 C. Supernatant containing

unentrapped fluconazole was withdrawn and measured

UV spectrophotometrically at 260 nm against phosphate

buffer saline (pH 7.4). The amount of fluconazole

entrapped in liposome was determined as follow

EE (%) = [(C -C )/C ] 100 (1)d f d

Where C is concentration detected of total fluconazole d

and C is concentration of free fluconazole. The f

entrapment efficiency was obtained by repeating the

experiment in triplicate and the values were expressed as

mean standard deviation.

Zeta potential ( ζ ) determination

Charge on empty and drug loaded vesicles surface was

determined using Zetasizer 300HSA (Malvern

Instruments, Malvern, UK). Analysis time was kept for

60 s and average zeta potential and charge on the

liposome was determined.

Skin permeation and drug deposition studies

Rat was sacrificed by exposing to excess chloroform. To

the abdominal skin, depilatory (Anne French, India) was

applied and kept for 10 m to remove the hair from the

skin. After 10 m of application, skin was washed with

water. Skin was excised from rat with scalpel and fatty

layer was removed by keeping the skin in warm water at 060 C. After 2 m, fatty layer was peeled off gently and skin

was washed with water and kept for saturation in

phosphate buffer saline pH 7.4 for about 30 m before it

was used for permeation studies. Fresh skin was used

every time. Skin permeation studies with fluconazole

containing liposome formulations were carried out using

abdominal rat skin, employing modified Franz-diffusion 12cells . The results obtained were compared with that of

non-liposomal formulations of fluconazole. The skin was

prepared by mounting on the receptor chamber with 2cross-sectional area of 3.91 cm exposed to the receptor

compartment. The receptor compartment was filled with


325

(22 ml) phosphate buffer pH 7.4. It was jacketed to 0maintain the temperature 37 + 0.5 C and was kept stirring

at 50 rpm. Prior to application of formulations, the skin

was allowed to equilibrate at this condition for 1 h.

Liposomal or non-liposomal fluconazole formulation

(amount equivalent to 5 mg of drug) was applied

uniformly on the dorsal side of skin. Aliquots of 2 ml

were withdrawn periodically and replaced with same

amount of saline solution to maintain the receptor phase

volume at a constant level. The samples were quantified

spectrophotometerically at a λ of 260 nm.max

For determination of drug deposited in skin, cell was

dismantled after a period of 8 h and skin was carefully

removed from the cell. The formulation applied on skin

surface was swabbed first with phosphate buffer pH 7.4

and then with methanol. The procedure was repeated

twice to ensure no traces of formulation are left onto skin

surface. The skin was then cut into small pieces and drug

present in skin was extracted in phosphate buffer pH 7.4

u s i n g b a t h s o n i c a t o r a n d d e t e r m i n e d

spectrophotometrically after suitable dilution and 2filtration .

Preparation of liposomal gel

On the basis of factorial design approach, liposome batch

(LPE) was selected for further formulation studies of 13 liposomal gel . Gel was prepared using carbopol® 934

NF (1, 1.5 and 2%). The appropriate quantity of carbopol

934 powder was dispersed into distilled water under

constant stirring with a glass rod, taking care to avoid the

formation of indispersible lumps and allowed to hydrate

for 24 h at room temperature for swelling. Topical

liposome gel formulations were prepared by

incorporation of liposome's containing fluconazole

(separated from the unentrapped drug) were mixed into

the carbopol gel with a mechanical stirrer (25 rpm, 2 m).

The dispersion was neutralized using triethanolamine

(0.5% w/w). Control gels were made under the same 14conditions .

Rheological studies

While considering the stable liposome dispersion or any

other delivery system they usually need to be

incorporated into convenient dosage for to obtain

formulation with desired semisolid consistency for ease

in topical and transdermal application. It is important and

controls the flow properties to ensure product quality and


15,16 effectiveness of the production . It helps in selection of

dermatological formulation that will progress to clinical

efficacy. In present study liposomal gels were prepared

using carbopol 934 as gelling agent.

Rheological analysis of liposome loaded carbopol gels

were performed using a stress control rheometer

(Viscotech Rheometer , Rheologica Instruments AB,

Lund, Sweden), equipped with stress rheologic basic

software, version 5, using cone-plate geometry with a

diameter of the cone being 25 mm and a cone angle of 10,

operating in the oscillation and static mode. Rheological

analysis was performed at room temperature. The

following parameters were carried out for rheology

measurement.

Oscillation stress sweep:

Dynamic oscillation stress sweep was performed to

determine the linear viscoelastic region (LVR). LVR is

the region where the elastic modulus (G') was

independent of applied stress because destruction in the 17 structure of gels occurs at high shear stress . Analysis of

viscoelastic material was designed not to destroy the

structure so that measurement can provide the

information about intermolecular and interparticle forces

in the material. This test gives idea about the critical stress

beyond which the sample may show significant structural

changes, and therefore the consequent choice of the stress

value to be used in other in other oscillation tests. The

samples were exposed to increasing stress (0.5 to 150 Pa)

at a constant frequency of 0.1 Hz.

The three main parameters determined in this test were

the storage modulus G', loss modulus G” and loss tangent

tan δ. The end point of the linear viscoelastic region was

determined as a stress, when the G' value was dropped

10% from the linear level that indicated a significant 18 change in the structure gel samples .

Oscillation frequency sweep:

The samples were exposed to stepwise increasing

frequency (0.1 to 100 Hz) at a constant stress in the field

of LVR and elastic moduli (G') as well as viscous 19 modulus (G?) were recorded against frequency .

Creep-recovery:

The creep recovery test was used to determine the

viscoelastic properties of the selected silk fibroin gel 20,21 samples . The samples were exposed to the selected

averaged stress of the stress sweep mode for 50 s. It was

326

followed by relaxation period for 100 s for recovery. The

creep compliance Jc (defined as the ratio of measured

strain to the applied stress) was monitored against time.

Drug content and content uniformity

The gel sample (100 mg) was withdrawn and drug

(fluconazole) content was determined using UV

spectrophotometer at 260 nm. Similarly, the content

uniformity was determined by analyzing drug

concentration in gel taken from 3 to 4 different points

from the container. In case of liposomal gel, it was shaken

with sufficient quantity of methanol to extract the drug

and then analyzed by using UV spectrophotometer at 260

nm.

Stability studies

The ability of vesicles to retain the drug (i.e., drug

retentive behavior) was assessed by keeping the

liposomal suspensions and liposomal gel at two different

temperature conditions, i.e., 4-8 °C (Refrigerator; RF),

25±2 °C (Room temperature; RT), for a period of 60 days.

Samples were withdrawn periodically and analyzed for

the drug content and particle size for liposomal

suspension and drug deposition for liposomal gel in the

manner described under entrapment efficiency and 22 particle size distribution studies .


Amount of PL 90H and CH were found to be critical in

preparation and stabilization of liposomes and hence 2selected as variables in the 3 factorial designs (Table 1).

In a preformulation study the optimum concentrations of

PL 90H, CH, and SA were determined to obtain stable

liposomes devoid of aggregation, fusion and

sedimentation. The 30 mg of SA was found to be

optimum to prevent aggregation of liposomes.

Liposomes were prepared using film hydration technique

and method was found to be well suited for the production

of liposomes without aggregation. Responses of different

batches were obtained by using factorial design (Table 2).

Obtained data were subjected to multiple regression

analysis using PCP Disso v3\Factorial-3^2.xls" software

and obtained data were fitted in Eq. (2).

Y = β0 + βX1 + β2X2 + β11X1X1 + β22X2X2 +

β12X1X2 (2)

Effect of variables on particle size

The most important parameter, which needs to monitor

during liposome preparation its best performance, is the

vesicle size and size distribution of liposomes. From the

number of reports, it was observed that the size and size

distribution of the liposome determines their in vivo or

ex-vivo performance. There are some reports, which

showed the effect of liposome size on the drug release as 5well as drug deposition in the skin . Thus for the effective

delivery, the selected method should result in optimum

size range and homogeneous population. In the present

study, film hydration technique found to produce

polydispersity index of less than 0.893 indicates obtained

liposome population have narrow size distribution (Table

2). It was observed that the relative amount of PL 90H and

CH was found to play important role in vesicle size

(Fig.1). Size of vesicles found to be in the range of 3.01 to

7.32 m. To understand the effect of lipid concentration on

vesicle size, coefficient observed for liposome size fitted

in Eq. (3)

Y = 4.50 + 1.038 X + 1.028 X (3)1 2

A positive correlation was observed for the both variables

X (PL 90H) and X (CH) in case of liposome vesicle size 1 2

2(Eq. 3; r = 0.8356015) Thus, with increase in the .

concentration of PL 90H & CH vesicle size was found to

be increased. The coefficient value of both the variables

observed that the effect of PL 90H was slightly more

prominent than effect of CH.

Size range of LPE liposome was found to be 2.17 to 5.56

m, with 90% of liposomal population equal or below 5.56

m. The mean vesicle diameter was found to be 3.712 m

(Fig. 1).

Effect of variables on entrapment efficiency

Determination of EE is an important parameter in case of

liposomes as it may affect the drug release and skin 5deposition . EE is expressed as the fraction of drug

incorporated into liposomes relative to total amount of

drug used. In the present study, the observed EE for all

batches were in the range of 57.78 – 66.64%. To

understand the effect of lipid concentration on vesicle

size, coefficient observed for EE fitted in Eq. is

Y = 64.15 + 2.91 X + 1.23 X – 2.1217X X (4)1 2 1 2

A positive correlation was observed for both variables X1

(PL 90H) and X (CH). Thus with increase in the 2

concentration of PL 90H and CH entrapment efficiency

found to be increased (Fig. 2). Among all the batches of


327

LPE, which had minimum vesicle size and intermediate

EE%, selected for the further study of gel formulation.

Small particle size liposomes can cover the skin surface

more compared to larger paricle size. Also, the

polydispersity index of LPI was higher compared to that

LPE which indicates wide particle size distribution,

which was not desirable. Hence, the batch with less

particle size and less polydispersity index was selected as

optimized batch ahead of the batch with more % EE

(Table 2).

Determination of zeta ?ζ potential

In the present study the ζ obtained for liposomes are

shown in Table 2. The values of ζ potential (-54.1 - 63.6

mV for vesicles) showed prepared liposome have

sufficient charge to avoid aggregation of vesicles.

Rheological study

The semisolid preparations should flow or deform after

applying the force and regain its elasticity as the force is

removed. Thus, to understand the rheological properties

of liposomal gels and for selection of optimum

concentration of carbopol having desired rheological

properties, different concentrations (1, 1.5, 2% w/w) of

carbopol 934 were used to prepare liposomal gels at 25 °C 15with neutralization method . The rheologies of all

samples were determined to identify the minimum

concentration of carbopol required for the formation of

gel with good viscoelastic properties.

Oscillation stress sweep:

During stress sweep, it was observed that there was

linearity between stress and strain produced all over the

applied stress range, which indicate that system was

working in correct range. Liposomal gel with 2% w/w of

carbopol had shown maximum G' and LVR as compared

to other system and it was comparatively more stable

over applied stress range. In oscillatory stress sweep test,

system with higher LVR and G' were considered as more 23elastic . Solid modulus (G') of other system has shown

greater dependence on applied stress. The degree of

dependence of G' over applied stress is critical parameter

to determine the gel strength; higher is degree, lower is

gel strength. Both 1% and 1.5% w/w systems were

presented lower G' and LVR and higher degree of

dependence of G' over applied stress indicating formation

of weak gel. It clearly indicated (Fig. 3) that for 2%

system, G' was greater than G'' over all applied stress,

while for 1% and 1.5% system, G' was higher than G'' at

lower stress (data not shown).The trend of G'>G'' is for 18gel and solid like materials has been studied . The phase

angle is good indicator of viscoelastic nature of system,

being measure of the lag in sine response after an

oscillatory stress has been applied to the sample. For

perfectly elastic system, phase degree must be close to 0° 24 and for viscous system it must be close to 90°. Both

liposomal gels containing 1% and 1.5% carbopol was

shown phase transition at higher stress (data not shown).

These systems have presented the change of behavior

from elastic to viscous one, thus proving the instability of

the system. Therefore, from stress sweep, it was

concluded that liposomal gel prepared with 2% w/w

carbopol was more stable and elastic one.

Oscillation frequency sweep:

Fig. 4, presents the behavior of G' over the applied

frequency range. It was concluded that 2% system had

shown independent behavior of G' over applied

frequency, while 1 and 1.5% system have exhibited a

monotonous increase in solid component. It is possible

that monotonic increase in G' at higher frequencies means

the partial breakage of the interconnected network,

inferred from the existence of the plateau region at lower

frequencies, which represents a true cross-linked

polymer gel network. It concluded that the gel network

was retained at low frequencies and, on the other hand,

destroyed by the more frequent changes of the

displacement at higher frequencies due to the formation 19 of too rigid and brittle structures . It was clear that 2%

system had shown low (Fig. 4) dependency on frequency

for viscous moduli and phase degree than 1 and 1.5%

system. The value of phase degree for 2% was well in

viscoelastic region. The increase in G'' and phase degree

clearly indicated the weak structure of breaking structure

for 1 and 1.5% systems.

In frequency sweep test a new terminology was

introduced i. e. loss tangent (tan δ). It is the ratio of loss

modulus (G'') and storage modulus (G''). The higher the

loss tangent less elastic is the material. Previous studies

shown that system with values of tan δ <1 characterize a

predominant elastic behavior and values of tan δ >1 23indicate a prevailing viscous behavior . For liposomal

gel containing 2% carbopol, tan δ was clearly below 1,


328

while for 1 and 1.5% system it was much higher than 1

(Fig. 5). With this prospective, only 2% system is elastic

and stable one. Torres, et al. (2007) has presented

guideline for determination of stability of gel structure.

According to his proposal, in frequency sweep, there

must be non-dependency of both G' and G'' over the

applied frequency and condition of G'>G'' must be 17prevalent over all range . Considering these criteria,

liposomal gel containing 2% carbopol was again

emerged as stable and elastic one.

Creep recovery:

The creep-recovery percent for liposomal gel containing

1%, 1.5% and 2% carbopol was 43.89%, 65.87%, 71.7%,

respectively. As the 2% has highest elastic recovery, it is

said to highly stable and easy to process, store and apply

system (Fig. 6).

From graph, it was clearly seen that 2% system had

lowest compliance value i. e. J. Tadros, (2007) defined

that lower the compliance, higher is elasticity. Therefore,

from creep recovery test, it was inferred that, all system

have shown considerable elastic and viscoelastic

recovery, liposomal gel with 2% w/w carbopol had

presented highest recovery and lowest compliance (J).

From oscillatory rheological measurement, it was

observed that liposomal gel prepared from 2% w/w

carbopol was highly elastic and stable. It was having high

recovery capacity. From these rheological properties, it

can be concluded that this system having good flow

properties, good spreadibility and applicability. Hence,

further studies were performed with liposomal gel with

2% w/w of carbopol.

Drug content uniformity and pH measurement

There was no significant difference observed in the %

drug at various locations, indicating that the method used

to disperse the liposomal dispersion in the gel base is

satisfactory. The pH of the developed formulations was in

accordance with that of human skin pH rendering them

more acceptable. The developed formulations had pH

near to that of skin. So, we can conclude that prepared

liposomal gel was suitable for topical application.

Skin permeation and drug deposition studies

Results obtained from in-vitro drug permeation studies

conducted with different formulations of fluconazole are

shown in Table 3. Significant augmentation in the skin

permeation of fluconazole has been observed (Fig. 7)

with liposomal formulations vis-à-vis aqueous solution

or plain carbopol gel. The amount of fluconazole

permeated in eight hour was found to be 32.47% and

30.46% from liposomal suspension and liposomal gel,

respectively, whereas only 27.2% and 24.68% of the drug

permeated in case of aqueous solution and plain Carbopol

gel, respectively. Higher values of flux obtained with 2liposomal suspension 0.1754 mg/cm /h, and liposomal

2gel 0.1871 mg/cm /h, than that obtained with marketed 2 2gel 0.1341 mg/cm /h, and carbopol gel 0.1531 mg/cm /h,

clearly vouch for the permeation enhancing effect of

vesiculation on the drug. Results of this study clearly

depict that the amount of drug retained in the skin was

considerably higher in case of liposomal preparations,

than with non-liposomal.

This gave an understanding that liposomes could not only

enhance the penetration of drug molecules but also 12helped localize the drug in the skin . Improved skin

permeation of fluconazole coupled with its enhanced

retention in the skin with liposomal formulation can be

ascribed to the lipo-solublized state of fluconazole

molecules. The latter was achieved in the presence of

aqueous and non-aqueous phase of bilayered systems, a

state most ideally suited for drug penetration. The

liposomal phospholipids (also one of the natural

constituent of skin lipids) helped generating and retaining

the required physico-chemical state of the skin for

enhanced permeation of the fluconazole. The

phospholipid-rich domains of vesicles might have helped

to produce the depot effect for drug molecules. The latter

has been reflected as higher amount of drug retention

within the skin layers in case of liposomal formulations.

Thus, the liposomal fluconazole formulation, with

desired characteristics for topical administration, could

be successfully prepared. The formulated fluconazole

liposomes have shown an appreciably enhanced skin

permeation as well as retention of drug molecules in the

skin.

Stability study

Stability of liposome dispersion (LPE) as well as 2% w/w

carbopol liposomal gel containg 0.5% fluconazole was

carried out for 60 months at 4-8 °C and room temperature.

Responses obtained for different parameters for

liposomal dispersion and liposomal gel during stability

period are as shown in Table 4.


329

LPE liposomes were found to be reasonably stable in

terms of aggregation, fusion and/or vesicle disruption

tendencies, over the studied storage period. From results

it can be concluded that at room temperature and freeze

temperature there was slightly but insignificantly

decrease in % entrapment efficiency and increase in

particle size for liposomal batch LPE. Deposition of

fluconazole in rat skin from liposomal gel was found

insignificantly decreased during stability period. Result

suggests that keeping the liposomal product in

refrigeration conditions minimizes stability problems of 22 liposomes .

CONCLUSION

Preparation of liposomes using factorial design was

found to be well suited and sound approach to obtain

stable liposomal formulation. Variables such as amount

of phospholipid, amount of stabilizer have a profound

effect on the vesicle size and entrapment efficiency.

Rheological studies of all liposomal gels prepared with

1%, 1.5%, and 2% w/w carbopol gave a clear idea of

concentration of carbopol required i.e. 2 % carbopol 934

NF. Liposomal dispersion and gels were found to

increase the skin permeation and deposition compared to

control. Stability studies performed for liposomal

dispersion (LPE batch) and Liposomal gel indicates the

prepared liposomes have more stability at freezing

temperature than that of room temperature. Fluconazole

molecules could be successfully entrapped in liposomes

with reasonable drug loading. Hence from results

obtained it can be concluded that liposomal gel containg

fluconazole has potential application in topical delivery.

Batch code Variable X1 Variable X2Amt. of PL 90 H (mg) Amt. of CHL (mg)

LPA 80 (-1) 20 (-1)

LPB 80 (-1) 30 ( 0 )

LPC 80 (-1) 40 (+1)

LPD 90 ( 0 ) 20 (-1)

LPE 90 ( 0 ) 30 ( 0 )

LPF 90 ( 0 ) 40 (+1)

LPG 100 (+1) 20 (-1)

LPH 100 (+1) 30 ( 0 )

LPI 100 (+1) 40 (+1)

Table No.1: Experimental design with coded levels of variables and actual values

Batch code Entrapment Vesicle size Polydispersity Zeta PotentialEfficiency (Size ± SD, µm) Index (ζ ± SD, mV)

(EE ± SD, %) (PdI ± SD)

LPA 57.78 ± 0.76 3.01 ± 1.38 0.412 - 55.5 ± 1.76

LPB 59.48 ± 1.59 3.98 ± 1.25 0.239 -54.1 ± 0.61

LPC 60.10 ± 0.83 4.17 ± 1.58 0.331 - 55.7 ± 1.49

LPD 62.62 ± 2.31 4.06 ± 1.40 0.249 - 63.6 ± 2.43

LPE 64.88 ± 0.93 3.50 ± 0.94 0.302 - 58.6 ± 1.19

LPF 64.97 ± 1.74 5.23 ± 1.36 0.632 - 55.9 ± 2.43

LPG 63.90 ± 2.76 4.28 ± 1.62 0.231 - 58.4 ± 3.80

LPH 64.31 ± 0.58 5.79 ± 1.39 0.893 - 54.7 ± 1.87

LPI 66.64 ± 2.27 7.32 ± 1.47 0.698 - 54.2 ± 2.51

Table No.2: Results obtained for all experimental batches.


330


Fluconazole Mean cumulative Permeation flux % Drug retainedformulations % drug permeated mg/ cm2 / h in skin

Liposomal gel 30.46 0.1754 8.21

Liposomal dispersion 32.47 0.1871 10.03

Marketed gel 23.41 0.1341 5.21

Carbopol plain gel 24.68 0.1531 3.64

Aqueous solution 27.2 0.1570 5.79

Table No. 3: In-vitro skin permeation and skin retention of fluconazole from different formulations.

No.of days Room Temp. 4-8 0 C Room Temp.

0 64.88 64.88 3.5 3.5 0.41074 0.41074

30 63.01 61.73 3.87 4.3 0.3981 0.3174

60 61.39 58.32 4.13 5.2 0.3127 0.2349

0 04-8 C 4-8 C Room Temp.

Entrapment efficiency % Vesicle size (µm) Drug deposition %

Table No. 4: Effect on entrapment efficiency, vesicle size for liposomal dispersion and drug deposition from liposomal gel during stability.

Fig.1: Effect of lipid concentration on vesicle size Fig.2: Effect of lipid concentration on % EE

Fig. 3. Stress sweep for G' and G'' for liposomal gel containing 2% carbopol.

Fig. 4. Frequency sweep for phase degree

331

tan

µ

Freq. Vs. tanµ

Fig. 5: Frequency sweep for tan µ Fig. 6. Creep recovery test

Fig.7. Permeation profile of different fluconazole containing systems


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22. Goindi S, Aggarwal N. Preparation of hydrogels of

griseofulvin for dermal application. Int. J. Pharm.

2006;326:20-24.

23. Korhonen M, Hellen L, Hirvonen J, Yliruusi J.

Rheological properties of creams with four different

surfactant combinations-effect of storage time and

conditions. Int. J. Pharm. 2001;221:187-196.

24. Lippacher A, Muller RH, Mader K. Liquid and

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rheological characterization. Eur. J. Pharm.

Biopharm. 2004;58:561-567.

333

Abstract

Glibenclamide (GLB) is a second generation anti-diabetic drug used for the treatment of Type –II Diabetes. The

drawback of the drug is, it is practically insoluble in water and so possesses poor solubility, GI absorption and

bioavailability. Hence the objective of the work is to develop fast dissolving tablets of Glibenclamide using

Crospovidone (CP) as super disintegrating agent. The kneading mixtures of glibenclamide were prepared with

Crospovidone in the weight ratios of 1:0.5, 1:1 and 1:1.5. The mixture, GLB : CP (1:1.5) (KM) exhibited highest

dissolution rate of 99.8% in 45 min. FTIR analysis of the selected kneading mixture indicated the absence of drug-

polymer interaction. Hence the tablets were prepared by using the kneading mixture GLB: CP (1:1.5) (KM) by wet

granulation method. Various formulations were tried and the FDT was selected which possessed optimum 2 characteristics of disintegration time of 20 sec, hardness of 3.5 kg/cm and friability of 0.34±0.61%.There is 99.9% of

drug release in 20 min. The FDTs were stable without any significant changes in their initial properties of hardness,

friability, disintegration time and % drug release upon storage for 4 weeks.

Key words: Glibenclamide, FDTs, Crospovidone, kneading method, Disintegration, Dissolution.


INTRODUCTION

Fast Dissolving tablets (FDTs) are the recent

developments to present viable dosage alternatives for

patients who have difficulty in swallowing. They are the

products that disintegrate rapidly in the saliva without the

need of water. Fast dissolving / disintegrating tablets are a 1perfect fit for all of the patients . Some tablets are

designed to dissolve in saliva remarkably fast, with in few 2 seconds and are true fast dissolving tablets They contain

agents to enhance the rate of tablet disintegration in the

oral cavity. When placed on tongue, this tablet

disintegrates instantaneously, releasing the drug, which

dissolves or disperses in the saliva. Some drugs are

absorbed from the mouth, pharynx and esophagus, as the

saliva passes down into the stomach. In such cases the

bioavailability of drug is significantly grater than those 3observed from conventional tablet dosage form .

The advantage of fast dissolving dosage forms are

increasingly being recognized in both industry and

academia. Their growing importance was underlined

recently when European Pharmacopoeia adopted the

.

term “Orodispesible tablet” as a tablet that to be placed in 4oral cavity where it disperse rapidly before Swallowing .

Zydis, the best known of fast dissolving / disintegrating

tablet preparations was the first marketed new

technology tablet. A zydis tablet is produced by

lyophilizing or freeze drying the drug in a matrix usually

consisting of gelatin. The product is very light in weight

and fragile, and must be dispensed in a special blister

pack. Several methods like lyophillization, granulation

methods using specific super disintegrating agents have

been utilized to produce the FDTs.

In view of the above information, we have selected

Glibenclamide (GLB), a second generation anti-diabetic

drug to develop as a fast dissolving tablet using

Crospovidone (CP) as a super disintegrating agent. 5Glibenclamide , is a sulphonyl urea derivative used for

the treatment of Type –II Diabetes mellitus. It is

practically insoluble in water and so possesses poor

solubility and subsequent poor GI absorption and

bioavailability. Several investigations revealed the rate

l imi t ing absorpt ion and b ioavai labi l i ty of 6-8Glibenclamide . Hence the objective of the present work

is to enhance the dissolution rate of Glibenclamide by

preparing its kneading mixtures with crospovidone, to


Development of Fast Dissolving Tablets of Glibenclamide Using Crospovidone and its Kneading Mixture

Jeevana Jyothi. B* and Suneela. GInstitute of Pharmaceutical Technology,

Sri Padmavathi Mahila Viswavidyalayam (Women's University), Tirupati(A.P) -517 502.*Author for Correspondence: jeevanajyothib @ yahoo.com

334

develop its fast dissolving tablet from the ideal kneading

mixture possessing faster dissolution and to evaluate the

FDTs.


Pure and certified sample of Glibenclamide was gifted by

M/s. Karnataka Antibiotics and Pharmaceuticals Ltd,

Banga lo re , INDIA. D ica l c ium Phospha t e ,

Crospovidone, Aspartame, and microcrystalline

cellulose Phosphate were obtained from SD Fine

chemicals Ltd, Mumbai. All other chemicals and reagents

were of analytical grade.

Preparation and evaluation of kneading mixtures of

Glibenclamide and crospovidone (GLB:CP) (KM):

Preparation of kneading mixtures of GLB:CP (KM):

The kneading mixtures of glibenclamide- crospovidone

were prepared by taking various weight ratios of drug to

CP as 1:0.5, 1:1 and 1:1.5 by using kneading method. The

accurately weighed amount of drug and polymer

mixtures were taken in the mortar and triturated by

adding small volume of methanol to get smooth moist

mass. The mass was kneaded for 45 minutes and then

dried in an oven at 35ºC till the constant weight is

reached. The dried mass was pulverized and sifted

through #100 and the collected powder fraction was

stored in 30ml glass vials. In each case, 5 gm of kneading

mixture was prepared. The mixtures were analyzed and 9confirmed for total absence of methanol for further use .

Evaluation of kneading mixtures of GLB: CP:

The prepared kneading mixtures of GLB: CP were

evaluated by

1. In-vitro dissolution studies

2. FTIR spectroscopy

3. X-Ray Diffractometry.

In vitro dissolution studies:

Dissolution studies of pure drugs and the Glibenclamide-

crospovidone Kneading mixtures were carried out in

triplicate with USP dissolution testing apparatus(Paddle

type) (Electrolab, India) using paddle method. The

stirring speed was maintained at of 50 rpm and 900 ml of

pH 7.2 phosphate buffer at 37±1ºC was used as

dissolution medium. Samples of each preparation

equivalent to 50 mg of the drug were added to the

dissolution medium. The sample aliquots each of 5ml

were withdrawn at appropriate time intervals, filtered

through a 0.45µ membrane filter (Millipore, nylon discs).

The initial volume of the dissolution medium was

maintained by replacing with 5ml of the medium to

maintain sink conditions. The filtered aliquots were

suitably diluted and assayed for its drug content

spectrophotemetrically at λ of 232 nm by measuring the max

absorbencies against blank. The mean percent of drug

dissolved and the S.D were calculated and the results are

given in Table.1 and represented in the Fig. 1.

FTIR Spectroscopy:

FTIR spectra of pure Glibenclamide and the selected

mixture containing Glibenclamide: crospovidone

(1:1.5)(KM) were obtained on a perkin-Elmer 841 model

FTIR Spectrometer equipped with a DTSG detector.

Samples were prepared by KBr pressed pellet technique. -1The scanning range was 500-4000 cm and the resolution

-1was 1 cm .The spectra are shown in Fig. 2.

X-Ray Diffractometry:

Powder X-Ray diffraction patterns of pure

Glibenclamide and the selected mixture containing

Glibenclamide: crospovidone (1:1.5)(KM) were

recorded using an automated X-ray diffractometer

(XGEN). The cross sections of the samples were held in

place on quartz plate and subjected to CuK radiations.

The samples were analyzed at room temperature over a 0range of 0-5 at an angle of 2 with sampling interval of

0 00.02 . The scanning rate was 2 C/min. The

diffractogram of the samples are shown in fig .3.

Formulation and Preparation of Fast dissolving

tablets of glibenclamide using selected solid

dispersion, GLB: CP (1:1.5)(KM):

Fast dissolving of Glibenclamide tablets were prepared

by using the selected kneading mixture, i.e. GLB: CP

(1:1.5) (KM) which exhibited a significant improvement

in the dissolution at in vitro level among all the

dispersions. The various formulations were tried to select

the tablet with ideal characteristics of FDTs. The selected

tablet formulation that satisfied all the official parameters

is given in the Table 2.

Glibenclamide and crospovidone kneading mixture

(1:1.5), MCCP, crospovidone and DCP was sifted

through the sieve no:30. Then the mix was granulated by

using 10% Starch solution as binding agent. The wet

mass was passed through # 30 to get the wet granules. The

wet granules were dried in an oven at 60°C for about 20

min. To the dried granules magnesium stearate, talc,


335

aspartame and pineapple flavor were added and mixed

well. The micomeritic properties of the blend were

satisfactory and are shown in the Table.3.Then the blend

was compressed with 7 mm flat round punches using 16-

station rotary punch machine (Clit Jemkay, CTD 3-16).

Evaluation of fast dissolving tablets of glibenclamide:10The prepared tablets were evaluated for disintegration ,

11 12 13hardness , friability and drug content . Five tablets

collected at random were determined for hardness by

using Monsanto hardness tester. The mean values are

presented in the Table.4. The disintegration time of

tablets was determined by using Thermionic tablet

disintegration test apparatus of USP standard. The results

are given in the Table.4. Friability test was carried out by

using Roche Friabilator (Veego Instruments Ltd., India).

Ten tablets were collected at random from a batch and

their initial average weight (W ) was denoted. Then the 1

tablets were placed in the rotating chamber and subjected

to combined effects of abrasion and shock with revolving

plastic chamber at 100rpm. After completion of

rotations, the tablets were reweighed (W ). The percent 2

loss in weight or friability (f) was determined.

Estimation of Drug content:

Drug content estimation was carried out by collecting ten

tablets from each batch at random. The tablets were

powdered and the fine powder equivalent to 10mg of the

drug was transferred into a 10 ml volumetric flasks. The

volume was made with water and sonicated for 10 min.

The sample was filtered through 0.45m micro pore filter

paper. The results are given in the Table .4.

In vitro dissolution studies of tablets:

In vitro dissolution studies of tablets was carried out

using USP dissolution testing apparatus (Basket type)

(Electrolab, India) using pH 7.2 phosphate buffer as

dissolution medium at 37±1ºC.The stirring speed was

maintained at of 50 rpm. Aliquot samples were

withdrawn at various time intervals, filtered, diluted and

assayed at 232 nm by measuring the absorbencies against

blank. The mean percent of drug dissolved and the S.D

were calculated and the results are given in Table.5 and

represented in the Fig .2.

Stability studies of prepared FDTs of glibenclamide:

The Fast dissolving tablets of glibenclamide were placed

in Stability chamber (Newtronics) at the temperature of 045 C and 75% RH. The tablets were taken after 4 weeks


and evaluated for hardness, Disintegration time and in-

vitro drug release. Student's t-test was applied to verify

any significant difference after storage.

RESULTS AND DISCUSSION:

The kneading mixtures of glibenclamide and

crosspovidone were prepared in the weight ratios of drug

to polymer as 1:0.5, 1:1 and 1:1.5 by using kneading

method.The dissolution studies were carried out for all

the Kneading mixtures. All the mixtures showed

enhanced dissolution compared to the pure drug as shown

in the Table.1 and Fig .1The pure drug released only

28.3% in 90 min. The mixtures GLB: CP (1:1.5) (KM),

GLB: CP (1:1)(KM) and GLB : CP (1:1.5) (KM) released

90.6%, 99.2% and 99.5% respectively in 90min.The

mixture GLB : CP (1:1.5) (KM) released faster amount of

95.4 in 20 min that is not the case with other two mixtures.

Hence GLB : CP (1:1.5) (KM) was selected was selected

for preparation of Fast dissolving tablets of

glibenclamide. The reason for enhanced dissolution of

Kneading mixtures may be due to effective

disaggregating capacity of crospovidone as a

superdisintegrant and the method used i.e., Kneading

technique, a method among the solid dispersion

techniques which were proved as successful methods to

enhance the dissolution of poorly soluble drugs. The

kneading mixtures were tested for the absence of

methanol as the presence of methanol is toxic and 9harmful . It was found that all the mixtures were totally

free from methanol content. Perhaps the drying of

Kneading mixtures would have led to the complete

removal of methanol from the mixtures.

The IR spectrum of pure glibenclamide in fig. 2(a) -1presents NH stretch at the wave number of 3314 cm , -IAr-H absorption peak at the wave number of 3116cm ,

-1 C=O absorption peaks at 1715 cm . The FTIR spectrum

of selected formulation (1:1.5) (KM) in fig. 2(b), also

presents all the absorption peaks produced by pure

glibenclamide. This indicates the absence of interaction

between the drug and crospovidone.

The X-Ray diffractogram of pure glibenclamide and

selected formulation GLB: CP (1:1.5) (KM) shown in

fig.3(a) and fig.3(b) did not exhibit much variation

revealing unaffected crystal nature of Glibenclamide

upon combination with crosspovidone. Thus it is

concluded that the enhanced solubility may be due to

336


enhanced wettability or due to the method of kneading

that entrapped the drug in the selected hydrophilic

polymer at deep molecular levels.

Therefore the Fast dissolving of Glibenclamide tablets

were prepared by using the promising mixture, i.e. GLB:

CP (1:1.5) (KM) which exhibited a significant

improvement in the dissolution rate of GLB at in vitro

level among all the mixtures. The tablets were produced

with ideal characteristics of FDTs especially the

disintegration time of 20 sec and 99.9% drug release in 20

min. The commercial conventional tablet (CT) of

Glibenclamide was also assessed for dissolution studies

for comparison. The CT released only 61.6% in 20 min.

The prepared FDTs of Glibenclamide complied the 2official compendia by possessing hardness of 3.5 kg/ cm ,

friability of 0.34±0.61% and %drug content of 99.26.

The tablets were stability tested for hardness, friability,

disintegration time and % drug release upon storage for 4

weeks. The t-test of significance at 5% L.S was applied to

observe any significant difference in values from the

initial values. It was observed that there is no significant

difference in the initial values after storage for weeks at 0temp of 45 C and 75% RH.

CONCLUSION:

The Fast dissolving tablets of Glibenclamide are

developed by using the super disintegrant, crospovidone

and its kneading mixtures prepared with crospovidone.

The mixture prepared in the ratio of drug to crospovidone

as 1:1.5 by kneading method is useful successfully to

prepare fast dissolving tablets by wet granulation

method. The Fast dissolving tablets possessed ideal and

reproducible characteristics of disintegration time of 20

sec and 99.9% drug release in 20 min.

ACKNOWLEDGEMENTS:

Authors thank M/S Karnataka Antibiotics &

Pharmaceuticals Limited, Peenya, Bangalore, INDIA,

for permitting to carry out the part of the present research

work in their industry and also for providing number of

excipients as gift samples.

Time (min) Pure drug KM(1:0.5) KM(1:1) KM(1:1.5)

0 0 0 0 0

5 5.59 ±0.26 26.8 ±0.91 34.2 ±0.34 55.4 ±0.92

10 8.92 ±0.64 34.1 ±1.56 46.8 ±0.24 69.1 ±0.76

15 11.2 ±0.52 47.5 ±1.32 56.1 ±0.72 86.7 ±0.82

20 17.3 ±0.59 59.4 ±1.64 61.2 ±0.59 95.4 ±1.48

30 19.5 ±0.63 66.9 ±1.06 77.7 ±0.64 97.9 ±1.05

45 21.7 ±1.01 77.4 ±0.85 84.6 ±1.32 99.8 ±0.64

60 25.4 ±1.51 88.2 ±0.95 95 ±1.25 99.0 ±0.85

90 28.3 ±0.96 90.6 ±0.64 99.2 ±1.04 99.5 ±0.69

Table.1: Percent drug release (n=3±SD) from pure glibenclamide and GLB:CP Kneading Mixtures.

Fig.1.: Comparative Dissolution Profiles of pure glibenclamide,KM(1:0.5), KM(1:1) and KM(1:1.5)

337

Fig.2:FTIR spectrum of a) pure Glibinclamide and b) GLB : CP (1:1.5) (KM)

(a) (a)

(a) (a)

Fig.3: X-ray diffractogram of a)pureGlibinclamide and b) GLB : CP (1:1.5) (KM)

Sl.No. Materials Quantity (mg) (%)

1. Glibenclamide: crospovidone

(1:1.5)(KM) 12.5 8.3

2 MCCP 101.3 67.5

3 DCP 15.2 3.46

4 Maize starch 10 6.6

5 Magneisum stearate 2 1.3

6 Talc 2 1.3

7 Aspartame 2 1.3

8 Pineapple flavour 2 1.5

9 Crospovidone 3 2

Table.2: Formula for fast dissolving Glibenclamide tablets

Average Weight : 150 mg, Tooling: 7 mm flat round punch


338


S.No. Parameter Result

1. Compressibility Index (%) 13

2. Hausner ’ s ratio 1.160

3. Angle of repose 22±0.8°

Table.3: Micromeritic properties of prepared blend.

S.No. Parameter Values (mean ± S.D)

1 Average weight (mg) 150±0.2

2 Drug content (%) 99.26±0.62

3 Hardness (kg/cm 2 ) 3.5±0.07

4 Friability (%) 0.34±0.61

5 Disintegration time (sec) 20±0.25

6 Thickness (mm) 4.6±0.15

Table.4: Evaluation Parameters of Fast dissolving Glibenclamide tablets

Time in Cumulative% Drug Release Cumulative% Drug Release

Minutes fromprepared FDTs from commercial tablets

0 0 0

5 76.7 ± 1.94 29.7±0.91

10 84.2 ± 1.34 42.3±0.78

15 99.7 ± 0.96 49.2±1.56

20 99.9 ± 1.14 61.6±1.66

30 98.9 ± 1.02 81.8±1.05

45 - 87.3±0.98

60 - 98.5±1.65

Table.5: Percent drug release (n=3±SD) from prepared fast dissolving tablets and commercial tablets of glibenclamide.

Fig. 2: Dissolution profiles of pure drug, prepared fast

dissolving and commercial tablets of Glibenclamide.

339

REFERENCES

1. Seager H. Drug Delivery Products and the Zydis

Fast-dissolving Dosage Form. J. Pharm and

Pharmacol. 1998; 50: 375-382.

2. Bradoo R, Shahani S, Poojary S, Deewan B,

Sundarshan. Ind. J. of Pharm. Sci, 2001; 4: 27-31.

3. Habib W, Khankari R, Hontz J. Fast dissolving drug

delivery system. Crit Rev Ther Drug Carrier Syst.

2000;17:61-72.

4. Chang RK, Guo X, Burnside B, Couch R. Fast

dissolving tablets. Pharm Technolo.2000; 24(6): 52-

58.

5. Martindale, The Complete drug reference, 3 rd

Edition Page No: 319

6. Kumar R, Gupta R.B, Betageri G.V. Formulation,

characterization, and in vitro release of glyburide

from proliposomal beads. Drug Delivery. 2001; 8:

25-27. nm

7. Tashtoush B.M, Al-Qashi Z.S, Najib N.M. In vitro

and in vivo evaluation of glibenclamide in solid

dispersion systems. Drug Dev Ind Pharm. 2004; 30:

601-607.

8. Valleri M, Mura P, Maestrelli F, Cirri M, Ballerini R.

Development and evaluation of glyburide fast

dissolving tablets using solid dispersion technique.

Drug Dev Ind Pharm, 2004; 30: 525-534.

9. Kasture AV, Mahadik KR, Wadodkar SG, More HN.

Pharmaceutical analysis.10 th ed. Pune: 2004.

10. Pharmacopoeia of India, Vol-II, 3 rd Ed., The

controller of publication, Delhi, 1985; 501.

11. Banker, G.S. and Anderson, N.R. In: Lachman, L.,

Lieberman, H.A. and Kanig J.L., Eds, Varghese

publishing house, Bombay, 1991; 297.

12. Marshall, K., In : Lachman, H.A. and Kanig, J.L.,

Eds., The theory and practice of Industrial

pharmacy, 3 rd Ed., 4 th Indian Reprint, Varghese

Publishing House, Bombay, 1991; 297.

13. Marshall, K., In : Lachman, H.A. and Kanig, J.L.,

Eds., The theory and practice of Industrial

pharmacy, 3 rd Ed., 4 th Indian Reprint, Varghese

Publishing House, Bombay, 1991; 88.


340

Abstract

A simple, selective, precise and stability-indicating high-performance thin layer chromatographic method for

analysis of , both as the bulk drug and in a tablet formulation, has been developed and validated.

Aluminium foil TLC plates precoated with silica gel 60F 254 were used as stationary phase and

as mobile phase. A compact band (R ) was obtained F

for . Densitometric analysis was performed in absorbance mode at 220 nm. Linear regression analysis 2revealed a good linear relationship (r = ) between peak area and concentration in the range

ng /spot. The mean values ± SD of the slope and intercept were and , respectively. The

method was validated for precision, recovery, and robustness. The limits of detection and quantitation were and

ng/spot, respectively. was subjected to acid and alkaline hydrolysis, oxidation, and photochemical

and thermal degradation and underwent degradation under all these conditions. Statistical analysis proved the

method enables repeatable, selective, and accurate analysis of the drug. It can be used for identification and

quantitative analysis of in the bulk drug and in tablet formulations.

Key Words: , HPTLC, validation, stability-indicating, degradation

trandolapril (TRA)

trandolapril

Trandolapril

trandolapril

trandolapril

toluene: ethyl

acetate: methanol: formic acid (2.5: 8: 1: 0.5 v/v/v/v) 0.51 ± 0.02

0.9994 ± 0.01 300–1800

2.8006 ± 1.23 71.67 ± 1.11

200

270


INTRODUCTION

Trandolapril (TRA) 1-[2-[(1-ethoxycarbonyl-3-phenyl-

propyl) amino] propanoyl]-2, 3, 3a, 4, 5, 6, 7,7a octa

hydroindole-2-carboxylic acid, is an angiotensin-

converting-enzyme inhibitor (ACE inhibitor) used to

lower high blood pressure. It is the ethyl ester prodrug of

a nonsulfhydryl angiotensin converting enzyme (ACE) 1 inhibitor, trandolaprilat. Trandolapril has been

determined in combination with other drugs using 2 3 4-5potentiometry , enantioselective analysis , HPLC ,

6- 7liquid chromatography-tandem mass spectrometry , in

pharmaceutical preparations. As far as we are aware, no

stability-indicating high-performance thin-layer

chromatographic (HPTLC) method for analysis of

trandolapril in pharmaceutical dosage forms has been

reported in the literature. The parent drug stability test

guidelines (Q1A) issued by the International Conference

on Harmonisation (ICH) requires that analytical test

procedures for stability samples should be fully validated

8-10and the assays should be stability-indicating .

Accordingly, the objective of this work was to put the

ICH recommendations into practice by subjecting

t to variety of suggested stress test conditions

to establish intrinsic stability of the drug and to develop a

validated stability-indicating HPTLC assay.

working standards was kindly supplied as a

gift sample by

; it was certified to contain 99.67 % (w/w) on dried

basis and was used without purification. All chemicals

and reagent used were of analytical grade and were

purchased from Merck Chemicals, Mumbai, India. Tablet

containing 2 mg of TRA was purchase from local market.

HPTLC Instrumentation and conditions:

Chromatography was performed on 20 cm × 20 cm on

aluminium foil plates precoated with 0.2-mm layers of

silica gel 60F254 (E. Merck, Germany). Before use the

plates were prewashed by development with methanol

then dried in the current of dry air and activated at 110 °C

randolapril

Trandolapril

MATERIALS AND METHOD

Chemicals and reagents:

Ind-Swift Laboratories Limited, Patiyala,

India


Stability Indicating HPTLC method for Estimation in the

Bulk Drug and Tablet Dosage Form

Department of Pharmaceutical Chemistry, Bharati Vidyapeeth University, Poona College of Pharmacy,

Pune – 411038, Maharashtra, India.


Trandolapril

Vikas, Rao J.R*, Sathiyanarayanan L and Yadav S.S.

341

for 5 min. Samples were applied as bands 6 mm wide, 6

mm apart, by use of a Camag (Switzerland) Linomat IV

equipped with a microlitre syringe. The mobile phase was

toluene: ethyl acetate: methanol: formic acid (2.5: 8:

1:0.5 v/v/v/v). Linear ascending development was

performed in a twin-trough glass chamber previously

saturated with mobile phase vapour for 30 min at room

temperature and relative humidity 60 ± 5%. The

development distance was approximately 80 mm. After

development the plates were dried in current of air by use

of an air dryer. Densitometric scanning, at 220 nm, was

performed with a Camag TLC scanner III in absorbance

mode. The source of radiation was a deuterium lamp

emitting a continuous UV spectrum in the range 190–400

nm. The slit dimensions were 5 mm × 0.45 mm.

Calibration:

A stock solution containing 1000 µg/ml TRA was

prepared in methanol. Different volumes of this solution

were applied to the plate resulting in application of 300-

1800 ng/spot to the plate. Each concentration was applied

seven times to the plate and the plate was developed as

described above. Peak areas were plotted against

corresponding concentrations to furnish the calibration

plot.

METHOD VALIDATION

Precision:

Repeatability of sample application and measurement of

peak area were assessed by chromatography of six

replicates of the same concentration (900 ng /spot TRA).

Intra-day and inter-day variation for determination of

TRA was measured at three different concentrations

(300, 900, 1500 ng/spot).

Robustness:

Small changes in the chromatographic conditions were

introduced and the effects on the results were examined.

Slight changes in the volume of formic acid ( ) and

Limits of Detection and Quantification:

To determine the limits of detection and quantification,

concentrations in the lower part of the linear range of the

calibration plot were used. Stock solution of TRA (1000

µg/ml) was prepared and different volumes in the range

± 0.1 ml

total volume of mobile phase (± 0.5 %) were made and the

peak areas were analysed. The time from spotting to

chromatography and time from chromatography to scan

was varied at 0 and 20 min.

200 to 600 ng were applied in triplicate. Amounts of TRA

per spot were plotted against average response (peak

area) and the regression equation was determined. The

standard deviations (SD) of responses and the average

standard deviations (ASD) were calculated. Detection

limit was calculated as (3.3 × ASD)/b and quantification

limit was calculated as (10 × ASD)/b, where 'b' denotes

the slope obtained in the linearity study.

Specificity:

The specificity of the method was determined by analysis

of drug standards and samples. The band for TRA in the

sample was identified by comparing the R value and F

spectrum of the band with those of the band from a

standard. The peak purity of TRA was assessed by

comparing spectra acquired at three different positions on

the peak, i.e. the peak start (S), peak apex (M), and peak

end (E) positions of the band.

Recovery:

To check the recovery of the drug at different levels in

formulations, analysed samples were spiked with an

extra 80, 100, and 120% of TRA standard and the

mixtures were reanalysed by the proposed method. The

experiment was conducted in triplicate.

Analysis of the Marketed Formulation:

To determine the TRA content of conventional tablets,

twenty tablets were weighed and powdered in a glass

mortar. An amount of powder equivalent to the average

weight of the one tablet TRA was transferred to a 10 ml

volumetric flask, extracted with methanol, sonicated for

30 min, diluted to volume with same solvent. The

resulting solution was filtered through a 0.45 µm filter

(Millifilter; Milford, MA, USA). The solution (800 ng

TRA) was applied to a plate for assay of TRA and the

TRA bands at R were observed in the densitogram F

obtained from tablets. There were no interferences from

the excipients commonly present in the tablets.

FORCED DEGRADATION OF

STANDARD

A stock solution containing 10 mg TRA in 10 ml

methanol was prepared. This solution was used for forced

degradation to provide an indication of the stability-

indicating property and specificity of the method. In all

degradation studies the average peak area of TRA after

application (5000 ng/spot) of seven replicates was

obtained after development and scanning of the plate as

0.51

TRANDOLAPRIL


342

described above.

Acid and Base-Induced Degradation:

TRA (10 mg) was separately dissolved in 10 ml of

methanolic solution of 1 N HCl and 0.01 N NaOH. These

solutions were refluxed at 80ºC for 6 h in the dark in order

to exclude the possible degradative effect of light. The

solutions (1 ml) were taken and neutralized and then

diluted up to 10 ml with methanol. The resultant solutions

were applied on TLC plate in triplicate (50 µl each, i.e.

5000 ng/spot). The plate was chromatographed as

described above.

Hydrogen Peroxide-Induced Degradation:

TRA (10 mg) was dissolved in 10 ml of methanolic

solution of hydrogen peroxide (30%, v/v) and the mixture

was kept for 48 h at room temperature in the dark, to

exclude the possible degradative effect of light. The

solution (1 ml) was then diluted to 10 ml with methanol

and treated as described for acid and base-induced

degradation.

Dry Heat Degradation:

The powdered drug was stored for 3 days under dry heat

conditions at 50°C. A solution of the treated powder was

then prepared and 5000 ng/spot was applied to a plate in

triplicate. The plate was then chromatographed and

treated as described above.

Photochemical Degradation:

TRA solution was left in sunlight for 7 days. The resultant

solution was treated as described for hydrogen peroxide-

induced degradation.


HPTLC Method Optimisation and Validation:

The TLC procedure was optimised to develop a stability-

indicating method. Both pure drug and the degraded

products were spotted on the plates and chromatographed

with different mobile phases. Initially toluene

methanol in different ratios was tried. The mobile

phase

enabled good resolution, and a sharp and

symmetrical peak of R 0.51 for TRA (Fig. 1) from a F

compact and non-diffuse band. It was observed that

prewashing of TLC plates with methanol (followed by

drying and activation) and pre-saturation of TLC

chamber with mobile phase for 30 min (the optimum

saturation time) ensured good reproducibility and peak

shape of TRA.

: ethyl

acetate:

toluene: ethyl acetate: methanol: formic acid (2.5:

8: 1: 0.5 v/v/v/v)


VALIDATION

Linearity and range:

The linear regression data for the calibration plots

revealed a good linear relationship over the concentration 2 range of 300–1800 ng/spot (correlation coefficient, r =

0.9994). The results are shown in Table I.

Precision:

The precision of the method was expressed as relative

standard deviation (RSD, %). The results obtained (Table

II) revealed the high precision of the method.

Robustness:

When the standard deviation of peak area was calculated

for each change of conditions RSD was found to be less

than 2%. These low RSD values (Table III) indicated the

method is robust.

Recovery:

When the method was used for extraction and subsequent

analysis of TRA from pharmaceutical dosage forms after

spiking with 80, 100, and 120% of additional drug,

recovery was 99.06-99.31 % (Table IV).

Limits of Detection and Quantification:

The limits of detection and quantification calculated as

described above were 200 and 270 ng/spot, respectively.

This indicates the sensitivity of the method is adequate.

The results obtained from validation of the method are

summarized in Table V.

Specificity

The peak purity was assessed by comparing the spectra at

peak start, middle and apex positions of the spot. ie)., r

(S,M) = 0.9992 and r (M,E) = 0.9997 . Good correlation 2

was observed r = 0.998 between standard and sample.

Assay of Marketed Formulation:

A single spot of R 0. 51 was observed in chromatograms F

obtained from drug samples extracted from conventional

tablets. There was no interference from the excipients

commonly present in the tablets. The drug content was

found to be 99.04 ± 0.3855 . It may, therefore, be

inferred that degradation of TRA had not occurred in the

marketed formulations analysed by this method. The low

value of RSD indicates the method is suitable for routine

analysis of TRA in pharmaceutical dosage forms.

FORCED DEGRADATION

Acid and Base-Induced Degradation:

The chromatograms obtained from TRA contained

additional 2 peaks at R 0.38 and R 0.42 in the acid-F F

%

343

degraded samples and at R 0.38 and R 0.41 in the base-F F

degraded samples (Figures 2 and 3). The concentration of

the drug was different from the initial concentration,

indicating that TRA undergoes degradation under acidic

and basic conditions. The similar Rf values of the

degradation products obtained in both acid and alkali

hydrolysis may lead to the assumption that, TRA

undergoes degradation in similar manner in both

hydrolysis. In a previous study, diacid degradate was 12shown as the degradant in acid and alkali hydrolysis

which can be correlated with one of the degradants of the

TRA in the present study. However further studies needed

to isolate and charcterise the degradants.

Hydrogen Peroxide-Induced Degradation:

The chromatograms obtained from samples degraded

with hydrogen peroxide (Fig. 4) contained an additional 2

peaks at R 0.13 and R 0.19. The spot of the degradation F F

product was well resolved from that of the drug.

Dry Heat Degradation:

There was no significant degradation observed after 3

days under dry heat conditions at 50°C.

Photochemical Degradation:

Significant degradation was not observed in standards

left in daylight for 7 days.

CONCLUSION

The developed HPTLC technique is precise, specific,

accurate and stability indicating. The developed method

is able to discriminate between TRA and its possible

degradation products. Statistical analysis proves that the

method is suitable for the analysis of TRA as bulk drug

and in pharmaceutical formulation without any

interference from the excipients. The method can be used

to determine the purity of drug available from various

sources by detecting any related impurities. Because the

method could effectively separate the drugs from their

degradation products, it can be regarded as stability

indicating.

ACKNOWLEDGEMENTS

The authors are grateful to

providing samples of

as gift. The authors are also grateful to

Bharati Vidyapeeth University, Poona College of

Pharmacy, Pune, India, for providing excellent facilities

for carrying out this research work.

Ind-Swift Laboratories

Limited, Patiyala, India,

trandolapril

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1. http://www.rxlist.com/cgi/generic/trandolapril.htm,

2009 (accessed on 10.09.2009).

2. Stefan RI, Staden JF, Hassan Y. Detection of S-

enantiomers of cilazapril, pentopril and trandolapril

using a potentiometric, enantioselective membrane

electrode. Electroanalysis 1999; 11 : 192-194.

3. Hassan Y, Stefan RI, Radu GL. Biosensor for

enantioselective analysis of S- Cilazapril, S-

Trandolapril, and S-Pentopril. Pharm Develop Tech

1999; 4: 251 – 255.

4. Gumieniczek A, Hopkala H. Development and

validation of a liquid chromatographic method for

the determination of trandolapril and verapamil in

capsules. J Liq Chromatograph Related Tech. 2001;

24: 393 – 400.

5. Gumieniczek A, Hopkala H. High-performance

liquid chromatographic assay of trandolapril in

capsules. Acta Pol Pharm. 2000; 57: 253-5.

6. Nirogi RV, Kandikere VN, Shrivastava W,

Mudigonda K. Quantification of trandolapril and its

metabolite trandolaprilat in human plasma by liquid

chromatography/tandem mass spectrometry using

solid-phase extraction. Rapid Commun Mass

Spectrom. 2006; 20: 3709- 3716.

7. Pistos C, Koutsopoulou M, Panderi I. Liquid

chromatographic tandem mass spectrometric

determination of trandolapril in human plasma. Anal

Chimica Acta. 2005; 540: 375-382.

8. International Conference on Harmonisation (ICH)

of Technical Requirements for the Registration of

Pharmaceuticals for Human Use (2005) Validation

of Analytical Procedures: Text and Methodology Q2

(R1), 2005, pp. 1–13

9. International Conference on Harmonisation (ICH)

of Technical Requirementsfor the Registration of

Pharmaceutical for Human Use (2003) Stability

Testing of New Drugs Substance and Products Q1A

(R2), 2003, pp. 1–18

10. Sethi PD. High performance thin layer

chromatography, quantitative analysis of

pharmaceutical formulations, 2 nd edition, CBS

Publishers and distributors, New Delhi, 1996, 56-63.


344

Abstract

Doxepin is an important antidepressant drug useful in the treatment of mild to moderate endogenous depression. The

synthesis of doxepin involves acid catalysis leading to the formation of E- and Z- isomers. So herein, we report the use

and effect of various acid catalysts on the formation E- and Z- isomers. Characterization of both the isomers was

achieved by using HPLC and NMR. Both NMR and HPLC analysis showed that E-isomer as the major component.

Key Words: Doxepin, E and Z isomer, NMR and HPLC


INTRODUCTION

Many important and widely used drugs are marketed as

mixture of optical isomers that often differ in

pharmacological, toxicological and pharmacokinetic 1 2,3properties . Doxepin is a tricyclic antidepressant

compound. It is N, N-dimethyl-3-(dibenz[b,e] oxepin-

11(6H)-ylidene) propylamine. Doxepin exists as a 15:85

mixture of Z- and E- isomers of N,N-dimethyl-3-

dibenz[b,e]-oxepin-11(6H)-ylidene-1-propanamine. The

isomers have been evaluated in various neurochemical

and behavioral tests (e.g., NE and 5-HT uptake in nerve

terminals, antagononistic effects of reserpine, histamine

and 5-HT; effects on amphetamine stereotypy; sleep

potentiation; anticholinergic effects) and in most tests,

the Z-isomer is the more potent. However, the E-isomer is

much more potent than the Z-isomer in inhibiting 5-HT 4,5uptake in platelets .

Doxepin was synthesized using different acid catalysts.

Using NMR technique we were able to identify the

isomers and confirm the E- and Z- isomer formation in

the experiments involving use of various acids. This

study was confirmed by HPLC to know the percentage of 6, 7, 8both the isomers in doxepin .

MATERIAL AND METHOD

The chemicals and reagents used in the present project

were of AR grade and LR grade and are purchased from

Lancaster, Sigma and NRChem. Melting points of the

synthesized compounds were determined in open 1capillary tubes and were found uncorrected. HNMR(400

MHz) spectra were recorded in deuterated chloroform in

Amx-200 liquid state NMR spectrometer (Astra Zeneca,

Bangalore) using TMS as internal standard. HPLC

chromatograms were recorded on Shimadzu SPD10 A

UV—Visible detector, Ray Chemicals Pvt .

Ltd.,Yelahanka, Bangalore.

STEP-1: General procedure for preparation E- & Z-

isomers of N,N-dimethyl-3- (dibenzo[b,e]oxepin-

11(6H) ylidene) propylamine (1a-g):

(11R,S)-11-[3-(Dimethylamino)propyl]-6 ,11-9dihydrodibenzo[b,e]oxepin-11-ol [Compound I] (10g,

0.0333 mol) was taken in a 500 ml round bottom flask, to

which acid (0.0404 mol) and toluene (100 ml) were added

with stirring at 110 °C. The reaction mixture was stirred

for a period of 6-8 hr. After the reaction, the reaction

mixture was extracted with toluene and poured in to 150

ml of water and made basic to pH-9. Basic and toluene

layers were separated. Toluene was distilled out to form a

semisolid product. Acetone was added and made acidic to

pH-2 to obtain the final product. -1IR (KBR) cm :3068(ArC-H str), 2954(aliph C-Hstr),

1602(C=C, str), 1219(C-O-C str), 1107(C-N str)

(Compound 1a).1HNMR (TMS) δ: 6.74-7.41{m, 8H,Ar-H};5.95 {t,1H,

Unsat. C=CH-E-isomer}; 5.88{ t,1H, Unsat. C=CH-Z-

isomer};3.01-3.08{t,2H, CH -O-C }; 2.71-2.76 {s, 6H, 2 2

х CH }[Compound 1c- obtained from Tartaric acidL(+)]3


Effect of Different Acids on the Formation of E and Z Isomers of

Doxepin 1 2 1 1G.K. Rao *, A.R. Ramesha , Amit Kumar Jain and B.V. AdaviRao

1Department of Pharmaceutical Chemistry, Al-Ameen College of Pharmacy-Bangalore2R.L.Fine Chemicals, Yelahanka, Bangalore


345

Compounds were obtained in the same manner by using

various acids (Table 1).

Determination of percentage of (Z) N,N-dimethyl-3-

(dibenzo[b,e]oxepin-11(6H) ylidene) propylamine

hydrochloride by High Performance Liquid

Chromatography:

Test solution: Dissolved 0.02 g of the N,N-dimethyl-3-

(dibenzo[b,e]oxepin-11(6H)ylidene) propylamine

hydrochloride in the mobile phase and dilute to 20 ml

with the mobile phase. 1 ml of this solution was diluted to

10 ml with the mobile phase.

Chromatographic Procedure:

Separation was carried out on ODS column of length 0.12

m and internal diameter 4mm. The column oven was set oat 50 C and detection was carried out at 254 nm. Mobile

phase comprised of a mixture of 30 volumes of methanol

and 70 volumes of a 30 g/l solution of sodium

dihydrogen phosphate, pH adjusted to 2.5 with

phosphoric acid.

Procedure:

20 µl of the test solution was injected. The system

sensitivity was adjusted so that the height of the principal

peak is at least 50 % of the full scale of the recorder. The

test is not valid unless the resolution between the first

peak (E-isomer) and the second peak (Z-isomer) is at

least 1.5.

The ratio of the area of the peak due to the E-isomer to the

area to the peak due to the Z-isomer is 4.4 to 6.7. The

percentage of both the isomers formed are tabulated in

Table 2.

RESULT AND DISCUSSION

The percentage of E-isomer is found to be higher than Z-

isomer in all the experiments. The conversion of (11R,S)-

1 1 - [ 3 - ( d i m e t h y l a m i n o ) p r o p y l ] - 6 , 1 1 -

dihydrodibenzo[b,e] oxepin-11-ol to E- & Z- N,N-

dimethyl-3-(dibenzo[b,e]oxepin-11(6H) ylidene)

propylamine was complete as shown by IR spectra with -1appearance of C=CH peak at 1602 cm .

1The H-NMR spectral analysis also revealed the

formation of the E- and Z- isomers, N,N-dimethyl-3-

(dibenzo[b,e]oxepin-11(6H) ylidene)propylamine at δ

value 5.95 and 5.88 respectively in all the experiments.

Further confirmation was done by HPLC, which showed

formation of E-isomer in the range of 80-85 % whereas Z-

isomer formation was in the range of 15-19 % for doxepin

Table 2.

Best results were obtained with Indian resin-220 for E-

isomer of doxepin (85%) while the other acids yielded

more than 80%.

CONCLUSION

The main focus of this research work was to understand

the effect of different acid catalysts on the formation of E-

and Z- isomers of Doxepin and to characterize the 1isomers by HNMR and HPLC methods. The yield of E-

isomer of doxepin was achieved in the range of 80-85%.

From the experiments conducted, the maleic acid (1a),

tartaric acid DL (1e), Indian resin-220 (1f) and p-toluyl

sulphonic acid (1g) were found to be ideal reagents for the

dehydration of the alcohol [compound-I] to form E- and

Z- isomers of doxepin. Ratio of E- and Z-isomers of

Doxepin obtained by NMR showed the range of 69:30

and 78:21 whereas in HPLC it was found to be in the

range of 80:19 and 85:15.

ACKNOWLEDGEMENT

The authors would like to thank Prof. B. G. Shivananda,

Principal, Al-Ameen College of Pharmacy, Bangalore

and Mr Anjan K. Roy, Managing Director, R.L.Fine

Chemicals, Bangalore for providing support and

necessary facilities, Department of Inorganic and

Physical Chemistry, Indian Institute of Science,

Bangalore, for providing help in obtaining the various

spectra.


346


SCHEME-1

Compound Acid Prac. % M.P.Code catalyst Yield Yield (ºC)

1a Maleic acid 8.5g 90 186-188

1b Oxalic acid 7.6g 81 186-188

1c Tartaric acid L(+) 9.2g 97 181-183

1d Phosphoric acid 8.2g 87 185-187

1e Tartaric acid DL 6.5g 70 187-189

1f Indian Resin-220 8.6g 92 187-189

1g p-Toluyl sulphonic acid 9.2g 98 188-190

Table 1: Percentage yield, Melting point of doxepin obtained with various acids

Sl. No. Compound Acid Used E:Z E:Z

1 1a Maleic acid 69:30 82:17 2 1b Oxalic acid 75:25 82:18

3 1c Tartaric acid L(+) 75:25 80:19

4 1d Phosphoric acid 75:25 82:18

5 1e Tartaric acid DL 78:21 83:17

6 1f Indian resin-220 75:25 85:15

7 1g p-Toluyl sulphonic acid 75:25 84:16

1( H-NMR) (HPLC)

Table 2: Percentage of Doxepin isomers evaluated by 1H-NMR and High performance liquid chromatography

347


1Fig. 1: H-NMR spectrum of (1e)

1H-NMR Spectrum (CDCl ) 6.74-7.41{m, 8H,Ar-H};5.95 {t,1H, Unsat. C=CH-E-3

isomer};5.88{t,1H, Unsat. C=CH-Z-isomer};3.04-3.09{t,2H, CH -O-C }; 2.71-2.76 {s, 2

6H, 2 х CH }(Compound 1e- obtained from Tartaric acid DL)3

1Fig 2: Calculation of percentage of isomers by H-NMR spectrum

348


Refrence

1. Greven J, Defrain W, Glaser K, Meywald K,

Heidenreich O. Studies with the optically active

isomers of the new diuretic drug ozolinone. Eur. J.

Pharm.Sci., 1996; 4: 57-60.

2. Kaiser C, Setler PE. Antidepressants: Burger’s

Medicinal Chemistry and Drug Discovery. Vol-6.

New York. John Wiley and Sons Ltd. 2007: 485-510.

3. Hajak G, Rodenbeck A, Voderholzer U. Doxepin in

the treatment of primary insomnia: a placebo-

controlled, double-blind, polysomnographic study.

J. Clin. Psychiatry. 2001; 62(6):453-463.

4. Lane RM,Baker GB. Chirality and drugs used in

psychiatry: Nice to know or need to know? Cellular

and molecular neurobiology, 1999; 19(3):355-67.

5. Haritos VS, Ghabrial H, Ahokas JT, Ching MS.

Stereoselective measurement of E- and Z- doxepin

and its N-desmethyl and hydroxylated metabolites

by gas chromatography- mass spectrometry. J

Chrom. B. 1999; 736:201-08.

6. Badenhorst D, Sutherland FCW, Jager AD, Scans T,

Hundt HKL, Swart KJ, et al., Determination of

doxepin and desmethyldoxepin in human plasma

using liquid chromatography- tandem mass

spectrometry. J Chrom. B. 2000; 742:91-98.

7. Park YH. Quantitative determination of doxepin and

nordoxepin in urine by high- performance liquid

chromatography. J Chrom. 1986; 375:202-06.

8. Kabra PM, Mar NA, Marton LJ. Simultaneous

liquid chromatographic analysis of amitriptyline,

nortriptyline, imipramine, desipramine, doxepin and

nordoxepin. Clinica Chimica Acta. 1981; 111:123-

32.

9. Lednicer D, Mitscher LA, The organic chemistry of

drug synthesis-Vol-1, John Wiley and Sons Ltd.

New York, 1977, 404.

349

Abstract

Optimization techniques represent analytical tools available for the best possible solution to constrained and

unconstrained problems involved in an experiment. Optimization techniques encompass experimental design,

mathematical model and graphical outcomes which help achieve optimum results. Choices of optimization

techniques depend on the number of responses that are required to be optimized. The purpose of this study was to

develop an optimized formula for microwave synthesis of Schiff's bases by using suitable optimization techniques. A 23 factorial design was employed using the software Statease 7.1.6 for the microwave assisted synthesis of Schiff's

bases with microwave power and molar ratio as independent variables and yield and time required for the reaction as

dependent variables. Reaction of 2-(1H-imidazol-1-yl) acetohydrazide (3) with appropriate aromatic aldehydes in

presence of catalytic amount of glacial acetic acid yielded corresponding derivatives of N'-Substituted-2-(1H-

imidazol-1-yl) acetohydrazide (4a-4h). The microwave assisted organic synthesis of these Schiff's bases made the

procedure economical and eco-friendly thus maintaining the sustainability of Green Synthesis. 2Keywords: Schiff's Bases, Optimization, 3 Factorial design, Statease 7.1.6.


INTRODUCTION

The search of novel heterocyclic compounds has led to

the discovery of many molecules having tremendous

potential. These heterocyclic compounds have been

proven to be backbone for the discovery of several

biologically active compounds. Imidazole has been

noticed as an important part of many pharmaceuticals.

Substituted imidazoles are reported to possess anti-1-3 4fungal activity along with anti-mycobacterial activity .

Schiff's bases of various heterocyclic scaffolds exhibits 5varieties of biological activities like anti-HIV , anti-

6 7 8 cancer , antibacterial , fungicidal and anti-9inflammatory . Optimization techniques require less

experimentation to achieve optimum results. It yields

“best” solution in presence of competing objectives and

makes problem tracing and rectification easy. In view of

this, various novel derivatives of imidazole based Schiff's

bases were synthesized under microwave radiations and

the reaction parameters were optimized so as to achieve

maximum yield in minimum time without compromising

the purity of the product.


Starting materials were obtained from commercial

suppliers and used without further purification. Synthesis

was performed using Catalyst Microwave Synthesizer.

All the melting points were determined by open capillary

method on a 'Veego' VMP-D apparatus and are

uncorrected. Silica gel G plates of 3x8 cm (Sigma-

Aldrich) were used for TLC and spots were located by

UV or in iodine chamber. The IR spectra were recorded in -1the 4000-400 cm range using KBr discs on FT-IR 8400

1SHIMADZU spectrometer. H NMR spectra were

recorded on Varian Mercury (300MHz) spectrometer in

CDCl with TMS as an internal standard and values are 3

expressed in δ ppm.

In the present study, imidazole was reacted with ethyl

bromoacetate to obtain ethyl – 2 – (1H-imidazol-1-yl) -

acetate (2) which was further converted to 2-(1H-

imidazol-1-yl) acetohydrazide (3) when reacted with

hydrazine hydrate. This hydrazide was treated with

various aromatic aldehydes in presence of catalytic

amount of glacial acetic acid to afford the Schiff's bases


Application of Factorial Design in Optimization of Synthetic Reactions:

a Novel Approach*Pal Tanushree, Somani Rakesh R . and Kadam Vilasrao J.

Department of Pharmaceutical Chemistry, Bharati Vidyapeeth's College of Pharmacy, Sector 8, C.B.D.,

Navi Mumbai 400614 India.


350

i.e. N'-Substituted-2-(1H-imidazol-1-yl) acetohydrazide

(4a-4h). Table 1 gives the details of the various

substituents used.

SCHEME

N'-Benzylidene-2-(1H-imidazol-1-yl) acetohydrazide 0(4a): Yellow crystals; yield 50%; m.p. 127 – 130 C; IR

-1(KBr, cm ): 3062 (-NH str), 2950 (-CH, str), 1602 (C=O

str, amide),1580 (-C=N, str), 1500 (NH, bend), 781-651 1(Ar-CH, bend); H NMR (CDCl ): δ 9.69 (s, 1H, CONH), 3

8.68 (d, 1H, N=CH), 8.68-7.66 (m, 3H, imidazolyl), 7.48-

7.45 and 7.29- 7.12 (m, 5H, phenyl), 7.00-6.90 (dd, 2H,

phenyl), 5.32 (s, 2H, NCH ).2

N ' -(3-Methoxy-4-hydroxy-benzylidene)-2-(1H-

imidazol-1-yl)-acetohydrazide (4b):0Yellow crystals; yield 65%; m.p. 174- 176 C; IR (KBr,

-1cm ): 3481 (OH, str), 3000 (NH, str), 2935 (-CH str),

1685 (C=O str, amide), 1598 (C=N, str) 1509 (NH, bend), 1813-752 (Ar-CH, bend); H NMR (CDCl ): δ 11.50 ( s, 3

1H, OH), 8.60 (d, 2H, N=CH and CONH), 7.60-7.50 (s,

1H, 2'-imidazolyl), 7.25-7.20 (m, 4H, imidazolyl and

phenyl), 7.00-6.90 (dd, 2H, phenyl), 6.00 (s, 2H, NCH ), 2

+ +4.00 (s, 3H, OCH ); MS: 150 (M - 124, 100%), 272 (M - 3

2, 15%), 152, 177.1, 301, 302.1.

N '-(4-Methoxybenzylidene)-2-(1H-imidazol-1-yl)

acetohydrazide (4c): Yellow crystals; yield 52 %; m.p. 0 -1171-174 C; IR (KBr, cm ): 3359 (-NH str), 3261.4 (-CH

str), 1652 (-C=0 str, amide), 1600-1558 (C=N, str), 1508 1( NH, bend) 817-615 (Ar-CH, bend); H NMR (CDCl ): δ 3

8.60 (d, 2H, N=CH and CONH), 7.60-7.50 ( s, 1H, 2'-

imidazolyl), 7.25-7.20 (m, 4H, imidazolyl and phenyl),

7.00-6.90 (dd, 2H, Phenyl), 6.00 (s, 2H, NCH ), 4.00 (s, 2

3H, OCH ).3

N '-(2-Chlorobenzylidene)-2-(1H-imidazol-1-yl)-

acetohydrazide (4d): Yellow crystals; yield 80 %; m.p. 0 -1138-142 C; IR (KBr, cm ): 3066 (NH, str), 3004 (-CH

str), 1701(-C=O str, amide), 1612 (C=N str) 1550 (NH,

bend), 748-642 (Ar-CH, bend).

N '-(2-Hydroxybenzylidene)-2-(1H-imidazol-1-yl)

acetohydrazide (4e): Yellow crystals; yield 72 % %; m.p. 0 -1213 - 216 C ; IR (KBr, cm ): 3357 (OH- str), 3261 (NH,

str), 3218-3178 (-CH, str), 1652 (-C=0 str, amide), 1573

(C=N, str) , 1429 (NH, bend), 821-815 (Ar-CH, bend).

N '-(3 nitrobenzylidene) -2-(1H-imidazol-1-yl)-

acetohydrazide (4f): Yellow crystals; yield 69 %; m.p. 0 -1195- 198 C; IR (KBr, cm ): 3085 (NH, str), 3020 (-CH

str), 1701(C=O str, amide), 1623 (C=N, str) , 1521 (NO , 2

str), 1438 (NH, bend), 748-642 (Ar-CH, bend).

N'-(4-Hydroxybenzylidene)-2-(1H-imidazol-1-yl)


acetate (2)

Synthesis of ethyl – 2 – (1H-imidazol-1-yl) -

In a solution of imidazole (6.8g, 0.1 mole) in dry

methanol, ethylbromoacetate (16.7g, 0.1 mole) was

slowly added under constant stirring in presence of 5 g of

anhydrous K CO . The resulting mixture was then 2 3

refluxed conventionally for about 8- 10 h. Then the it was

cooled, filtered and filtrate was distilled to obtain the 0 -1desired ester (65 %), b.p: 132-135 C; IR (KBr, cm ):

2983-2929 (-CH str), 1748 (C=O str, ester), 1580 (C=N,

str), 1217 (C-O-C), 749 (Ar-CH bend).

Synthesis of 2-(1H-imidazol-1-yl) acetohydrazide (3)

Ethyl – 2 – (1H-imidazol-1-yl) - acetate (2), (15.4g, 0.1

mole) was dissolved in 15 mL anhydrous methanol and to

this solution; hydrazine hydrate (7.65 g, 0.15 mole) was

added slowly under constant stirring. It was refluxed on

water bath for about 9-10 h. The removal of excess of

solvent under vacuum afforded the required product (70 0 -1%), b.p.: 160 - 162 C; IR (KBr, cm ): 3100 (NH str) 2950

(-CH, str), 1685 (C=O str amide), 1589 (C=N str) 1512

(NH, bend) 676 (Ar-CH, bend).

General procedure for Microwave Assisted Synthesis of

N'-Substituted-2-(1H-imidazol-1-yl) acetohydrazide

(4a-4h)

2-(1H-imidazol-1-yl) acetohydrazide (2, 14g, 0.1.mole)

was dissolved in anhydrous ethanol and to this solution

aromatic aldehyde (0.11 mole) was added, in presence of

catalytic amount of glacial acetic acid (2-3 mL). The

reaction mixture was refluxed under microwave for 15-

30 minutes, as the case may be. It was then poured into ice

cold water to afford the corresponding Schiff's bases (4a-

4h). All of them were recrystallized using methanol.

351

acetohydrazide (4g): Yellow crystals; yield 72 %; m.p. 0 -1179 - 182 C; IR (KBr, cm ): 3342 (OH, str), 3100 (NH,

str), 2600-2900 (-CH str), 1623 (-C=O str, amide), 1515 (

C=N, str), 1452 (NH, bend), 748-642 (Ar-CH, bend).

N'-(3-phenylprop-2-en-1-ylidene)-2-(1H-imidazol-1-

yl)acetohydrazide (4h): Yellow crystals; yield 72 %; m.p. 0 -1155-157 C; IR (KBr, cm ): 3037 (-CH str), 1629 (-C=O

str, amide), 1585 (C=N, str), 1550 (NH, bend) 750-690 1(Ar-CH, bend); H NMR (CDCl ): δ 8.38- 8.35 (d, 2H, 3

N=CH and CONH), 7.55-7.33 (m, 3H, imidazolyl), 7.15-

7.02 (m, 7H, NCH andphenyl).2

Optimization

For optimization of the reactions it is important to

consider the various factors that dictate the over all

reaction. In the present microwave assisted synthesis, the

microwave power and the molar ratio of the reactants are

considered as the two significant independent factors.

The response would be the two dependent factors namely

the percentage yield and the time required to attain the

optimum yield. The 2 independent factors were

considered at 3 levels i.e. high, intermediate and low 2 levels. Thus 3 factorial design was used to optimize the

reactions.

Optimization of Microwave Assisted Synthesis of

compound 4b

The parameters considered in the optimization of

microwave assisted synthesis of 4b were the molar ratio

(A) of hydrazide (3) and aldehyde (Vanillin) and the

microwave power (B). Table 2 denotes the different

levels of the experimental procedure and the factors used.

The various combinations of factors and levels were used

after appropriate permutation and grouping as mentioned

in Table 3. Then the time required for the completion of

the reaction was noted along with the percentage yield.

Based on the responses and the factors Table 4 was

constructed using the software Statease 7.1.6. Based on

the summary sheet time analysis and yield analysis was

performed. In time analysis, the Quadratic model was

found to be significant. Thus the ANOVA was generated

as demonstrated in Table 5. This was followed by

determination of Standard error and coefficient as

mentioned in Table 6. This generated a regression

Equation 1 as given below;

Equation 1: Time = 509.4365 -163.619 x A - 0.93741 x B 2 2+ 0.167347 x AB + 12.66667 x A + 0.000408 x B


Once the model was found to be significant, the graphical

plots were generated. The Fig.1 gives a three dimensional

view of the effect of each independent parameter,

(number of moles and microwave power) on dependent

parameters (time required for the completion of the

reaction). The overview denotes regions in the graph

where the time required for the reaction ranges from 15

min to 45 min. A similar data is projected through a 2-D

Contour Plot as shown in Fig.2.

Yield analysis was done in the same pattern. Table 7

gives the ANOVA data for 4b. Table 8 demonstrates the

Standard error and coefficient. This generated another

equation for yield analysis as follows;

Equation 2: Yield = -300.204 - 27.4898 x A + 1.388194 x 2 2B + 0.01398 x AB + 12.52667 x A - 0.00139 x B

Similarly 3-D Surface Response Curve (Fig. 3) and 2-D

Contour Plot (Fig.4) were plotted. The curves gave an

overview of the effect of each independent parameter

(number of moles and microwave power) on the

dependent parameter (percentage yield of the product

formed). The overview denotes regions in the graph

where the percentage yield of the compound ranges from

51.28 % to 95.72 %.

Similarly such analyses were performed for 4a, 4c and 4f

separately using the same parameters.


According to Equation 1, the coefficient of A or B

bearing negative sign imply that they are of no impact

when considered alone. As observed in Table 4, 2.5

moles show no significant difference in the time required

for the completion of the reaction. Thus molar ratio alone

cannot govern the time analysis. Similarly, for different

powers when considered alone, showed no specific

changes in the time required for the reaction to complete.

But the statistical analysis states that, when the combined

effect of molar ratio and power are considered,

substantial change is observed in the time required for the

reaction as given in Table 9. Therefore an interaction

between the two parameters; molar ratio and microwave 2 2power was noticed. The square terms A and B appear

since the significant model is quadratic.

Similarly, Equation 2 implicated that due to the negative

sign designated to the coefficient of molar ratio (A), it

alone did not show any extreme change in the yield of the

product formed but the microwave power when

352

considered alone could create noticeable changes in the

percentage yield because the equation denotes a positive

sign for the power B. And the interaction of A and B

together shows considerable difference in the results of

the percentage yield observed.

The optimized solutions obtained for 4b (Table 9) were

compared and it was found that Solution 7 gave the

highest possible yield. But the time required for the

completion of the reaction was more than that of the other

solutions and the power required was less as compared to

others. But the desirability of the reaction is not 1. Thus

the closeness of the response to the target value is not

precise. Looking through all the 7 solutions, solution 5 is

the best possible optimized result and the others were

near about the best possible optimized condition for the

synthesis of the Schiff's base (4b). Thus, molar fraction of

1:1.96- 1:2 with microwave power ranging somewhere

between 535-560 watts gave the best percentage yield of

4b. This yield was achievable within 14.9- 16.22 min,

which made the entire procedure economical and less

time consuming.

Optimized solutions suggested by the software for

microwave assisted synthesis of 4a were use of molar

proportion of 1:2 of the reactants at 560 watts which

afforded maximum yield of 60.41 % within minimum

time of 17.76 min. For 4c, yield ranging from 62-64 %

using molar proportion of 1:1.7-1:1.8 and microwave

power of 552-560 watts in 14.1-14.9 min were the best

possible optimized conditions for the microwave assisted

synthesis. Finally for 4f, the molar proportion ranging

from 1:1.9-1:2 and the power – 510-535 watts helps

achieving the optimum responses of yield 78-79% in

16.4-16.9 min.

CONCLUSION

Traditional method of synthesis is comparatively slow,

non-uniform and inefficient method for transferring

energy into the system because it depends on thermal

conductivity of the various materials. By contrast

microwave irradiation produces efficient internal heating

by direct coupling of microwave energy with polar

molecules. Optimizing the various variables of the

microwave helped achieve an economical and eco-

friendly methodology for the synthesis of the mentioned

azo compounds. Experiments performed showed that

maximum yield was attainable in minimum time when

molar proportion of the reactants was 1:2 (Hydrazide:

aldehyde) used along with the microwave power of 560

watts. The results obtained through the software

confirmed that the optimum condition for the scheme of

reaction lies in the range of molar proportion 1:1.7-1:1.9

and microwave power of 513-560 watts. The results were

more refined in the case of software. Thus it can be

concluded that the optimum solutions are super-

imposable and pose to be the best conditions to

synthesize the series of Schiff's bases under microwave

energy.

4a 4e

4f

HO

CI

2NO

4b

4c

OH

3OCH

3OCH OH4g

4h4d


Table 1: Various substituents in titled compounds (4a-4h):

353

Factor 2: Response 1:Factor 1: Power Time Response 2:

Std Run No. of moles (Watts) (minutes) % Yield

5 1 2.5 490 23 76.92

9 2 3 560 15 95.72

1 3 2 4 55 45 51.28

8 4 2.5 560 20 64.95

6 5 3 490 18 94

7 6 2 560 22 52.99

2 7 2.5 455 25 67.52

4 8 2 490 35 51.28

3 9 3 455 20 92.3

Indian J.Pharm. Educ. Res. 44(4), Oct-Dec, 2010

HIGH (2) INTERMEDIATE (1) LOW ( 0)

MOLAR RATIO (A) 1:2 1:1.5 1:1

POWER (B) 560 490 455

FACTORSLEVELS

Table 2: Different Levels and Factors Used In Experimental Model:

2Table 3: 3 factorial design for 4b:

0 1 2

0 00 10 20

1 01 11 21

2 02 12 22

FACTOR B FACTOR A

Table 4: Data Sheet for 4b:

Source Sum of df Mean F P-Value Square Square Value Prob > F Remark

Model 230.6667 5 46.13333 14.82857 0.0251 Significant

A-No. of Moles 23.57895 1 23.57895 7.578947 0.0706

B-Power 192.6667 1 192.6667 61.92857 0.0043

AB 9.333333 1 9.333333 3 0.1817

A^2 0.5 1 0.5 0.160714 0.7153

B^2 6.880952 1 6.880952 2.211735 0.2337

Residual 9.333333 3 3.111111

Cor Total 240 8

Table 5: Analysis of variance (ANOVA) table for time analysis of 4b:

df – Degree of freedom

Cor total – Corrected total

F-Value- Fixation Indice or Fisher-Snedecor distribution

354


Factor Coefficient df Standard 95% CI 95% CIEstimate Error Low High VIF

Intercept 21.875 1 1.44391 17.27983 26.47017

A-no. of moles -2 1 0.726483 -4.312 0.311996 1.017857

B-Power -5.66667 1 0.720082 -7.95829 -3.37504 1.037037

AB 1.5 1 0.866025 -1.25608 4.256082 1.017857

A^2 0.5 1 1.247219 -3.46921 4.469211 1

B^2 2.125 1 1.428869 -2.4223 6.672303 1.037037

Table 6: Details of Coefficient and Standard Error for time analysis of 4b :


CI- Confidence Interval

VIF- Variance Inflation Factor

Source Sum of df Mean F P-Value Square Square Value Prob > F Remark

Model 1944.228 5 388.8455 10.27646 0.0418 Significant

A-No. of Moles 1668.261 1 1668.261 44.08903 0.0070

B-Power 80.44682 1 80.44682 2.12606 0.2409

AB 13.60048 1 13.60048 0.359435 0.5911

A^2 174.16 1 174.16 4.602725 0.1212

B^2 6.302688 1 6.302688 0.166568 0.7106

Residual 113.5154 3 37.83846

Cor Total 2057.743 8

df – Degree of freedomCor total – Corrected totalF-Value- Fixation Indice or Fisher-Snedecor distribution

Table 7: Analysis of variance (ANOVA) table for yield analysis of 4b:

Factor Coefficient df Standard 95% CI 95% CIEstimate Error Low High VIF

Intercept 42.69569 1 5.035576 26.67023 58.72116

A-no. of moles 16.82286 1 2.533579 8.75987 24.88584 1.017857

B-Power 3.661667 1 2.511257 -4.33028 11.65361 1.037037

AB 1.810714 1 3.020227 -7.801 11.42243 1.017857

A^2 -9.33167 1 4.349624 -23.1741 4.51079 1

B^2 2.03375 1 4.98312 -13.8248 17.89228 1.037037


CI- Confidence Interval

VIF- Variance Inflation Factor

Table 8: Details of Coefficient and Standard Error for yield analysis of 4b:

355


Solution No. of Power Time %Yield

1 2.996047 559.8151 14.99174 93.555 1

2 2.998895 559.0205 14.98958 93.81591 1

3 3 560 14.92857 93.74996 1

4 2.998514 559.4075 14.97674 93.74483 1

5 2.999939 558.573 14.99638 93.93107 1

6 2.964736 559.9996 15.40871 91.8085 0.972753

7 2.979851 535.8595 16.22988 94.99997 0.918008

Desirability

Moles (Watts) Req. Min.

Table 9: Solution for optimum conditions required for microwave assisted

synthesis of N'-(3-Methoxy-4-hydroxy benzylidene)-2-(1H-imidazol-1-yl) acetohydrazide (4b):

Fig.1: 3D Surface Response curve for time analysis of 4b: Fig. 2: 2D Contour Plot for time analysis of 4b:

Fig. 3: 3D Surface Response curve for yield analysis of 4b: Fig. 4: 2D Contour Plot for yield analysis of 4b

356


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substituted nitroimidazole derivatives as potential

antioxidant and antifungal agents, Eur. J. Med.

Chem. 2009; 44: 645-652.

2. Hori K, Csakaguchi A, Kudoh M, Ishida K,

Aoyama Y, Yoshida Y, Structure-Activity

Relationships of a New Antifungal Imidazole, AFK-

108, and Related Compounds. Chem Pharm Bull.

2000; 48(1):60-64.

3. Andriole VT. Current and future antifungal therapy:

new targets for antifungal agents. J Antimicrob

Chemother 1999; 44:151–62.

4. Daniele Z, Maria GM, Luciano V, Elena B, Giuditta

S, Maurizio F, Marco F, Sabrina P. Synthesis,

antifungal and antimycobacterial activities of new

bis-imidazole derivatives, and prediction of their

binding to P450 by molecular docking and 14DM

MM/PBSA method. Bioorg. Med. Chem. 2007;

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5. Pandeya SN, Sriram D, Nath G, Clercq E.D.

Synthesis, antibacterial, antifungal and anti-HIV

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derivatives with 3-amino-2-methylmercapto

quinazolin-4(3H)-one. Pharm. Acta Helvetiae.

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6. Kuz'min VE, Lozitsky VP, Kamalov GL, Lozitskaya

RN, Zheltvay AI, Fedtchouk AS, Kryzhanovsky

DN. Analysis of the structure-anticancer activity

relationship in a set of Schiff bases of macrocyclic

2,6-bis(2- and 4-formylaryloxymethyl)pyridines.

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antibacterial activity of some Schiff base complexes.

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Triazolethione Schiff Base. Chin. J. Chem. 2007;

25(10): 1573-1576.

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357

Abstract

A series of pyrazole derivatives of benzimidazole were synthesized. Condensation of substituted o-phenylene diamine

with lactic acid under microwave irradiation and further oxidation of the product with potassium dichromate gave

intermediate 2-acetyl benzimidazole which was then reacted with aromatic aldehydes and finally the product was

cyclized with hydrazine to form pyrazole derivatives of benzimidazoles. Compounds IVa,b,c,d,e have shown

significant anti bacterial and compounds IV b,g have shown significant anti cancer activity when compared with

standard drugs.

Key words: benzimidazole, pyrazole, anti cancer and antibacterial


INTRODUCTION

The benzimidazole and pyrazole nucleus are an

important structures in numerous natural and synthetic

compounds and in medicinal chemistry. Benzimidazole

derivatives are known to possess antifungal, anti-

bacterial, antiviral anthelmintic analgesic, anti-, ,

inflammatory, anti-neoplastic, depressive, hypnotic, 1-8.anti-pyretic and anti-spasmolytic, activities Thus, we

became interested in the synthesis of pyrazole-

substituted benzimidazoles by using microwave

technique and also evaluation of their biological

activities. Microwave assisted reactions have received

great interest because of their simplicity in operation,

enhanced reaction rates, products with high purity and

better yields compared to those conducted by

conventional heating.

MATERIALS AND METHODS9,10Antibacterial activity

The antibacterial activity of the synthesized compounds

was determined by cup plate method. The test organism

chosen were S.aureus (ATCC 25923), E.coli (ATCC

25922) and p.aeruginosa (ATCC 27853) and B.subtillis,.

The concentration of the sample compounds was

100mcg/ml. Gentamycin was used as a standard drug.

The results are reported in the Table no.4.

11Anticancer activity

The anticancer activity of the synthesized compounds

was determined by SRB assay method, against Human

Breast cancer cell line –MCF7. The concentration of the

sample compounds was 100mcg/ml. Doxorubicin

(Adriamycin, ADR) was used as a standard drug. This

study was carried out at TATA memorial centre

(ACTREC), Navi Mumbai and the results are reported in

the Table no.5.

EXPERIMENTAL

Melting points were determined in open capillary method

and are uncorrected. The compounds were routinely

checked by TLC on silica gel G plates for their purity. IR

Spectra (Shimadzu 8400S FTIR Spectrometer) was 1recorded using KBr disc method. H NMR spectrum was

recorded (Bruker NRC 400MHz spectrometer) using

DMSO as a solvent and mass spectra was recorded on a

Shimadzu 2010A LC-MS spectrophotometer. The

reactions were carried out in catalyst synthetic

microwave oven. The physical and spectral data of the

synthesized compounds are reported in the Table no. 2

and 3 respectively.

Synthesis of 1-(1H-Benzo [D] Imidazol-2-Yl) ethanol

(Compound-I)

1-(1H-benzo[d]imidazol-2-yl) ethanol was prepared by

the reaction of 0.1 mol (10g) of o-phenylenediamine

with 0.1 mol (9 ml) of lactic acid. The mixture was Indian Journal of Pharmaceutical Education and ResearchReceived on 21/7/2009; Modified on 11/6/2010Accepted on 23/7/2010 © APTI All rights reserved

Microwave Assisted Synthesis and Biological Evaluation of Pyrazole

Derivatives of Benzimidazoles

R. Kalirajan*, Leela Rathore, S. Jubie, B.Gowramma, S. Gomathy, S. Sankar

and K. Elango 1Department of Pharmaceutical Chemistry, JSS College of Pharmacy, Ooty - 643001, Tamilnadu

*Author for Correspondence: [email protected], [email protected]

358

refluxed in microwave oven in the intensity of 20 %( 210

Watts) for 7 min. The mixture was cooled and added with

10% NaOH until basicity to litmus paper. Then the

product was filtered, dried and recrystallized from hot

water.

Synthesis of 2-Acetyl Benzimidazole (Compound-II)

To a solution of the compound-I (10mmol) in aqueous

acetic acid (5% v/v, 10 ml) was added at room

temperature with the solution of potassium di chromate

(10mmol) in aqueous acetic acid (5% v/v,10 ml) and the

mixture was stirred for 15 min. The separated product

was filtered, washed with water and dried. The dried

product was recrystallized with ethanol.

Synthesis of Aromatic Aldehydes Derivatives of

Benzimidazole (Compounds -IIIa-h)

To a solution of Compound - II (10mmol) in aq.NaOH

(10 %, 30ml) was added with the respective aldehydes

(10mmol) at room temperature. The reaction mixture was

stirred for 30 min and the separated solid was filtered,

washed with water and the crude product was

recrystallized from ethanol.

Synthesis of pyrazole Derivatives of benzimidazoles

(Compounds IV a-h)

A mixture of compound III a-h (0.03 mol), hydrazine

(0.03 mol) were mixed in ethanolic sodium acetate

(25ml) was refluxed under microwave in different

conditions (Table No.1). The mixture was concentrated

in water bath and poured into ice cold water. The

precipitate obtained was filtered, washed and

recrystallized from ethanol.


All the synthesized final compounds were first analyzed

by performing TLC and melting point determination.

Then the synthesized compounds were subjected to 1spectral analysis such as IR, mass and HNMR Spectra to

confirm their structure. All the analytical data showed

satisfactory results.

All the synthesized compounds were screened for their

anti-bacterial activity against two Gram-Positive

bacteria such as B. Subtilis, S. aureus as well as two

Gram-negative bacteria such as P.aeruginosa, E. coli by

the cup and plate method.

Compounds (IV-a,d,e) showed significant activity

against S. aureus as well as B. Subtilis. Compounds (IV-

b,c,d) showed significant activity against E. coli and

Compound (IV-b) showed significant activity against P.

aeruginosa.

All the eight compounds were screened for their

anticancer activity against MCF7 human breast cell line

used in in-vitro methods. Only the Compounds (IV-b, g)

have significant activity when compared with standard

drug.

S.No. Compounds Intensity Power in Watts Reaction time

1 IV-a 20% 210 30 min

2 IV-b 10% 140 32 min

3 IV-c 10% 140 35 min

4 IV-d 20% 210 30 min

5 IV-e 20% 210 30 min

6 IV-f 10% 140 34 min

7 IV-g 20% 210 30 min

8 IV-h 20% 210 30 min


Table 1: Microwave conditions for synthesis of compounds IV a-h

359


CH CH(OH)COOH3NH2

NH2

O-Phenylene diamine

M.W, 20%,7min

N

NH

CH3

CH

OH

1-(1H-benzo[d]imidazol-2-yl)ethanol(I)

K Cr O2 2 7 Acetic acid

Ar-CHO

10% NaOH

N

NH

O

C CH CH

Ar(IIIa-h)

N

NH

C

O

CH3

2-Acetyl Benzimidazole(II)

R-NHNH2 CH3COONa / C H OH2 5

N

NH

NN

H

Ar

IV a-h

Compound,IVa- Ar =

Compound,IVb- Ar =

Compound,IVc- Ar =

Compound,IVd, Vid - Ar =

Compound,IVe, - Ar =

Compound,IVf, - Ar =

Compound,IVg, - Ar =

Compound,IVh, - Ar =

CI

CI

CI

CI

NO2

OCH3

OH

H CO3

HC HC

SCHEME

IVa C H N 260.11 160 63 73.83 4.65 21.52 --- --- 16 12 4

IVb C H ClN 294.74 134 71 65.20 3.76 19.01 --- 12.03 16 11 4

IVc C H ClN 294.74 134 70 65.20 3.76 19.01 --- 12.03 16 11 4

IVd C H Cl N 329.18 170 61 58.38 3.06 17.02 --- 21.53 16 10 2 4

IVe C H N O 305.29 220 59 62.95 3.63 22.94 10.48 --- 16 11 5 2

IVf C H N O 290.32 147 58 70.33 4.46 19.29 5.51 --- 16 14 4 2

IVg C H N O 306.32 207 63 66.66 4.61 18.29 10.45 ---17 14 4 2

IVh C H N 286.33 140 60 75.50 4.93 19.57 --- ---18 14 4

Compd Mol. Form. Mol. Melting Yield Elemental Analysis (found)(%)

Wt. point % C H N O Cl

Table 2: Physical data of the Synthesized compounds

360


3363(N-NH),1604(C=N),1500(C=C),906(CH=CH),

759(ArC-H)

3336(N-NH),1600(C=N),1508(C=C),837(CH=CH),

7596(ArC-H)

3367(N-NH),1608(C=N),1473(C=C),795(CH=CH),

688(ArC-H)

3354(N-NH),1604(C=N),1456(C=C),840(CH=CH),

759(ArC-H)

3329(N-NH),1602(C=N),1456(C=C,CH ),1359(ph-OH,C-O),3

956(CH=CH),759(ArC-H)

3335(N-NH), 1398(ph-OH,CO), 1475(C=C,O-CH ),3

906(CH=CH), 1604(C=N)

3388(N-NH),1604(C=N),1417(C=C), 906(CH=CH),

759(ArC-H)

3396(N-NH),1608(C=N),1521(Ar-NO ), 1456(C=C), 2

840(CH=CH), 756(ArC-H)

3.5(NH),5.0(sN-H),7.22-7.62(mArC-H),

8.05(C-H)

3.5(NH),5.0(sN-H),7.22-7.62(mArC-H),

7.73(sC-H

3.5(NH),5.0(sN-H),7.22-7.62(mArC-H),

7.98(C-H)

3.5(NH), 5.0(sN-H),

7.22-7.62(mArC-H)

3.5(NH), 3.83(CH ),3

5.0(sN-H), 7.22-7.62 (mArC-H)

3.5(NH), 3.83(sCH ),3

5.0(sN-H),5.53(OH),

7.22-7.62(mArC-H)

3.5(NH), 5.0(sN-H),

7.22-7.62(mArC-H)

3.5(NH), 5.0(sN-H),

7.22-7.62(mArC-H)

IVa

IVb

IVc

IVd

IVf

IVg

IVh

Mol.wt. 260.11

m/z =259.2

Mol.wt. 294.74

m/z = 296.7

Mol.wt. 294.74

m/z = 296.7

Mol.wt. 329.18

m/z = 328.1

Mol.wt. 290.32

m/z = 291.4

Mol.wt. 306.2

m/z = 305.4

Mol.wt. 286.33

m/z = 287.3

Mol.wt. 305.29

m/z = 306.1

Compd Mass spectra IR spectra(KBr) NMR spectra(in DMSO)

Table 3: Spectral data of the Synthesized compounds

IVa --- --- 12 15

IVb 14 13 --- ---

IVc 15 --- --- ---

IVd 12 --- 16 12

IVe --- --- 12 16

Ivf --- --- --- ---

IVg --- --- --- ---

IVh --- --- --- ---

Std 20 19 18 18

CompdZone of Inhibition (in mm)

E.coli P.aeruginosa S.Aureus B.Subtillis

Table 4: Anti bacterial activity of the Synthesized compounds

361


IVa >80 >80 26.0

IVb >80 61.6 15.3 *

IVc >80 >80 47.1

IVd >80 >80 61.8

IVe >80 >80 73.8

Ivf >80 >80 >80

IVg >80 61.8 12.2 *

IVh >80 >80 46.1

Std 66.8 25.5 <10

Compdµg/ml values for

Lc50 TGI Gi50

Table 5: Anti cancer activity of the Synthesized compounds against Human Breast cancer cell line –MCF7

* significant activity

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362

Abstract

The present study was an endeavor to evaluate anti-inflammatory and antioxidant activity of methanolic extract of

Buchanania lanzan kernel (BLK-ME). The in vivo anti-inflammatory activity was evaluated in rats by using

carrageenan-induced paw edema, as an acute model and formaldehyde induced arthritis as a chronic model,

whereas in vitro antioxidant activity was performed by 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) and reducing power

method. Quantitative estimation of total polyphenolic content of the (BLK-ME) was estimated by Folin-Ciocalteu

method. BLK-ME (200 mg/kg body wt) significantly decreased paw volume, after oral administration of BLK-ME in

carrageenan and formaldehyde injection. BLK-ME also exhibit significant antioxidant activity. Total polyphenolic

content was found to be 16.82 %± 23 mg of GAE/100. Presence of phytochemicals like triterpenoids, saponins and

tannins in the BLK-ME might contribute to the observed anti-inflammatory and antioxidant activity.

Keywords: Buchanania lanzan kernel, Antioxidant, Anti-inflammatory, Polyphenols, DPPH


Anti-inflammatory and Antioxidant Activities of Methanolic extract of Buchanania Lanzan Kernel

Warokar A. S. *, Ghante M. H., Duragkar N. J. and Bhusari K. P.Sharad Pawar College of Pharmacy, Wanadongari, Hingana road, Nagpur- 441 110.

Author for correspondence: [email protected], [email protected]

INTRODUCTION

Buchanania lanzan Spreng., family Anacardiaceae,

commonly known as Char, Chirauli. It is widely

distributed in hot and dry parts of India. In the Jhansi

district of India, the Buchanania lanzan kernel (BLK) is

used into an ointment, for skin diseases. In Berar, kernels

are pulverized and applied as a remedy for itching. It is

used to apply on glandular swellings of the neck. It is

believed to cure pimples and prickly heat. It is also

employed by women to remove spots and blemishes from

the face. In the Bombay presidency, the kernels were

employed as a tonic and being substituted for the almond.

The oil extracted from the kernels is used as a substitute

for almond oil in native medicinal preparations and 1confectionery.

Although it is a popular traditional medicine in Indian

subcontinent, literature surveys reveals that anti-

inflammatory and antioxidant activity of BLK has not yet

been documented. Its ethnobotanical claim prompted us

to undertake this investigation. Current drugs for

inflammation such as NSAIDs and opiates are not

beneficial in all cases, due to their side effects and 2potency. Hence search for other alternatives seems

necessary and beneficial. Antioxidants help to deal with

3oxidative stress which is caused by free radical damage.

Reactive oxygen species (ROS) and reactive nitrogen

species (RNS), contribute significantly to tissue injury 4and in pathogenesis of asthma, rheumatisms and burns.

Moreover, several studies suggest that natural

antioxidant and anti-inflammatory agents could be

beneficial in the prevention and treatment of these 5, 6 pathologies. This study aimed to investigate in vivo

anti-inflammatory and in vitro antioxidant potential of

methanolic extracts from BLK. Moreover, total

polyphenolic content BLK-ME was determined as per

standardized parameter. Therefore an effort has been

made to corroborate and establish scientific evidence for

its ethnobotanical uses.


Collection of plant material and extraction

The Buchanania lanzan kernels (BLK) were collected

from the Sakoli, district Bhandara The herbarium was

prepared (Voucher no. 9137) and authenticated by Prof.

Alka Chaturvedi, Department of Botany, R. T. M. Nagpur

University, Nagpur.

Kernels were cleaned and dried well under shade which

was further pulverized using Willey's mill. 500 gm of

coarse powdered BLK was extracted in soxhelet °apparatus exhaustively with petroleum ether (60- 80) C

and finally with methanol to obtain brown sticky residue.

Indian Journal of Pharmaceutical Education and ResearchReceived on 12/6/2009 ; Modified on 8/9/2009Accepted on 9/2/2010

363

Chemicals

DPPH and gallic acid were purchased from Sigma

–Aldrich, USA. Indomethacin was courteously gifted by

Zim laboratories, Nagpur. All other reagents used were of

analytical grade.

Animals

Animal study protocol was approved by institutional and

animal ethical committee, CPCSEA and Wistar rats

(150–200 g) were acclimatized to laboratory condition

for 7 days before commencement of experiment. The

animals were grouped and housed in polyacrylic cages

with not more than six animals per cage and maintained

under standard laboratory conditions of 12h light and o dark cycle with relative humidity 55±5% and at 25±2 C.

They were allowed to free access for standard dry pellet

diet (Trimurti Feeds, Nagpur) and water ad libitum. 7Phytochemical Screening

The extract was subjected to preliminary phytochemical

screening, for evaluation of major phytochemical

constituents such as alkaloids, steroids, polyphenols,

saponins, anthroquoinones, coumarins and glycosides. 8Acute oral toxicity studies

Acute oral toxicity studies were performed according to

OECD No. 423 guidelines (acute toxic class method).

Three rats of either sex (2 female and 1 male) were

selected for the study. The BLK-ME methanolic extract

(suspended with 0.5%, w/v, CMC) was administered

with higher dose 2000 mg/kg (p.o.). The rats were fasted

over-night for food with free access for water prior to test

extract. Individual rat was observed after dosing at least

once during the first 30 min, periodically during the first

24 h, with special attention given during the first 4 h and

daily thereafter, for a total of 14 days. The rats were

observed for systemic and behavioral toxicity patterns as

described in OECD/OCDE Test Guidelines. At the end of

toxicity study, all surviving animals were scarified.

Screening of anti-inflammatory activity

Wistar rats of either sex were divided into four groups,

six animals in each for carrageenan and formaldehyde

induced paw inflammation method. Group I (control;

vehicle treated), group II (BLK- ME 100 mg/kg), group

III (BLK- ME 200 mg/kg) and Group IV was

(Indomethacin 10 mg/kg). In all animals, vehicle, test

extract and standard drug was administered p.o.9, 10Carrageenan induced paw edema

In this study, vehicle, BLK-ME (100 and 200 mg/kg) and

Indomethacin was administered 1 h before the injection

of carrageenan. Inflammation was induced in all rats by

single sub plantar injection of 0.1 ml freshly prepared 1%

carrageenan in normal saline. The change in paw

thickness (mm) was measured using digital calibrated

vernier caliper (Model 2061, Mututoyo Digimatic

Caliper, Japan) at 0, 1, 2 and 3 h after carrageenan

injection. Change in paw thickness was considered as a

measure of inflammation and was calculated as %

inflammation inhibition. [Control group mean-Test grop mean]

% inflammation inhibition = ----------------------------------------- x 100

(Control group mean)11Formaldehyde induced paw oedema

Experimental arthritis was induced in rats of either sex

according to the Seyele et al. The animals groups were

same as described in above study. A subplantar injection

of 0.1 ml formaldehyde [2% (v/v)] was administered to

the right hind paw on the first and third day of the

experiment. BLK-ME (100 and 200 mg/kg),

Indomethacin 10 mg/kg and vehicle (0.5%, w/v, CMC)

was administered once daily for 10 days. The change in

paw thickness (mm) of each rat was measured for 10 days

and percent inhibition of inflammation was determined

as above.

Antioxidant Assay

In vitro models based on reactions between unstable

radicals such as DPPH and ferricyanides with plant based

antioxidants were used to evaluate antioxidant potential

of BLK-ME. Ascorbic acid was used as standard for both

methods.12DPPH assay

BLK-ME was dissolved in methanol to obtain range of

concentrations (100-1000 g/ml). To a set of test tubes,

freshly prepared DPPH solution (2.9 ml of 100 g/ml in

methanol) and 0.1 ml of different concentrations of BLK-

ME sample were added. The mixture was then mixed

well and allowed to stand in dark for 30 min and

absorbance at 517 nm was measured using Shimadzu

1170 UV–Vis spectrophotometer. A control solution

consisted of 0.1 ml of methanol only and 2.9 ml of DPPH

radical solution. Ascorbic acid was used as standard.

Percentage scavenging of DPPH by BLK-ME was

calculated by comparing the absorbance between the test


364

mixture and control Percentage scavenging of DPPH was

calculated by using formula.

% scavenging of DPPH = (A - A ) / A X 100cont test cont

13Reducing power method

The reducing power of BLK-ME was evaluated

according to the method of Oyaizu et al 1986. Various

concentrations of BLK-ME (100- 1000 µg/ ml) were

taken. 1.0 ml of test sample was mixed with 2.5 ml

phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of 1%

potassium ferricynide. The mixture was made

homogeneous and incubated at 50º C for 20 min; aliquots

of trichloroacetic acid (2.5 ml, 10%) were added to the

mixture, which was then centrifuged at 1500 rpm for 10

min (2.5 ml) and finally freshly prepared FeCl solution 3

(0.5ml, 1%) was added to this and mixed uniformly. The

absorbance of supernatant was measured at 700 nm.

Increased absorbance of the reaction mixture indicated

increased reducing power. 14Quantitative Estimation of Total Phenolic content

The total phenolic content of BLK-ME was determined

using a modified Folin-Ciocalteu method. A 125 µl of

BLK-ME of known concentrations (100-1000 µg/ml)

was added to the set test of tube and mixed with 0.5 ml of

Folin–Ciocalteu reagent. The tubes were vortexed for 5 0 min and then allowed to stand for 5 min at 20 C. To this

mixture 1.25 ml of sodium carbonate (7%) solution was

added and the mixture was diluted to 3 ml with distilled

water. Absorbance of bluish green coloured solution

developed after 60 min was measured at 760 nm using

Shimadzu 1170 UV–Vis spectrophotometer. The

measured absorbance was compared to a standard curve

of gallic acid solutions and expressed as gallic acid

equivalents (GAE) in milligrams. Triplicate

determinations were performed of each sample.

Statistical analysis

Statistical analysis was performed by using 2 way

ANOVA. Experimental data were expressed as mean

±SD. Student's t-test was applied for expressing the

significance and P value.

RESULTS

Phytochemical screening

BLK-ME showed the presence of alkaloids, triterpenods,

saponins, glycosides and tannins.

Acute toxicity study

BLK-ME did not produce any mortality at the highest

dose employed. Selected doses of BLK-ME were found

to be safe. Two doses (100 and 200 mg/kg, p.o.) of BLK-

ME were selected for further pharmacological studies.

The results obtained with BLK-ME and indomethacin in

the carrageenan- induced edema test are shown in (Table

1)

Table 2).

Among several traditional claims, the utility of

Buchanania lanzan in inflammation and pain has been 1emphasized only in literature. Hence results of present

investigations might give scientific authentication to the

traditional claims. At concentration 125 µg/ml of BLK-

ME, exhibited 83.11 and 88.34 of % scavenging DPPH

and % reducing power respectively which found to be

near to standard antioxidant ascorbic acid

ROS) and reactive nitrogen species (RNS) have been

implicated to failure in the defense mechanisms, damage

to cell structures, DNA, lipids and proteins in tissue.

Furthermore, these reactive species (ROS and RNS) have

been known to cause several inflammatory disorders

such as asthma, rheumatoid arthritis atherosclerosis, 4inflammatory bowel disease . Recent study showed that

several polyphenolic compounds act as a potent

cyclooxygenase inhibitor. Moreover polyphenolics

combat with oxidative stress in the body and maintain

balance between oxidants and antioxidants to improve 1617, 18 human health. Antioxidants have been reported to

work by single or combined mechanisms viz. free

radical-scavenging, reducing activity, complexing of

pro-oxidant, scavenging lipid peroxyl radicals and

quenching of singlet oxygen. This suggests possibility to

reduce the risks of chronic diseases and prevent disease

progression by either enhancing the body's natural

antioxidant defenses or by supplementing with proven 19dietary antiaccident . Antioxidants of natural origin such

as polyphenols (tannins, flavonoids and chalcones), act

by donating electron to the intermediate radicals formed

in oxidative stress or tissue damage which help in

inhibition of lipid peroxidation. The computational

studies also supports that the compounds having more

Anti-inflammatory study

BLK-ME extract exhibited significant inhibition of

inflammation at 200 mg/kg but not shown any significant

effect at 100 mg/kg, the results were same in case of

formaldehyde induced arthritis (

DISCUSSION

(Table-3).

Literature survey suggest that reactive oxygen species

(

,

365


electron donating potentials are better inhibitors of

hydroperoxides which suggests many of the antioxidant

agents are found to be effectively exhibit anti-20, 21inflammatory activity.

The DPPH approach is simple and widely applied for the 22measurement of antioxidant activity of polyphenolics.

Therefore total polyphenolic content and in vitro DPPH

assay for antioxidant activity were evaluated. The

carrageenan test is long accepted as a useful tool for

investigating new anti-inflammatory drugs similarly

formaldehyde-induced paw edema in rats is widely used

to screen anti-arthritic and anti-inflammatory agents as it 23, 24 closely resemble human arthritis.

BLK-ME in two different

models reduced the paw edema significantly (Tables 1

and 2). Inhibitions of the paw edema in both

inflammatory models were comparable to the standard

reference drug indomethacin . BLK-ME

showed scavenging of DPPH and ferricyanide radicals

near to that of ascorbic acid. In the free radical

scavenging assay, BLK-ME was found to interact with

the DPPH free radical and reduced it. While the reducing

power observed at different concentration of extracts was

Anti-inflammatory

activities of (200 mg/kg)

(10 mg/kg)

satisfactory. Antioxidant capacity of extract was found to

be concentration dependant (Table 3). Quantitative

estimation of total phenolic content of BLK-ME by

Folin-Ciocalteu was found to be 16.82% ± 23 mg of

GAE/100 g. Furthermore, the free radical scavenging

capacity of BLK-ME in the DPPH assay and reducing

power methods suggests that the antioxidant activity may

be one of the mechanisms of its anti-inflammatory

property, because anti-inflammatory processes are

related to an increase of free radicals. Observed results

may be due to the presence of phytochemical constituents

like polyphenols, saponins, triterpenoids, and glycosides.

Thus the results from present study indicate the efficacy

of the BLK-ME as a therapeutic agent in acute as well as

chronic inflammatory conditions and oxidative stress.

Studies are in progress in order to determine in vivo

antioxidant activity identify and isolate the bioactive

Phytoconstituents.

ACKNOWLEDGEMENT:

Authors are greatly thankful to the Principal, Sharad

Pawar College of Pharmacy, Wanadongri, Nagpur for

providing free access to their facilities to carry out

research work.

Control 2.87 ± 0.023 5.94 ± 0.012 3.99±0.020

Indomethacin (10 2.43 ± 0.029 2.29 ± 0.013 1.65±0.009

mg/kg) [15.33]* [61.44] ** [58.64] **

BLK-ME 2.89 ± 0.025 2.91 ± 0.009 1.93 ± 0.007

(100 mg/kg) [51.01] ** [51.62] **

BLK-ME 2.49 ± 0.019 2.34±0.016 [60.60] 1.56 ± 0.011

(200 mg/kg) [13.24] ** ** [60.90] **

Treatment Groups

Change in paw thickness (mm) at [h]

1 3 5

Table 1: Acute anti-inflammatory activity of BLK-ME in carrageenan induced inflammation

Values are expressed as mean ± SD, *P<0.01 and **P<0.001 values are compared with control; square brackets indicates the percent inhibition

(n=6)

Indian J.Pharm. Educ. Res. 44(3), Oct - Dec, 2010Indian J.Pharm. Educ. Res. 44(4), Oct - Dec, 2010

366

Control 3.51±0.023 3.02±0.031 2.43±0.027 3.24±0.035 2.85±0.015 2.51±0.035 2.21±0.023 1.92±0.015 1.85±0.031 1.74±0.020Indomethacin 2.45±0.015 1.87±0.024 1.55±0.018 1.88±0.035 1.47±0.025 1.42±0.022 1.35±0.029 1.27±0.007 1.12±0.029 1.07±0.012

[10 mg/kg] [22.22] * [38.07] ** [36.21] ** [41.97] ** [48.42] ** [43.42] ** [38.94] ** [33.85] ** [39.45] ** [38.50] ** BLK-ME 2.84±0.016 2.26±0.023 2.08±0.018 2.25±0.027 2.18±0.017 2.05±0.015 1.75.±0.02 2.31±0.019 2.23±0.02 1.73±0.014

(100 mg/kg) [19.08] * [25.16] ** [14.40] * [30.55] ** [23.50] ** [18.32] * [20.81] * [20.31] * [20.54] * [23.56] * BLK-ME 2.38±0.044 2.10±0.012 1.76±0.026 2.15±0.028 1.80±0.023 1.72±0.015 1.57±0.019 1.40±0.022 1.27±0.033 1.16±0.033

(200 mg/kg) [21.19] * [30.46] ** [27.57] ** [33.64] ** [36.84] ** [31.47] ** [28.95] ** [27.08] ** [31.35] ** [33.33] **

GroupsPaw thickness (mm) at days

1 2 3 4 5 6 7 8 9 10

Table 2: chronic anti-inflammatory activity of BLK-ME in formaldehyde induced inflammation

Values are expressed as mean ± SD, *P<0.001 and **P<0.001 values are compared with control; square brackets indicates the percent inhibition

25 22.97 39.28 20.17 30.9650 45.68 58.71 39.63 45.08 75 61.24 76.12 48.15 62.35

100 72.29 89.34 68.97 79.30 125 83.11 99.58 88.34 98.92

Concentration (µg/ml)

% Scavenging of DPPH % Reducing powerBLK-ME Ascorbic acid BLK-ME Ascorbic acid

Table 3: Antioxidant activity of BLK-ME in DPPH Assay and reducing power

(n=6)

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368

Abstract

Eupatorium odoratum Linn is found in the tribal area of Koraput district and extensively used traditionally by the

tribal people as anthelmintic, antimicrobial, antifungal and wound healing. The present study is an attempt to

preliminary investigation of phytochemical constituent and to explore the anthelmintic activity of different extracts of

leaves of plant Eupatorium odoratum using petroleum ether, ethanol and chloroform as solvents. The various doses of

extracts were screened for phytochemical constituent and evaluated for their anthelmintic activities on adult Indian

earthworms, Pheretima posthuma. Tests for alkaloid and tannins were positive in all extracts except tannin was

absent in petroleum ether. Tests for saponin, protein, aminoacid and anthraquinone glycoside were negative in all

extracts. All extracts were able to show anthelmintic activity at 2.5 mg/ml concentration. The activities are

comparable with the standard drugs, piperazine citrate and albendazole. All the doses of petroleum ether, ethanol and

chloroform extracts of Eupatorium odoratum showed better anthelmintic activity than the standard drug albendazole

except petroleum ether extract at 2.5 mg/ml of concentration. The extracts of three solvents at concentration of 2.5

and 5.0 mg/ml showed lesser anthelmintic activity than the standard drug piperazine citrate. When the dose of the

extract is increased, a gradual increase in anthelmintic activity was observed. The ethanolic extract showed better

anthelmintic activity in comparison with petroleum ether and chloroform extracts. The data were verified as

statistically significant by using one way ANOVA at 5 % level of significance (p < 0.05).

Key Words: Eupatorium odoratum; Asteraceae; Anthelmintic; Piperazine citrate; Albindazole.


INTRODUCTION

Helminthiasis, or worm infestation, is one of the most

prevalent disease and one of the most serious public

health problems in the world. Hundreds of millions if not

billions of human infections by helminthes exist

worldwide and with increased world travel and 1immigration from the developing countries .

Eupatorium odoratum Linn. (Asteraceae) is also

commonly called as Christmas bush (English). The plant

is mostly perennial herbs or shrubs, sometimes climbers.

Leaves opposite. There is 15 to 25 tubular florets per

head, white, lavender, or purple colour, cylindrical

glandular often hairy. It is a scrambling shrub. The seeds

are a brownish gray to black achene that is 4mm long with 2,3a pale brown pappus 5 or 6 mm long .

It is used as a traditional medicine in Indonesia. The

young leaves are crushed, and the resulting liquid can be

used to treat skin wounds. In herbal medicine, leaf

extracts with salt are used as a gargle for sore throats and

colds. Extracts of Christmas bush have been shown to

inhibit or kill Neisseriagonorrhoeae (the organism that 4causes gonorrhoea) in vitro In the southern part of .

Nigeria, the leaves are used for wound dressing, skin 5infection and to stop bleeding . The literature survey

reveals that previously work has been done on flower and

leaves in water extract for antimicrobial and anti-6,7inflamatory activity but there were no reports on the


Phytochemical investigation and evaluation of anthelmintic activity of

extract from leaves of Eupatorium odoratum linn. 1 1 1Debidani Mishra *, Deb Kumar Sarkar , Bhabani Shankar Nayak ,

1 1 2Prasant Kumar Rout , P. Ellaiah and S. Ramakrishna .1Department of Pharmacognosy, Faculty of Pharmacy, Jeypore College of Pharmacy, Rondapalli, Jeypore -

764002, Koraput, Orissa, India. 2Department of Pharmacognosy, Faculty of Pharmacy, College of Pharmaceutical Sciences, Mohuda,

Berhampur – 760002, Odisha, India.

*Author for Correspondence: [email protected],

369

anthelmintic activity of the leaves extracts of Eupatorium

odoratum. This prompted us to investigate the

anthelmintic activity of Eupatorium odoratum leaves

extracts.


Drugs and Chemicals

Albendazole (Micro Lab. Ltd., Goa), piperazine citrate

(Burroughs Wellcome Ltd., Mumbai), petroleum ether

AR (60-80C, Thomas Baker Chemical Pvt. Ltd.),

chloroform GR (Loba Chemicals), ethanol AR (Merck

Pvt. Ltd. Mumbai), normal saline water was used as

control.

Plant material

The leaves of Eupatorium odoratum Linn. (Asteraceae)

were collected from local area of Koraput district (India)

in the month of June 2008. The plant was identified and

authenticated by the Biju Pattnayak Medicinal Plants

Garden and Research Centre, Dr. M.S. Swami Nathan

Research Foundation, Jeypore, Koraput (District), Orissa

(Letter no. MR08/DBT/115, date. 12.06.2008). The

leaves were shade dried under normal environmental

condition. The dried leaves were powdered and stored in

a closed container for further use.

Preparation of Extract

The leaves of Eupatorium odoratum was extracted by

using Soxhlet extraction apparatus. In the extraction

procedure a total amount of 1550 gm powdered leaves

were extracted with each solvent. The solvents are

petroleum ether, chloroform, and ethanol. For each

solvent, 50 cycles were run. Each extract was filtered and

then it was concentrated by distilling of the solvent to

obtain the crude extract. Then it was dried by rotary

evaporator. On successive solvent extraction of leaves of

Eupatorium odoratum with different solvents resulted in

separation of constituents of different polarity range in

different solvent extracts. The percentage yield of

petroleum ether, chloroform and ethanol extract of

Eupatorium odoratum was found to be 0.555 %, 1.052 %

and 0.855 % respectively.

Phytochemical screening

Chemical tests were carried out on all the extracts for the

quali tat ive determination of phytochemical 5,8-11constituents . Total phenolic content was determined

12using Folin-Ciocalteau reagent . Total phenol value was

expressed as mg/g gallic acid equivalent.

Thin Layer Chromatography (TLC) study.

A TLC study of chloroform extract was carried out using

Silica gel GF as stationary phase. Spots were observed 254

under UV-Visible light. Chloroform crude extract of

Eupatorium odoratum was found to contain five spots in

petroleum ether: chloroform (50:50) mobile phase, four

spots in chloroform: ethyl acetate (90:10), two spots in

chloroform: ethyl acetate (80:20), three spots in

chloroform: ethyl acetate (90:10) and two spots in

methanol: ethyl acetate (5:95) respectively.

Animals

Healthy adult Indian earthworm, Pheretima posthuma

(Annelida, Megascolecidae) was used for evaluating the

anthelmintic activity due to its anatomical and

physiological resembles with the intestinal roundworm 13-15parasites of human beings . All earthworms were of

approximately equal size. They were collected from local

place, washed and kept in water.

Anthelmintic Activity

The anthelmintic activity was evaluated on adult Indian

earthworms by the reported methods with slight 16,17modification . Eighteen groups of approximately equal

sized Indian earthworms consisting of six earthworms in

each group were released in to 50 ml of respective

formulation as follows: vehicle (1% gum acacia in

normal saline), piperazine citrate (15 mg/ml),

albendazole (10 mg/ml), petroleum ether extracts (2.5, 5,

10, 25 and 50 mg/ml), chloroform (2.5, 5, 10, 25 and 50

mg/ml), ethanol 2.5, 5, 10, 25 and 50 mg/ml). The wide

range of dose was taken to establish the relationship

between dose and pharmacological activity and also to

find out the minimum and maximum dose that can be

better therapeutically effective in comparison to standard

drug. Observations were made for the time taken to

paralysis and/or death of individual worms. Paralysis was

said to occur when the worms do not revive even in

normal saline water. Death was concluded when the

worms lose their motility followed with fading away of

their body color.

Statistical analysis

The data on biological studies were reported as mean ±

Standard deviation (n = 5). For determining the statistical

significance, standard error mean and analysis of

variance (ANOVA) at 5 % level significance was 18employed. P values < 0.05 were considered significant .


370


From the TLC study profile, it is concluded that the

mixtures of bioactive fraction of ethanol extract of

Eupatorium odoratum has exhibited the anthelmintic

activity. Table 1 shows the phytochemicals detected in

Eupatorium odoratum leaf extract. Tests for alkaloid and

tannins were positive in all extracts except tannin was

absent in petroleum ether. Tests for saponin, protein,

aminoacid and anthraquinone glycoside were negative in

all extracts. Triterpenoid, sterols, steroids, flavons and

flavonoids are present in petroleum ether and chloroform

extracts but absent in ethanol extracts. Cardiac

glycosides present in ethanol extracts but absent in

petroleum ether and chloroform extracts. Gum mucilage

present in ethanol extracts but absent in petroleum ether

and chloroform extracts. The phytochemicals detected in

the extracts are acting as major role to possess medicinal

properties. Each leaves extracts containing 2.5, 5, 10, 25,

50 mg/ml, produced dose-dependent paralysis ranging

from loss of motility to loss of response to external

stimuli, which gradually progressed to death as shown in

Table 1. The Petroleum-ether extracts of dose 2.5, 5, 10

and 25 mg/ml, produced paralysis within 37.815, 24.841,

17.39, 16.788 min. respectively. Death was noted within

8.877 min. Ethanolic extract at concentration 2.5, 5, 10

and 25 mg/ml produced paralysis within 26.79, 18.83,

15.56, 13.716 min. respectively. The death was noted

with 50 mg/ml concentration within 7.42 minutes.

Chloroform extract also showed dose-dependent

paralysis at concentration of 2.5, 5, 10 and 25 mg/ml,

paralysis was noted at 27.683, 21.433, 17.65, 16.58

minutes respectively, while 50 mg/ml concentration

produced death within 8.11 minutes. The standard drug

Piperazine citrate of concentration 10 mg/ml and

Albendazole of concentration 15 mg/ml shows paralysis


at 18.50 min and 34.66 min and death occurred after

63.83 min incase of Albendazole. The higher

concentration of each extract produced paralytic effect

more quickly and the time taken to death was shorter. By

employing one-way ANOVA, all data were verified and

found to be statistically significant at 5 % level of

significant (p<0.05).

From the above results, it was observed that the ethanolic

extract was more potent than the other two extracts

(petroleum ether and chloroform) even though all the

three extracts were endowed with anthelmintic property.

The order of activity was ethanol extract greater than

chloroform extract greater than petroleum ether extract.

The activity revealed concentration dependence nature of

the different extracts. Potency of the extracts was found

to be inversely proportional to the time taken for

paralysis/death of the worms.

CONCLUSION

It could be concluded that the ethanolic extract showed

most potent anthelmintic activity. The other two extracts

e.g., petroleum ether and chloroform extracts, exhibited

lesser anthelmintic activity than the ethanolic extract.

The present study revealed that the anthelmintic activity

increases with increasing polarity. Further studies are

required to identify the actual chemical constituents that

are present in the crude extracts of this plant which are

responsible for anthelmintic activity and to establish the

effectiveness and pharmacological rationale for the use

of Eupatorium odoratum as an anthelmintic drug.

ACKNOWLEDGEMENT

Authors wish to thank to local people of Koraput and Biju

Pattnayak Medicinal Plants Garden and Research Centre,

Dr. M.S. Swami Nathan Research Foundation, Jeypore,

Koraput (Dt), Orissa, for providing valuable information

about the plant and its identification.

371


Phytochemicals Pet. Ether extract Chloroform extract Ethanol extract

Alkaloids + + +

Cardiac glycoside - - +

Anthraquinone glycosides - - -

Gums mucilage - - +

Proteins, amino acids - - -

Tanins-Phenolic compound - + +

Triterpenoid + + -

Steroids & sterols + + -

Saponin - - -

Flavones & flavonoids + + -

Table 1. Phytochemicals detected in extracts of Eupatorium odoratum.

+ = Present and - = Absent

Groups Treatments Concentration Time taken for Time taken for

used (mg/ml) paralysis (min) death (min)

(X±S.D.) (X±S.D.)

1 Vehicle - - -

2 Piperizine citrate 10 18.50 ± 0.31 -

3 Albendazole 15 34.66 ± 0.72 63.83 ± 0.79

4 Pet-ether extract 2.5 37.815 ± 0.81 -

5 Pet-ether extract 5 24.841 ± 1.85 33.08 ± 3.098




9 Ethanol extract 2.5 26.79 ± 1.33 -

10 Ethanol extract 5 18.83 ± 0.805 25.84 ± 0.776

11 Ethanol extract 10 15.56 ± 1.752 25.57 ± 0.6979

12 Ethanol extract 25

13 Ethanol extract 50 7.42 ± 0.589 14.588 ± 0.2733

14 Chloroform extract 2.5 27.683 ± 0.811 -

15 Chloroform extract 5 21.433 ± 0.854 27.783 ± 0.331




13.7160.596 19.230.855

ANOVA Data df F P-value F crit

50 10.947 0.04896 3.190727

Table 2. Anthelmintic activity of leaves extracts of Eupatorium odoratum.

Each values is represented as mean ± standard deviation (n = 5). Standard error mean < 0.492. Data are found to be significant by testing through one way ANOVA at 5 % level of significance (p < 0.05).

372

Fig 1. Anthelmintic activities of various extracts of leaves of Eupatorium odoratum on Indian Earthworm Pheretima posthuma.

Each bar is represented as mean ± standard deviation (n = 5). Group 1 – Control (Normal saline water), group 2-Standard-1 (Piperazine citrate-10 mg/ml),

group 3-Standard-2 (Albendazole-15mg/ml), group 4 to 8 – Pet-ether extract 2.5, 5,10, 25 and 50 mg/ml respectively, group 9 to 13 – Ethanol extract 2.5, 5,10, 25 and 50 mg/ml respectively,

group 14 to 18 – Chloroform extract 2.5, 5,10, 25 and 50 mg/ml respectively.

REFERENCES

1. Williams DA, Lemke TL. Parasitic Infections –

Helminthes In: Foye ’ s Principal of Medicinal

Chemistry. 5th ed. New York: Lippincott William

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ML, Pai KS, Synthesis and pharmacological

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374

Abstract

The present study was designed to study the sunscreen activity of herbal formulation. There is no evidence regarding

the sun protection factor studies on essential oil of Curry leaf oil (Murraya koenigii L. Spring., Rutaceae). This study

investigates its in vitro sun protection factor (SPF) by COLIPA method of Curry leaf oil in a cream formulation. The

sun protection factor were analysed by using Optometrics LLC, SPF- 290S is a recording ultraviolet – visible (UV-

VIS) spectrophotometer using samples exposed to Xenon arc lamp. The sun protection factor of Curry leaf oil cream

exhibited less activity (SPF= 2.04±0.02) suggesting it can be used to maintain the natural pigmentation of the skin or

can be used as an adjuent in other formulations to enhance the activity.

Key Words: Essential oil, Murraya koenigii, Curry leaf oil formulation, Sun Protection Factor, UV.

Abbreviations: UV: Ultraviolet, 290-400 nm; UV-B, 280-320 nm; UV-A, 320-400 nm; BED, Biologically Efficient

Dose; MED, Minimal Erythemal Dose; SPF: Sun Protection Factor.


INTRODUCTION

The purpose of sunscreen product is to prevent the skin

from tanning and burning by screening out the UV-A and 1UV-B radiations in sunlight .The proof of sunscreen

products efficacy is of high importance for the protection

of public health as the UV-B fraction of solar radiation is

t h e m a i n c o n t r i b u t o r t o s k i n s u n b u r n ,

immunosuppression and skin cancer. Evaluation of SPF

has been performed for a long time in vivo on human 2volunteers according to the COLIPA method . It is based

on the minimal erythemal dose (MED) related mainly to 3the biological effect of UV-B irradiation .

Murraya koenigii L. Spring. (Rutaceae) is an aromatic

pubescent shrub or small tree commonly known as curry

leaf in India. It often forms undergrowth in forests

throughout India and in Andaman Island, growing up to 4an altitude of 1500m . In traditional system of medicine,

Murraya koenigii L. Spring. is used as antiemetic,

antidiarrhoeal, in dysentery, febrifuge, blood purifier,

tonic, stomachic, flavouring agent in curries and chutney. 5-8The oil is used externally for bruises, eruption .

The essential oil from leaves of Murraya koenigii L.

9-exhibit strong antibacterial as well as antifungal activity 10. On commercial basis Curry leaf oil may find use as

4fixative for a heavy type of soap and perfume industry .

In aromatherapy, aromatherapists have used Curry leaf

essential oil against diabetes, hair loss and a means of

helping the skin maintain its natural pigmentation.

The present study deals with the determination of sun

protection factor (SPF) as cosmetic use of Murraya

koenigii L. leaf essential oil. The carbapol base cream

formulation containing isolated Curry leaf oil and its in

vitro evaluation for sun protection factor (SPF) by using

COLIPA method.

The study was designed with an objective to in vitro

determination of sun protection factor, using

Optometrics LLC, SPF- 290S, of the investigational

sunscreen cream sample. The SPF 290S is a recording

UV-VIS spectrophotometer designed and optimized for

the in vitro determination of SPF values, on a variety of

sunscreen and cosmetic products.

MATERIAL AND METHODS

2.1 Plant Material:

The plant specimens for the proposed study were

collected from in-house garden in Nashik; district

Nashik, Maharashtra, India. The specimens were

identified and authenticated by Botanical Survey of


Determination of In-vitro Sun Protection Factor (SPF) of Murraya

Koenigii L. (Rutaceae) Essential oil Formulation1 1 1 2Rekha B Patil , Shantanu Kale *, Devanshi M Badiyani and A.V. Yadav

1MGV's Pharmacy College, University of Pune, Pune, Maharashtra, India. 2 Government Pharmacy College, Karad, Shivaji University, Kolhapur, Maharashtra, India.


375

I n d i a , P u n e , M a h a r a s h t r a , I n d i a . ( R e f . thBSI/WC/TECH/2008/501 Date 15 October, 2008). The

herbarium of the plant was deposited in the BSI against

voucher no. RBMUK1 10/2008.

2.2 Isolation of essential oil:

Fresh Curry leaves (voucher specimen deposited) were

obtained from the in-house garden, were cleaned and

washed thoroughly under running water. The excess

water was drained out completely and the leaves were

dried under shade for 7-8 days. Dried leaves of M.

koenigii L. were coarsely powdered in grinder. Weighed

(550 g) were macerated in alcohoh for 7 days with

frequently shaking, distilled the solvent up to

concentrate. That concentrated mass were subjected to

hydrodistillation in a Clavenger-type apparatus for 8 h.

The distillate was extracted with Diethyl ether, the

ethereal layer was dried over anhydrous sodium sulphate

and ether distilled of on gently heated on water bath. The

yield of the oil obtained was found to be 0.95 %.

2.3 Physico-chemical properties of oil:

After isolation of oil, the physico-chemical parameters

for oil were tested for the identity of oil. Specific gravity: 0 250.8858 g/ml at 25 C, relative density n : 0.993 g/ml, D

0optical rotation: -5.3 , viscosity by Brookfield

viscometer: 13 Cp, acid number: 1.15, saponification

value: 122.8, ester value: 121.03.

2.4 Preparation of cream formulation:

Sun protection formulation is a main issue for

formulators nowadays. Considering the need of the

market, formulations are more difficult to make than for

standard care products. That is a challenge, which is

achieved by preparing a stable formulation by

considering the things: the difficulty to stabilize

components, the interaction between the ingredients,

water resistance, easy spreadibility, pleasant feeling

during and after application, stable formulation and non

irritant. Carbopol 940 has been selected the gel forming

polymer for the preparation of the semi-solid formulation

of curry leaf oil, because the rheological property of

Carbopols have extensively been studied as a function of

Concentration, pH and cross-linked density.

Cream formulation of isolated Curry leaf oil from leaves

of Murraya koenigii L. were prepared using formulae

given in (Table No. 1). For the formulation to prepare

plain gel Carbopol-940 was soaked in Water (80%). After

0that, it was homogenized and then heated to 80 C. In

another 5% quantity of Water was added Disodium

EDTA, Sodium methyl paraben and kept aside. In another

5% quantity of Water was added Triethanolamine and

kept aside. Other is oil phase, was prepared by collecting

and heating Sodium propyl paraben, Cetostearyl alcohol,

Stearic alcohol, Cetomacrogol-1000, along with Cetyl 0alcohol at 80 C. Homogenized heated Carbapol and

added Disodium EDTA, Sodium methyl paraben, in

water and quickly added oil phase and homogenized for

15 minutes. The cream was kept at room temperature.

Then Murraya koenigii L. oil was weighed (5%) and

added in the formed emulsion under constant

homogenization. The cream was once more neutralized

with Triethanolamine under constant stirring. The final

pH of the formulation was 6.0. Whole formulation was

stored in well closed amber coloured glass bottle and was

compounded fresh for all studies.

2.5 Physical parameter of cream tested:

The finished product was evaluated for its safe use, by

studying its sensory evaluation, pH, spreadibility,

specific gravity, heavy metal testing for Lead (Pb), total

aerobic microbial count and patch testing for irritancy:

etc. as per official methods and results are mentioned in

(Table No.2).

2.6 Determination of the in vitro sun protection factor:

Approximately 100 mg of the investigational sample was

applied and spread on 50 sq.cm area to obtain a sample 2film thickness of 2 l/cm on Transpore Tape® to get an

even film as suggested in the operation manual of

Optometrics LLC for the sample preparation and

application technique. The samples thus prepared were

exposed to Xenon arc lamp for determining the sun

protection factor. Scan of the sample were run from 290

nm to 400 nm. This study was performed by

Transmittance Measurement of the sample. The

Optometrics Model SPF–290 Analyzer is a computer-

controlled instrument that is designed to measure the

sunscreen protection factor of sunscreen preparations.

For US FDA standards the protection factor is calculated

over the wavelength range from 290 to 400 nm.

The SPF-290 software uses Trapezoidal Approx.

calculation technique to approximate the integral for SPF

and Erythemal UVA protection factor. These include

UVA/UVB ratio, critical wavelength, cumulative


376

absorbance, etc. The Average Absorbance method is used

for calculating average protection factor; this method

averages and computes the standard deviation based on

the absorbance scan data. This method of calculation

gives a better average value assuming that sample

thickness is the largest variable in performing a

protection factor measurement. For the calculation of

standard deviation, Diffey's method is used, is based on

B. L. Diffey's paper on using Transpore Tape® as the 11substrate for SPF measurements . Diffey's equation

applies weighting by recognizing that the MPF

(Monochromatic protection factor) measurements for a

set of scans have some distribution. Therefore, the

standard deviations of the MPF measurements at each

wavelength are factored in to the Diffey SPF standard

deviation calculation.

Win SPF has used the following equation for calculating

SPF.

RESULT

The Formulation of essential oil of Murraya koenigii L.

was studied for all parameters of cream as well as for in

vitro sun protection factor. The results of cream and sun

protection factor test summarized in (Table No. 2 and 3)

showed that cream parameters complies as per official

acceptance criteria's and SPF for Curry leaf oil cream

formulation 2.04±0.02 shows minimum sun protection

activity for sunlight and erythema.

DISCUSSION

Determination of SPF in the Curry leaf oil cream

formulation

To initiate an analysis, a reference scan (which consists of

data from the 23 wavelengths) was acquired with the

blank substrate in the incident beam. The samples were

then applied to the substrate and the first sample scan was

made. Data was collected in the same manner as the

reference data, ratioed to the reference and plotted as a

MPF. Ratioing the sample signal to the reference signal

negates any effect of wavelength dependent variables in

the optical system (source, monochromator, and

detector). Up to 6 sample scans were made to compensate

for variables in the substrate and sample application.

Three readings were taken consecutively and its average

value is shown in Table No.2.

There is no evidence regarding the sun protection factor

studies on essential oil of Murraya koenigii L. from the

literature survey it could be found that aromatherapists

have used Curry leaf oil in helping the skin maintain its

natural pigmentation. It is a discovery that Curry leaf oil

shows SPF activity.

CONCLUSION

This study has shown that Curry leaf oil cream shows the

low sun protection factor so the cream can be used in

maintaining the natural skin pigmentation or it can be

used as additives in other formulations to enhance the

activity.

ACKNOWLEDGEMENTS

The author wish to thank the Dr. Mrs. Anupama Wagale,

In charge, The Kelkar Education Trust's, Scientific

Research Center, Mulund (E), Mumbai for technical

guidance about Sun Protection Factor of Formulation.


377

Sr. No Ingredients Weight (%w/w)

1. Cetostearyl Alcohol 05

2. Stearic Acid 02

3. Cetomacrogol 02

4. Cetyl Alcohol 01

5. Carbopol 940 0.5

6. Disodium EDTA 0.02

7. Sodium Methyl Paraben 0.3

8. Sodium Propyl Paraben 0.06

9. Triethanolamine q.s. (to pH 5-6)

10. Demineralised Water Up to 100.00

11. Murraya koenigii L. Oil 05


Table No. 1. Formulation content

Sr. No. Parameters Observations

1 Colour Off-White

2 Odour Spicy

3 Spreadibility Good and uniform

4 pH 6.5

5 Specific gravity 0.97

6 Limit test for Lead Passes

7 Viscosity (26 0 C)S 251646.3 (2 rpm)

8 Total microbial count Nil

9 Patch test for irritancy No Irritation Reaction Persist

Table No.2 Physical parameters of Murraya oil Cream formulation

Test Sample Parameters Average value

Curry Leaf Oil SPF 2.04

Cream Standerd Deviation ±0.02

Critical Wavelength 38.37

Table No. 3. Result expressed as the average and S.D. of three determinations replicated of SPF values with critical wavelength.

378

REFERENCES

1. Mitsui T. New Cosmetic Science. 1st Edition;

Netherlands, Amsterdam AE, Elsevier Science BV

1000. 1997;460.

2. COLIPA - Bertil H, CTFA-SA – Jill Gardiner, JCIA

–Toshitaka Makino. International Sun Protection

Factor, (SPF) test method (COLIPA - The European

Cosmetic Toiletry and Perfumery Association;

CTFA-SA - Cosmetic, Toiletry & Fragrance

Association of South Africa; JCIA - Japan Cosmetic

Industry Association) 2006; 5.

3. Bendova H, Akram J, Krejici A, et. al., In vitro

approaches to evaluation of Sun Protection Factor.

Toxicology in vitro 2007;21:1268-75.

4. Anonymous. Wealth of India. CSIR, New Delhi:

NISCAIR press Ltd.; 2005;6:446-8.

5. Kirthikar KR and Basu BD. Indian Medicinal

Plants. 2 nd Edition. Dehra Dun: Bishen Singh

Mahendra Pal Singh; 1935;1:474-5.

6. Drury HC. The Useful Plants of India. 2 nd Edition.

London: Allen; 1978;78.

7. Peter KV. Curry leaf - A good ingredient for

vegetable preparations. Indian Farmers Digest

1978;10:13-4.

8. Prajapati ND, Purohit SS, Sharma AK and Kumar T.

A Handbook of Medicinal Plants. Jodhpur: Agro

bios, 2003;352-3.

9. Goutam MP and Purohit RM. Antimicrobial activity

of the essential oil of the leaves of Murraya koenigii

(Linn) Spreng (Indian Curry leaf). Indian J. Pharm.

1974;36(1);11–12.

10. Kishore N, Dubey NK, Tripathi RD and Singh SK.

Fungitoxic activity of leaves of some higher plants.

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11. Diffey BL, Robson J. J. Soc. Cosmet. Chem

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379

Abstract

Stimuli sensitive hydrogels are hydrophilic , three dimensional networks, which are able to imbibe large amount of

water or biological fluids and undergoes a phase transition after receiving a specific stimulus.

Ophthalmic drug delivery is often impaired by removal mechanism (blinking, tears) and by the barriers of the pre

corneal areas and further patients do not seek medical attention until the disorder is well established because of lack

of symptoms. Conventional topical treatments have measure drawbacks including poor ocular bioavailability i.e. less

than 5 % of administered active drug is absorbed or becomes available at the site of physiological activity. The

reduced therapeutic responses and the poor bioavailability exhibited by the conventional dosage forms are due to

rapid pre corneal elimination of the drug, tear turnover, lacrimal drainage and degradation by enzymes. Low

absorption results in short duration of action and high frequency of eye drop instillation is associated with discomfort

of patients. This problem was solved by using stimuli sensitive hydrogels that are instilled as drops in eye and undergo

a phase transition in cul-de-sac. These hydrogels are able to prolong the residence time of drug in pre corneal cavity

due to enhanced viscosity by stimulation of pH. The developed hydrogels was therapeutically effacious, stable, non-

irritant and In-vitro drug release for 8 hours was observed.

Keywords:Timolol maleate, Stimuli Sensitive Hydrogels , viscosity , carbopols.


associated with patient non compliance. After instillation

of eye drop, the effective tear drainage and blinking

action of eye results in a 10 times reduction in the drug 4concentration with in 4- 20 min . Due to the tear

drainage, most of the administered dose passes via

nasolacrimal duct into the Gastro Intestinal tract leading

to the side effects . Rapid elimination of the eye drops

often results in a short duration of the therapeutic effect

.The normal volume of tear in the eye is 7 µl whereas a

non blinking eye can accommodate a maximum of 30 5µl of the fluid, the usual single drop size of an instilled

eye drop is upto 50 µl and thus most of the drug instilled

as eye drop is drained away or lost.

Ophthalmic therapy can be improved by increasing the

corneal residence time of drugs. Several drug delivery 6systems are widely used such as ocular inserts, collagen

7shields etc. These systems are able to prolong the

contact time of vehicle on the ocular surface and also

slow down drug elimination. However these systems are

having some disadvantage such as poor compliance,

INTRODUCTION

Stimuli sensitive hydrogels are hydrophilic , three

dimensional networks, which are able to imbibe large

amount of water or biological fluids and undergoes a 1phase transition after receiving a specific stimulus .This

hydrogels approach can be used for the treatment of

Glaucoma in ophthalmic drug delivery. Glaucoma

comprises a group of chronic conditions that is

characterized by progressive deformation of the optic

nerve head and elevated intraocular pressure (IOP), a risk

factor. It affects primarily the middle aged and elderly, the

glaucoma currently constitute second most common 2,3cause of treatable blindness worldwide.

In the ophthalmic drug delivery systems, the

physiologicals constraints imposed by the protective

mechanism of the eye leads to the low absorption of drugs

and results in a short duration of therapeutic action.

Excessive frequency of the eye drops instillation is


In vitro and In Vivo evaluation of Stimuli Sensitive Hydrogel for

ophthalmic drug deliverya* b c d e Vinod Singh , S.S. Busheetti S Appala Raju , Rizwan Ahmad , Mamta Singh,

bLuqman College of Pharmacy, Gulbarga. Karnataka(India).a,d,eSBS PG Institute of Biomedical Sciences & Research , Dehradun, Uttrankhand.(India ).

c HKE's College of Pharmacy, Gulbarga, Karnataka (India).


380

uncomfort, especially by the elderly people and many

patients sometimes may lose or drop it without noticing

it.

This problem can be overcome by using stimuli sensitive

hydrogels prepared from the polymers that exhibit

reversible phase transition. Stimuli sensitive hydrogels

can be formulated in liquid phase suitable to be

administrated by instillation into the eye cavity and upon

exposure to the stimuli such as pH, temperature, ion

activated etc, changes to the gel phase of high viscosity

and thus improves the corneal residence time and

bioavailability of the drug .

There are various methods used to cause reversible

phase transition on ocular surface such as temp 8,9dependent concept (pluronics) , pH triggered systems

10, (including cellulose acetate hydrogen phthalate latex 11 12 13, carbopols , ion activated systems including gelrite ,

14 15gellan , carbopol /pluronics .

The purpose of the designed work was to develop a

Stimuli sensitive hydrogels which can be used for

ophthalmic therapy and was able to provide a sustained

effect and helps in reduction of frequency of

administration of dose.

In the present work, ophthalmic stimuli sensitive

hydrogels were prepared and evaluated for glaucoma

treatment.

2. MATERIALS AND METHODS:

2.1 MATERIALS & ANIMALS

Timolol maleate is provided by the FDC pvt .ltd,

Mumbai.

Carbopols 934p was provided by the Noveon polymers,

Arihant trading Co. Mumbai. Viscolozers i.e Hydroxyl

propyl methyl cellulose was made available by S.d fine

chem. ltd, Biosar. Triethanolamine. Sodium chloride was

provided by S.d fine chem. pvt. ltd. mumbai. All the

reagents were of the analytical grade. Albino rabbits of

both sexes, weighing between 1.8 kg to 2.2 kg were used

for the study.

The procedure involving animals were reviewed

approved by the animal ethics committee (No.

273/CPCSEA).

2.2 FORMULATIONS DETAILS OF HYDROGELS

Hydrogels were formulated by using Timolol maleate 16(anti glaucoma agent), benzalkonium chloride

(preservative), ethylene diamine tetraacetic acid

17(chelating agent ), sodium chloride (tonicity

contributors ) & viscolizer i.e Hydroxy propyl methyl

cellulose.

Weighed quanties (Table 1 ) of Timolol maleate,

Benzalkonium chloride, EDTA, NaCl, were dissolved in

the pH 4 phosphate buffer under aseptic conditions.

Then poly acrylic acid was slowly added with

continous stirring with digital remi stirrer at speed of

1500- 2000 rpm to minimize the formation of the lumps,

then viscolizers was added with a slow stirring to avoid

the foam formation. Stirring was continued until a clear

dispersion was formed. The prepared hydrogels were

evaluated for the viscosity study in order to identify the

composition suitable for the use.

2.3 EVALUATION OF THE HYDROGELS

2.3.1 RHEOLOGICAL STUDIES

Viscosity determination of the prepared formulation were +determined using Brookfields viscometer LVDV II . The

viscosity of the hydrogel was measured at different rpm.

The correct viscosity of the hydrogel was noted at

particular spindle at which it shows maximum percent

torque value.(Table 2).

Viscosity results indicate that at acidic pH 4 phosphate

buffer, hydrogel were less viscous and at pH 7.4

phosphate buffer (equivalent to pH of eye cavity) it

changes into a highly viscous preparation . The

Literature also suggests that the viscosity value in the 18range of 15 cps to 50 cps significantly improves the

contact time of the formulation on the corneal surface

and higher viscosity values offers no significant

advantage and have a tendency to leave a noticeable

residue on the lid margin .

2.3.2 DRUG POLYMER INTERACTION STUDIES

Drug polymer interaction studies was carried out by

Infrared spectral analysis . Infrared spectra of Timolol

maleate pure drug & formulation were scanned by

using Perkin elenmeyer FTIR 1600, by a thin film

method . The drug Timolol maleate in its infrared -1spectrum exhibited a strong peak at 3445 cm

indicating the presence of -OH group. The absorption

due to the –NH group present molecule is supported

by exhibiton of a shoulder to the main peak around -12100 cm .The drug contain more than one C=N

absorbtion present in the thiadizole moiety of the

heterocylic ring system. When pure drug was


381

formulated with the carbopol 934p & viscolizers , the

spectrum obtained for this formulated product exhibit a -1 -1 broad absorption peak from 3050 cm to 3500 cm

indicating the participation of the alkali hydroxyl in

forming gel preparation. The increased viscosity leads to

a broadening of peak . The spectral data suggest s that

the inactness of the thiadizole ring structure of the

timolol maleate indicated by the absence of the

additional peaks which confirm the opening of the

thiadizole ring is not taking place. Hence the drug

timolol maleate was not reacting with the polymers used

in the formulation .

2.3.3 INVITRO RELEASE STUDY

Invitro release rate of the timolol maleate from the

stimuli sensitive hydrogels was determined by the

diffusion process. 1 ml of the formulation was kept in

the donar compartment over a cellophane membrane

which was rinsed and soaked for the 24 hours in the

diffusion medium. The donar compartment was

immersed in the receptor compartment containing 50ml

of the phosphate buffer of pH 7.4, the beaker containing

diffusion medium (receptor compartment) was o 19 maintained at 37 C with the constant stirring at 22 rpm

using the magnetic stirrer. One ml aliquots were

withdrawn from the diffusion medium every hour for the

8 hours and same quantity of fresh, prewarmed diffusion

medium was replaced for the amount withdrawn. The

s a m p l e s w i t h d r a w n w e r e a n a l y s e d 20spectrophotometrically at 294 nm for the timolol

maleate using Shimazdu Double beam UV-Visible

spectrophotometer.

2.3.4 STERLITY TESTING

The sterlity testing of the hydrogels were performed for

the aerobic, anaerobic bacteria and fungi by using

alternative thioglycolate medium and soyabean casein

digest medium. The positive control (growth promotion),

negative control (sterlity ) test were also carried out.

Bacillus subtilis was used as a test organism in the case

of aerobic bacteria test. Bacteriodes vulgatus was used

as a test organism in case of anaerobic bacteria test &

candida albicans in fungi test .

Incubation was carried in all cases and growth was

checked. The overall results of the sterlity test showed

that ophthalmic formulation prepared passes the sterlity

test as there was no evidence of the growth found in the


negative control test tubes. Thus the hydrogels are

sterile in nature.

2.3.5 INVIVO EVALUATION

Glaucoma was induced in the rabbit by the method of 21Bonomi l et. al . In this method, six albino rabbits of both

sexes weighing between 1.8 kg to 2.2 kg were used in

the study & they were checked for normotensiveness,

care was taken to acclimatize the rabbit to the

laboratory conditions. During the experiment food and

water was provided ad libitum. The increase in the

intraocular pressure was achieved by the a

subconjuctival injection of the Betamethasone 4 mg/ml 22every weeks for the 4 weeks . The formulation X2 was

instilled in the conjuctival Cul de sac and the lowering in

the intraocular was measured by using schiotz

tonometer. Statistical evaluation was performed and it

was found significant i.e ( p < 0.05).

Comparative evaluation was also performed with

commercial available eyedrops. The marketed eyedrops

suddenly lowers the intraocular pressure to the minimum

and afterwards there was a sudden increase in the

intraocular pressure to the original reading , where as the

hydrogels lowers the intraocular pressure slowly to the

minimum and there after a gradual increase in the intra

ocular pressure . Thus a sustained effect was maintained

with this stimuli sensitive hydrogels.

2.3.6 OCULAR EYE IRRITATION

In the measurement of injury to the eye, a modification of

the scoring system of Friedenwald, Hughes and

Herrmann (Modified Draize Technique) was used.

Six albino rabbits of both sexes weighing 1.8 to 2.2 Kgs

were used for the study. The method of the study of

ocular irritation was based on a modification of the

Draize technique. 0.1 ml of selected formulation was

instilled in the conjunctival sac of each rabbit and

readings were made at 1, 24 and 48 hours after instillation

of the formulation into the eye and were evaluated on the

guidelines of scale of weighted scores for grading the

severity of ocular lesions.

CONCLUSION

In the present research work, stimuli sensitive hydrogels

was successfully formulated and evaluated for drug

release studies, infra red studies, sterlity studies and in-

vivo studies. The hydrogels provide a sustained drug

release upto 90% upto 8 hours period. Viscosity of the

382

prepared hydrogels lies in the optimum range i.e 25 cps to

50 cps.

Infra red studies shows that there was no interaction of

the thiadizole ring which shows that the drug and

polymer was not reacting together . All the hydrogels

passes the test for the sterlity as growth was not observed

in test tubes. Invivo results clearly shows that the

hydrogels provide a better sustained release of the

drug in comparison to the marketed conventional

dosage form. Thus the hydrogels are safe and provide

therapeutically effacious and provide increased

bioavaliabilty and therapeutic response.

ACKNOWLEDGEMENT

Authors are thankful to the Luqman College of

Pharmacy, and HKE's college of pharmacy, Gulbarga,

Karnataka for providing the help during this work..

Authors are also thankful to SBS Post graduate Institute

of Biomedical Sciences and Research, Balawala,

Dehradun.

Concentration ( %w/v)

Sl.no Ingredients X1 X2 X3 X4 X5 X6

1 Timolol maleate 0.25 0.25 0.25 0.25 0.25 0.25

2 Benzalkonium chloride 0.01 0.01 0.01 0.01 0.01 0.01

3 EDTA 0.1 0.1 0.1 0.1 0.1 0.1

4 NaCl 0.9 0.9 0.9 0.9 0.9 0.9

5 Poly acrylic acid - C 934p 0.3 0.3 0.3 0.35 0.35 0.35

6 HPMC - 0.4 0.5 - 0.4 0.5

7 pH 4 buffer 150 ml 150 ml 150 ml 150 ml 150 ml 150ml

Table 1: Formulation details


pH X1 X2 X3 X4 X5 X6

4.0 3.84 19.2 30.7 4.02 19.7 33.1

7.4 6.72 34.3 53.9 7.50 41.6 68.4

Viscosity(cps)

Table 2 .Viscosity details of the formulations at pH 4.0 and pH 7.4

Time Log Time Sq. Root Cum % Drug Released Log cum % Drug Released

(Hrs) Time X1 X2 X3 X4 X5 X6 X1 X2 X3 X4 X5 X6

1 0.0000 1.00 35.1 32.7 30.3 35.8 32.6 32.9 1.54 1.51 1.48 1.55 1.51 1.51

2 0.3010 1.4142 54.6 51.1 48.6 50.6 49.3 48.5 1.73 1.73 1.68 1.70 1.69 1.68

3 0.4471 1.7320 68.0 61.1 65.0 62.8 61.5 60.3 1.83 1.83 1.81 1.79 1.78 1.78

4 0.6020 2.0 74.6 67.5 68.0 67.0 66.2 68.2 1.87 1.87 1.83 1.82 1.82 1.83

5 0.6989 2.236 76.8 73.6 74.6 74.0 72.8 74.2 1.88 1.88 1.87 1.86 1.86 1.87

6 0.7781 2.4494 80.56 79.0 79.7 80.3 76.2 78.6 1.90 1.90 1.90 1.90 1.88 1.89

7 0.8450 2.6457 86.81 80.8 82.9 86.8 81.5 80.8 1.93 1.93 1.91 1.93 1.91 1.90

8 0.9030 2.8284 85.31 83.7 84.8 83.7 82.5 81.8 1.93 1.93 1.92 1.922 1.91 1.91

Table 3. Invitro drug release studies of prepared hydrogels

383

0 30 60 90 120 150 180 210 240 270 300R 18.5 18.5 18.5 18.5 18.5 18.5 18.5 18.5 18.5 18.5 18.5L 23.8 23.8 21.9 17.0 15.6 17.0 21.9 22.8 23.8 23.8 23.8 R 18.0 18.0 18.0 18.0 18.0 18.0 18.0 18.0 18.0 18.0 18.0L 21.9 21.9 1 4.6 17.3 21.9 21.9 21.9 21.9 21.9 21.9 21.9

Time (Min)

Hydrogel

Marketed

Table 4. Intra-ocular Pressure (mmHg) measurement in rabbit eye

Figure 1. Viscosity details of hydrogels at different pH . Figure 2. In-vivo study

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385

Abstract

Total quality management (TQM) is a management strategy which is aimed to embed quality awareness in all the

organizational processes. It is undisputed that TQM is necessary in pedagogy, however, how to go about achieving

this is a larger question. In pedagogy, TQM involves improvement of teaching quality and learning processes. The

paper describes the methods to achieve teaching quality, evaluation of teaching quality by peer-reviewing, student

feedback and evaluation of learning process. The authors propose a new concept of 'teacher-accreditation', which

may be more important over the other accreditations. A scheme is suggested for such accreditation, which may be

refined to make it robust, accurate and acceptable to all parties.

Key Words: Total quality management, pedagogy, professional education, teacher accreditation


INTRODUCTION

In the recorded history of human civilization, never

before was there so much of scientific and technological

advancements and innovations, all over the world. Vast

knowledge means that it can go out of the grip of mankind

if not absorbed properly with an effective command.

There have not been sufficient efforts on improving the

human capacity for absorbing and utilizing knowledge

effectively. Hence, it is necessary to develop advanced

methods, which can systematize the teaching and

learning process in a dynamic manner, by identifying

bottlenecks and analyzing the reasons for bottlenecks.

Essentially, this needs the integration of scientific

analyses and methods into the art of teaching and

learning. Such an attempt to increase the knowledge

absorption through improvement of teaching and

learning process is the basis for total quality management

in pedagogy (TQM_P).

Total Quality Management (TQM) is a management

strategy aimed at embedding awareness of quality in all 1organizational processes . In any organization, there are

workers, investors, producers, distributors, sellers and

customers, which forms a kind of supply chain. Here,

they are conceived as servers and customers. However,

when TQM philosophy is applied, all participants

become servers and customers at the same time.

The awareness of total quality management (TQM) in

professional and higher education started in early 1980s

and gained a quick popularity. TQM could serve as a

paradigm for improving every aspect of collegiate

functioning from fiscal administration to classroom

instruction. Thus, when TQM is applied to pedagogy, the

governing body, the administrative departments, the

heads of departments, the teaching force and students –

all of them become important parts of the teaching-

learning process, with 'same overall objectives'. It would

be necessary to translate individual objectives to be in

harmony with the overall objectives of the system.

ROLE OF TQM IN PROFESSIONAL AND

HIGHER EDUCATION

Deming (1994) linked the quality management principles 2and education . He had the view that 'improvement of

education and management of education require the

application of the same principles that must be used for

the improvement of any process, manufacturing or

service'. Traditionally, teaching and learning are

considered as arts. But, they can also be subjected to

scientific scrutiny (without causing any harm to the art-

sense), which would require appropriate quantifications

of important qualities of the art and this quantification Indian Journal of Pharmaceutical Education and ResearchReceived on 15/04/2009; Modified on 29/09/2009Accepted on 13/04/2010 © APTI All rights reserved

Total Quality Management in Pedagogy (TQM_P): An Update1 2Ram Chakka and G.T. Kulkarni *

1Department of Information Technology, Meerut Institute of Engineering and

Technology, NH-58 Bypass, Baghpat Crossing, Meerut 250 005 (UP)2Laureate Institute of Pharmacy, Kathog 177 101 (Teh Dehra, Dist. Kangra, HP)


386

forms the basis for TQM_P.

Many academic programs have tried applying quality

principles in their work. Many quality based models have

been described, which include models for classroom

instructions, department program planning and

administration. Even after more than a decade of such

efforts, TQM has not yet established itself as 'the way'

educational institutions operate. However, there have

been excellent results, particularly in the west, even with 2partial application of the TQM principles .

Total quality management in pedagogy is directly related

to teaching and learning quality. Hence, basic step

towards TQM is to improve the effectiveness of 3teaching . This can be done by adopting teaching

strategies which have been validated by extensive

research. Improvement in teaching requires

identification of problems in the existing academic

practices, followed by utilization of a combination of

sound educational and psychological principles, along

with feedback, to devise better approaches dynamically.

This would, in turn, lead to improvement of institutional

teaching programs, which is a major step towards

achievement of total quality.

Achievement of Teaching Quality through Action

Research

A recent advancement in assurance of education quality 3is Action Research . Action research is a type of applied

experimental research, which is conducted by the teacher

himself / herself for improving his / her own action

(teaching) strategy. It aims at the development of

alternative teaching strategies and changes in the

classroom system that improves the learning skills of the

students. The nature of action research involves aspects

such as - felt need, quick and correct feedback, immediate

result, improvement in levels of knowledge and skill,

experimental approach, localized approach, and 3immediate applicability .

In formulating action research projects, the teachers may

identify a problem, discuss the problem with peers to

sharpen it, formulate and test the hypotheses and draw

conclusions and apply the findings. The problems may be

identified by an individual or a group. The effectiveness

can be observed immediately through improvement in

students' learning and 'feel'.

EVALUATION OF TEACHING PROCESS

Traditionally, teaching is considered as knowledge

transmission. Hence, in the traditional teacher-centered

concept of teaching, only the quality of the knowledge

that the teacher transmits and the quality of the mode of

transmission of knowledge to the students are

considered. Once the focus is shifted from the teacher to

the learner, the above concept gets a radical shift, which

results in measurement of the quality of the learning and

enhanced learning capability, resulting from teaching.

The quality of teaching can be assessed on the basis of

basic qualities of a teacher, which include competence of

the teacher in imparting knowledge, teacher's ability to

facilitate independent thinking and life-long learning,

growth and innovativeness as a teacher and educationist.

Further, inspiring and attracting the students by making

the process of learning an enjoyable experience, can also

be added as another benchmark.

Student Ratings of Teaching: A Tool for Evaluation of

Teaching Quality

Student rating is the most popular method, which gives a 4valid assessment of teaching quality . Student

evaluations have higher levels of reliability, and should

always be a part of the TQM process in education. But, it

cannot be the sole source of teaching assessment, since

students cannot evaluate some of the aspects of course

such as whether course learning objectives are

appropriate, content is current with the state of the art in

the field, and the particular course prepares the students

for subsequent courses in the curriculum. Such aspects

should be evaluated by knowledgeable peers. Hence,

student rating can only be a supplement to the peer 4,5rating .

How to Make the Student Ratings Effective?

The following recommendations from different

educationists need to be considered to improve the 6effectiveness of the student ratings :

1. The rating form should be developed with the

assistance of a person who is knowledgeable in

educational quality.

2. The ratings should be collected from a large number

of students, which should not be less than 2/3 of a class.

3. Decisions should not be made on the basis of ratings

from a single semester. There should be seeming

consistency in the ratings obtained in at least three

semesters.


387

4. The rating form should be designed so that the

evaluation is affected, only slightly, by factors other than

quality of teaching, such as class size, gender of

instructor, nature of course, etc.

5. Before taking the evaluation forms, students have to

be informed that their ratings will be considered carefully

and may have an impact on teaching assignments. By

this, students will consider the rating seriously.

In many cases, modifications of the criterion for better

evaluation of teaching quality can be done. Some of such

modifications include the following:

1. In certain cases, 'normalization' factors may also be

considered in order to achieve uniformity among

different subjects. That is because some subjects may be

'highly scoring' compared other subjects.

2. Steps may be taken to eliminate certain possible

'outliers' from the students' evaluation results of the

teacher. 6,7LEARNING PROCESS

Traditionally, the learning objectives have been confined

to learning the subject material sufficient enough to get

good marks, complete the program of study successfully,

and to get a good job. In a student-centered program

where TQM principles are applied, different techniques

to improve the learning process are to be applied. Some of

them are given below:1Active Learning : It is the course-related stuff that

students do besides listening to a lecture. They may write,

reflect, discuss, solve problems, assignments, prepare for

tests, etc. They may do it individually or together. As long

as it is something other than watching and listening to the

teaching, it is all active learning.1,2Cooperative Learning : It is a more formal kind of

activity where students work in 'teams' that stay together

for extended periods of time. This involves the following

five criteria:

1. Positive Interdependence: The team members have

to count on one another to do what they are supposed to

do, otherwise everyone loses.

2. Individual Accountability: This means everyone is

held responsible for understanding both their part of the

work and everyone else's parts.

3. Face-to-face Promotive Interaction: In cooperative

learning, although some of the group work may be done


individually, some must be done interactively, with team

members providing mutual feedback and guidance,

challenging one another, and working towards

consensus.

4. Development of Interpersonal Skills: These are

needed to work effectively in teams, which include the

knowledge of conflict-resolution, communication,

leadership, time management, and so forth.

5. Regular Self-assessment of Group Functioning:

Periodically, teams have to stand back from what they are

doing and ask themselves, 'Well, what are we doing as a

team? Let us list them. What could we be doing better?

What are we going to do differently next time?'

The extent to which a group work has all these five

elements in place, is the extent to which it qualifies as

'cooperative learning'. In a single phrase, cooperative

learning develops the team-spirit of an individual.

Inductive Teaching and Learning: It is the technique in

which students are first presented with challenges

(questions or problems) and learn the course material in

the context of addressing the challenges. Inductive

methods include inquiry-based learning, case-based

instruction, problem-based learning, project-based

learning, discovery learning, and just-in-time teaching.

The best possible learning experience can be offered to

the students not only through participation in lectures,

seminars, tutorials and other formal teaching

arrangements, but also by using library and computer

facilities to maximum advantage by implementing active

and cooperative learning.

Most of the world's great achievements are the successes

of teams and team spirit. Unfortunately, sufficient

emphasis has not been given to the development of team

spirit. Most often, students are tested in these aspects

without even giving formal training. For example, a

student faces a group discussion as part of the

examination procedure required for getting a job. Most of

the times, the students are absolutely not trained in the

basic formalities that must be observed in these group

discussions. The net result is that some one who has been

acquainted with these formalities would get the job and

not the one who is best equipped to do the technical work

concerning the appointment. Hence, as a part of the

process to apply TQM principles, the above mentioned

learning techniques must be applied to prepare the

388

students for team work, which in turn would give the

individual many folds of productivity along with the

qualities such as self-esteem, joy of learning and joy of

working.

WHERE ARE WE IN ALL THIS?

In developed countries, the concept of quality

management entered education system only very

recently, that is, during 1980s. Almost at the same time,

UGC, an Indian statutory body for higher education,

started efforts about improvement of quality of higher

education in the country. After inception of AICTE,

efforts began to improve the technical higher education in

the country. Since then, many efforts have been made by

these two bodies, and some of them proved to be fruitful,

whereas, a lot of areas still need improvement. Recently,

Knowledge Commission has suggested many measures

for the improvement of quality of education.

Another important development that has taken place in

the education system is introduction of accreditation

process. UGC-NAAC and AICTE-NBA are the bodies

that accredit institutions based on their academic quality.

Accreditation process helps in improving the basic

structure of education, introduce the suggested policy

measures in practice and motivate the teachers as well as

educational planners to install the right level of

educational excellence. But, in reality, the situation of

higher and professional education in India is still grim.

The overall improvement in the educational quality has

remained a dream yet, and the education institutions of

the country have to transform this dream into reality.

WHERE DO WE GO? HOW BEST TO GO ABOUT?

TQM_P should not get confined to just a few traditional

methods of teaching and learning, but should involve all

the influencing factors. Data analyses software can be

utilized in pedagogy, for the analysis of the influence of

various external, internal and dynamically changing

factors, on the quality of the teaching and learning

process.

Traditional methods of quality assessment are designed

for the accreditation of the overall teaching process of a

program, which do not cover all components in a

systematic way. TQM_P should involve quality

assessment of all the important and involved

components. Some of them include evaluation of the

teacher, evaluation of the teaching process, teaching

facilities, what is being taught (syllabus, curriculum, etc.)

and quality of the end product (e.g, job-worthiness). Each

of these parameters is complex and has its own

influencing factors. The evaluation should be

accomplished at affordable and minimized cost, should

be dependable and acceptable to all.

Teacher Accreditation: A New Concept towards

TQM_P

The authors are of the opinion that Accreditation of

Teacher is a very important component and should be

introduced in TQM_P. Among the different

accreditations such as accreditation of an institution, a

program of study, teacher, etc., the most dependable and

robust method is the accreditation of teacher. That is

because, a good institution may turn bad if badly

managed; a good program of study may become obsolete

if not updated in pace with changing academics and

industry. However, the probability of a consistently good

teacher turning into a bad teacher is much less probable.

In this context, it is important to induct “accreditation of

teachers” program by the technical education ministry

and concerned governing bodies. In the next paragraphs,

the authors propose a skeleton of one such scheme.

How should one go on evaluating a teacher? The common

methods include peer-reviews and student-evaluations.

The conditions under which these reviews and

evaluations are done, and hence their effectiveness are

indeed somewhat disputable. For example, this kind of

evaluation strategy has given rise to teachers giving more

marks to students just in order to receive back good

ratings. Hence, the methods used for teacher

accreditation should be accurate, easy and cost-effective,

easy to standardize, easy to improve and should be

enjoyed by all the participants. In a country like India,

standard classrooms may first be conceived. This can, for

example, be accomplished in the following way. In case

of technical and pharmacy education, there are perhaps

over 5000 colleges in India, including top-level

institutions like IISc, IITs, NITs, NIPERs, and high

quality private institutions like Birla Institute of

Technology and Science (BITS), Manipal University and

Dhirubhai Ambani Institute of Information and

Communication Technology (DA-IICT). All these

institutions may be divided, say, into 10 levels of merit.

The factors for merit-evaluation can be the percentage of


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Indian J.Pharm. Educ. Res. 44(4), Oct-Dec, 2010

students obtaining reasonably good jobs or going for

higher education at high quality institutions, after

completion of their undergraduate course. The factors for

merit-evaluation need not be static; can be dynamically

upgraded utilizing proper feedback. In order to evaluate a

given teacher, one classroom from each level should be

chosen by the random number generator. The teacher

under evaluation would go to each of these 10 classrooms

and teach the students for a substantial time (say 2 or 3

sessions of lecturing, each for an hour) and gets evaluated

by those students. The results (in the form of peer-reviews

and these student-evaluations) obtained by this teacher

would demonstrate his technical skills as well as the

presentation skills, not just one level but at all levels of

standards. These results are obviously 'unbiased' since

the students and peer-reviewers are previously not

connected with the teacher being evaluated. The result is

essentially a vector quantity. If the evaluations obtained

by the teacher are not good enough, one or two more

chances (with different random selections of the

classrooms) may be given, preceded by some special

training if needed. Only upon failing all the evaluations,

the concerned teacher may be asked to look for another

profession. Those who do exceptionally may become part

of a standard pool of Exceptionally Good Teachers of the

Nation.

The results of teacher accreditation would be very useful

for the institutes during recruitment. The rating of an

institution largely depends on the quality levels of the

teachers – this fact must get registered deeply in the

minds of the governing bodies.

CONCLUSIONS

TQM_P systematizes the teaching-learning process in a

dynamic manner. In order to apply TQM, the goals have

to be defined clearly, responsibilities are to be identified

at all levels and all these must be updated dynamically

using the feedback information and according to the

changing conditions and scenarios. Emphasis is needed

towards a student-centered teaching-learning process,

which has repeatedly been shown to be superior to the

traditional teacher-centered approach. A new concept of

teacher-accreditation, suggested by the authors, may also

be given emphasis, which might be more important over

many other accreditations. If all these principles are

applied, education scenario in India would change

dramatically within a short span of time. Hence, it is

strongly recommended to consider these ideas.

REFERENCES

1. Felder RM, Brent R. How to improve teaching

quality?. Quality Management Journal. 1999; 6(2):

9-21.

2. Deming WE. The new economics. 2nd ed.

Cambridge: MIT Center for Advanced Engineering

Studies; 1997.

3. Shabaraya RA, Kulkarni GT, Gowthamarajan K.

Effective Teaching Through Action Research.

Proceedings of the 7th APTI Convention; 2002 Sep

20-22; Panaji, India. Bangalore; APTI; 2002.

4. Kaur K, Singh S, Kaur M. Evaluation of teachers by

students. University News. 2001; 39(15): 5-8.

5. Mathew G. Teachers ’ attitude towards evaluation of

teachers by students. University News. 2001;

39(16): 1-9.

6. Fedler RM, Brent R. Student ratings of teaching –

myths, facts, and good practices. Chemical

Engineering Education. 2008; 42(1): 33-4.

7. Prince MJ, Felder RM, Brent R. Does faculty

research improve undergraduate teaching? an

analysis of existing and potential synergies. Journal

of Engineering Education. 2007; 96(4): 283-94.

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