FORMULATION AND EVALUATION OF MUPIROCIN GEL AGAINST ...

www.wjpps.com Vol 8, Issue 11, 2019.

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Sweety et al. World Journal of Pharmacy and Pharmaceutical Sciences

FORMULATION AND EVALUATION OF MUPIROCIN GEL AGAINST

METHICILLIN RESISTANT STAPHYLOCOCCUS IN BURN WOUND

INFECTION

Sudhir, Navneet Kaur and Sweety Birla*

*Department of Pharmaceutics Swami Vivekanand College of Pharmacy, Banur.

ABSTRACT

The aim of present work is to develop and evaluate mupirocin gel

against methicillin resistant staphylococcus in burn wound infection.

Gel were prepared with the help carbopol gel base which was produced

by hot method. Mupirocin were incorporated into carbopol gel base by

magnetic sterring. Mupirocin gel shows pH 7.2. The viscosity of

mupirocin gel was found to be 73,200 cp. The spreadability of

mupirocin was found to be 12.66 g.cm2 /sec. The % yield of mupirocin

gel was found to be 95.68%. In vitro release study show 97.4% release

of drug, stability is good with effective antibacterial activity. It was

observed that the first order model was found to be best suited with R2

values of 0.9826. First order model be used to describe the drug dissolution for, several types

of modified release pharmaceutical dosage forms.

KEYWORDS: Mupirocin, carbopol gel, antibacterial activity, staphylococcus aureus.

INTRODUCTION

Over the last decades the treatment of illness has been accomplished by various drug delivery

systems like liposomes, proliposomes, microspheres, gels, prodrugs, cyclodextrins, for

administrating drugs to human body. via. Various routes. But patient compliance is more in

the oral drug delivery system due to easy administration of formulation.

The goal of any drug therapy is to provide a therapeutic concentration of the drug to desired

site in the body that elicits the desired pharmacological action and to minimize the incidences

of unwanted adverse effects.

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 7.632

Volume 8, Issue 11, 939-956 Research Article ISSN 2278 – 4357

Article Received on

24 August 2019,

Revised on 14 Sept. 2019,

Accepted on 04 Oct. 2019,

DOI: 10.20959/wjpps201911-14931

*Corresponding Author

Sweety Birla

Department of

Pharmaceutics Swami

Vivekanand College of

Pharmacy, Banur.


940


Topical drug delivery can be defined as the application of a drug containing formulation to

the skin to treat cutaneous disorders (e.g. psoriasis, acne) with the intent of containing the

pharmacological or another effect of the drug to the surface of the skin or with in the skin.

The topical drug delivery system is used where other systems of drug administration fails to

deliver the drug on target site. Topical preparations provide localized effect at the site of

application by virtue of drug penetration into the underlying layers of skin.

Advantage of topical drug delivery system

Convenient and easy.

Improve patient compliance.

Provide suitability for self-medication.

Avoidance of first pass metabolism.

Avoidance of the risk and inconveniences of intravenous therapy.

Avoids fluctuation in drug levels, inter and intra patient variations.

Ability to easily terminate the medications, when needed.

A relatively large area of application in comparison with buccal or nasal cavity.

Ability to deliver drug more selectively to a specific site.

Avoidance of gastro-intestinal incompatibility.

Providing utilization of drugs with short biological half-life and narrow therapeutic

window.

Burns

Approximately a one fourth of a million people in the India every year experience an injury

due to a burn. Of these, about 175,000 will visit an accident and emergency department and

about 13,000 are admitted to hospital. One in thirteen of these will have severe burns and

have the chances of survival from a severe burn have improved steadily over the last 25years.

The injuries from a burn may be multiple. In addition to physical problems such as shock,

pain, infection and multi-organ failure, there are psychological issues such as post-traumatic

stress disorder and dealing with disfigurement. Additionally there are often social problems

such as rehousing after a house-fire.

Risk factors for infection

A patient with burns is at high risk of infection primarily because the burn wound provides a

route of entry into the body but also because of the patient immuno compromised state,


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mechanical ventilation, prolonged time in hospital, gastrointestinal translocation, invasive

procedures and urinary catheterisation. The risk of developing burn wound infection depends

on both patient factors such age and size of burn, and microbial factors such as the type and

density of microorganisms invading the wound.

MATERIALS AND METHODS

Materials: The following chemicals were used: Mupirocin (Belcom, India), Methanol

(LOBA CHEMIE, India), Ethanol (LOBA CHEMIE, India), Carbopol 940 (Lubrizol

advanced material, Belgium), Methyl paraben (Lubrizol advanced material, Belgium), Propyl

paraben (Lubrizol advanced material, Belgium), Triethanolamine (Nice chemicals, India).

METHODS

1. Method of preparation of formulation 1.1Preparation of the carbopol gel

Carbopol 940 forms very good consistency transparent gel at low concentration. 2% carbopol

gel base was prepared by dispersing 2g carbopol 940 in 86ml hot distilled water. 0.6g of

propyl paraben was dissolved in ethanol. 0.3g of methyl paraben was dissolved in 10ml of

propylene glycol. The mixture was stirred until thickening occurred and then neutralized by

the drop wise addition of 50% (w/w) triethanolamine to maintain pH 6-7.

The drug was slowly added in carbopol 940gel base and mixed by using a mechanical stirrer

for 5min.

Determination of melting point: Small quantity of drug was placed into a sealed capillary

tube. The tube was placed in the melting point apparatus. The temperature in the apparatus

was gradually increased and the temperature at which entire drug gets melted was noted.

Solubility studies: The solubility study of Mupirocin was performed in ethanol, acetone,

chloroform, 0.1N HCL, separately by keeping the drug containing test tube on vortex

mixture.

Determination of partition co-efficient

The known quantity of mupirocin was added into 20ml of octanol and it was mixed with

20ml of phosphate buffer pH 7.4 in a separating funnel. Then two phases were allowed to

equilibrate at 370C for 2 hours with intermittent shaking. The concentration of drug in the

aqueous phase and organic phase was determined by UV spectroscopic method at λmax

220nm after necessary dilution. The apparent partition coefficient was calculated as the ratio


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of drug concentration in each phase by the following equation:

Kp: C organic

C aqueous

C organic us concentration of drug in organic phase C aqueous is concentration of drug in

aqueous phase.

Preparation of standard curve in methanol: Accurately weighed 100mg of mupirocin and

was dissolved in 100ml of methanol, from this stock solution 10ml was withdrawn and

transferred into 100ml volumetric flask. Volume was made with methanol in order to get

standard stock solution containing 100μg/ml.

Standard graph of mupirocin

Form this standard stock solution, a series of dilution were prepared using methanol. The

absorbance of these solutions was measured spectrophotometrically against blank of

methanol at 220nm for mupirocin. Absorbance of drug at different concentrations was

calculated and graph was plotted.

Infrared spectroscopic analysis

The FTIR spectrums of moisture free samples of mupirocin, carbopol, methyl paraben,

propyl paraben and mixture of mupirocin, carbopol, methyl paraben, propyl paraben were

recorded on IR spectrophotometer. The scanning range varies from 4000 – 400 cm-1 and

resolution was 1 cm-1.

1. Physical evaluation of mupirocin gel

The mupirocin gel formulation of was evaluated for organoleptic characteristics,

occlusiveness and washability.

Measurement of pH of the mupirocin gel

1g mupirocin gel was mixed in 100ml distilled water with homogenizer. Then the electrode

was immersed in the prepared gel solution and readings were recorded from digital pH meter

in triplicate and average value was calculated.

Viscosity study

Viscosity measurements were done on Brookfield viscometer by selecting suitable spindle

number and rpm. 50g of preparation was kept in 50ml beaker which was set till spindle

groove was dipped and rpm was set and dial reading was measured after three minutes. From


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the reading obtained, viscosity was calculated by using factor. The procedure was repeated

three times and observations are recorded as mean.

Spreadability

It is the term expressed to denote the extent of area to which gel readily spreads on

application to skin or affected part. The therapeutic efficacy of a formulation also depends

upon its spreading value. Spreadability is expressed in terms of time in seconds taken by two

slides to slip off from gel and placed in between the slides under the direction of certain load.

Lesser the time taken for separation of two slides, better the spreadability. It is calculated by

using the formula:

S: M. L / T

Where M: wt. tied to upper slide L: length of glass slides

T: time taken to separate the slides

0.1g of mupirocin gel was pressed between two slides (divided into squares of 5mm sides)

and left for about 5minutes where no more spreading was expected. Diameters of spreaded

circles were measured in cm and were taken as comparative values for spreadability. The

standardized weight tied on the upper slide was 125g. The results obtained are average of

three determinations.

Extrudability study

The extrudability of mupirocin gel was determined by filling mupirocin gel in the collapsible

tubes. The extrudability of the mupirocin gel determined in terms of weight in grams required

to extrude a 0.5cm ribbon of gel in 10second.

Entrapment Efficiency

The empty container was weighed in which the gel formulation was stored then again the

container was weighed gel formulation. Then subtracted the empty container weighed with

the container with gel formulation then it gives the practical yield. Then the Entrapment

Efficiency was calculated by the formula.

Entrapment Efficiency: Practical yield x 100 Theoretical yield.

Homogeneity and grittiness

A small quantity of mupirocin gel was pressed between the thumb and the index finger. The

consistency of the mupirocin gel is noticed (whether homogeneous or not), if there is any


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coarse particles appeared on fingers. Also, the homogeneity can be detected when a small

quantity of the mupirocin gel is rubbed on the skin of the back of the hand. The grittiness of

prepared mupirocin gel is also observed in the same manner.

3 In vitro release studies 3.1Skin permeation studies

Franz diffusion cell was used for permeation studies. Study was conducted using a rat skin.

200ml of PBS 7.4 was taken in receptor compartment and was continuously stirred with a

magnetic stirrer and equilibrated at with a recirculating water bath. The prepared rat skin was

mounted facing stratum corneum upward into the donor compartment. 1g of mupirocin gel

formulation was taken in donor compartment and covered with parafilm to avoid any

evaporation process. 5ml sample was withdrawn through the sampling port at predetermined

intervals over 8 hours and each sample is replaced with equal volume of fresh dissolution

medium. Then the samples are analyzed for drug content by using phosphate buffer as blank

with UV-Visible double beam spectrophotometer at 220nm. Similar study was performed

with marketed mupirocin gel.

Franz diffusion cell

In vitro absorption studies are generally carried out in vertical Franz diffusion cell. According

to Food and Drug Administration (FDA) regulations, it is an ideal tool for quality control of

topical preparations. It has a receptor and a donor chamber, which is filled with phosphate

sandwiched between the two chambers and clamped in place tightly.

The donor chamber is filled with a known volume of and the permeation of solute through the

membrane is monitored by periodic sampling of the solution from the receptor chamber. The

jacketed cell embodied is stirred throughout the study at 500rpm employing a magnetic

stirrer.

Drug release kinetics

The release kinetic was studied by various kinetic models as zero order plot, first order plot,

higuchi plot and korsmeyer-peppas plot. To study the release kinetics of the mupirocin gel

data obtained from in-vitro drug release studies were plotted in various kinetic models: zero

order as cumulative amount of drug releases vs. time, First order as long cumulative % of

drug remaining vs. time, higuchi model as cumulative % of drug released vs. square root of

time and korsmeyer-peppas model as log cumulative % drug release Vs. long time. The best

fit model was confirmed by the value of correlation coefficient near to 1.


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Stability studies

The Optimized formulation was stored at 40ºC/75% relative humidity (RH) in closed glass

vials for 6weeks. Gel was analyzed at specified time intervals (0, 2, 4, 6 weeks) for the %

drug content and in vitro study.

Antimicrobial activity studies

Antimicrobial activity has been assayed against two different of bacteria (one gram-positive

and one gram-negative) by agar diffusion method. Generally, the antibiotics activity of a

compound is expressed in terms of its ability to inhibit the growth of bacteria in nutrient broth

or agar. The bacterial inhibition can be measured by Cup-plate method. In this method, cups

or discs of standard diameter are made in the nutrient agar medium, containing standard

bacterial inoculums. The test compounds are introduced into the disc and the diameter of the

zone of inhibition was measured. All the test compounds were evaluated for antibiotics

activity against Staphylococcus aureus (gram-positive).

Here the drug is allowed to diffuse through a solid medium so that a gradient is established,

the concentration being highest near the site of application of the drug and decreasing with

distance. The test bacterium is seeded on the medium and its sensitivity to the drug

determined from the inhibition of its growth. Several methods have been used for the

application of the drug. It may be added to ditches or holes cut in the medium or to hollow

cylinders placed on it. Ditches or hole is 6mm in diameter and charged with appropriate

concentrations of the drugs. The discs are stored dry in the cold. A suitable dilution of a broth

culture or a broth suspension of the test bacterium is flooded on the surface of a solid medium

(Mueller—Hinton agar or nutrient agar). The plate is tilted to ensure uniform spreading and

the excess broth pipette off. Inoculation may also be performed by spreading with swabs.

After drying the plate (37 °C for 30 min.), antibiotic discs (four or five per 10cm plate) are

applied with sterile forceps. After overnight incubation, tile degree of sensitivity is

determined by measuring the zones of inhibition of growth around the disc.

RESULTS AND DISCUSSION

Melting point determination: Melting point of mupirocin was found to be 78 0C. Melting

point was measured three times and mean was noted.

Solubility studies: Solubility studies are performed to determine the solubility of drug in

different solvents.


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Table 1: Solubility profile of mupirocin.

S.NO. Solvent Solubility(mg/ml) (mean±SD)

1. Acetone 8.426±0.030

2. Ethanol 5.180±0.021

3. 0.1 N HCL 2.180±0.004

4. Chloroform 0.412±0.055

5.1.6 FTIR analysis: FTIR spectroscopic analysis was carried out to characterize drug. The

FTIR spectra obtained was compared with that given in pharmacopoeia for mupirocin.

Diagnostic peaks and finger print regions were found identical. These characteristics peaks

are useful in identification of drug. FTIR of carbopol 940 and mixture containing mupirocin

and carbopol 940 was done for drug compatibility studies. The results obtained showed that

there occur no interactions between the components when taken together.

3614 cm-1

for OH group, 2941 cm-1

and 2883 cm-1

for C-H stretch of CH3, CH2 & 2503,

1716 cm-1

for C=O stretch of carboxylic acid & 1390, 1338 cm-1

for C-H bending of CH3,

CH2 & 1073 cm-1 for C=O stretch of ether.


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Figure 1: FTIR of Mupirocin.

Figure 2: FTIR of Carbopol 940.


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2955cm-1 for OH stretching vibrtation, 1706 cm

-1 for C=O stretching band, 835 cm-1

for outplane bending of C=CH.

Figure 3: FTIR of Physical mixture.

Table 2: Comparison between peaks obtained in drug and in mixture.

Description Peak obtained in mixture (frequency cm-1)

OH group 3613.95

C-H stretch of CH3, CH2 2947, 2893

C=O stretch of carboxylic acid 2593, 1728

C-H bending of CH3, CH2 1389, 1362

C=O stretch of ether 1059

5.2 Analysis by UV-Visible spectrophotometry

5.2.1 Preparation of standard graph

Stock solution of Mupirocin: Stock solution of 100µg/ml was prepared by dissolving

100mg of mupirocin in 100ml of methanol. Dilution in the range of 10 of 100 µg/ml were

scanned for determining λmax from 200-400 through UV spectrophotometer and λmax was

found to be at 220 nm for mupirocin.


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Table 3: Absorbance different dilutions of drug at 220 nm in methanol.

S.NO. Concentration( µg/ml) Absorbance

1 0 0.000±0.000

2 2 0.084±0.002

3 4 0.138±0.000

4 6 0.207±0.003

5 8 0.315±0.003

6 10 0.483±0.002

7 12 0.503±0.007

8 14 0.639±0.002

9 16 0.751±0.006

10 18 0.823±0.002

11 20 0.910±0.013

Figure 4: Standard calibration curve of mupirocin at 220nm.

4. Physical Evaluations of Mupirocin Gel

The gel formulation of mupirocin was evaluated for the following:

Organoleptic characteristics Colour = pale yellow

Odour = characteristic

Appearance = translucent

Phase separation = no

Occlusiveness = yes

Washability = washable


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Determination of pH of gel base and mupirocin gel

The pH of gel base and freshly prepared mupirocin gel was found to be 6.8 and 7.2

respectively.

Viscosity

The viscosity of carbopol 940gel base and mupirocin gel by Brookfield viscometer was found

to be 744,00 and 73,200 cP respectively.

Spreadability

The spreadability of mupirocin gel was found to be 12.66 g.cm2/sec. The spreadability results

showed that mupirocin gel was most effective i.e. it showed best results for spreadability.

Extrudability study

The extrudability of mupirocin gel was found to be positive.

Entrappment Efficiency

The % Entrapment Efficiency of mupirocin gel was found to be 95.68%.

Homogeneity and grittiness

Mupirocin gel was found to be homogeneous and no grittiness was noted.

5. In vitro release study

In vitro release study was performed to determine amount of drug released at different

interval of time.

Table 4: Release of drug from formulation.

Time (min.) % cumulative release of mupirocin

gel in 7.4 pH PBS

0 0

30 5.6

60 10.3

120 19.6

240 35.4

480 54.3

960 75.9

1440 90.6

2880 97.4


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Figure 5: Release of drug from formulation in PBS at pH 7.4 6.1Kinetics of drug

release.

The release kinetic of formulation in PBS of pH 7.4 was studied by various kinetic models.

The following data was obtained.

Table 5: Drug release data of formulation in PBS at pH 7.4.

Time

(min.)

Log

time

Square root

of time

% cumulative release

of ormulation

Log% cumulative

release of ormulation

% cumulative

remaining

Log% cumulative

remaining

0 0 0 0 0 100 2

30 1.48 5.48 5.6 0.75 94.4 1.97

60 1.75 7.75 10.3 1.01 89.7 1.95

120 2.08 10.95 19.6 1.29 80.4 1.91

240 2.38 15.49 35.4 1.55 64.6 1.81

480 2.68 21.91 54.3 1.73 45.7 1.66

960 2.98 30.98 75.9 1.88 24.1 1.38

1440 3.15 37.95 90.6 1.96 9.4 0.97

2880 3.46 53.67 97.4 1.99 2.6 0.41

Zero Order Plot

Graph was plotted between % cumulative drug release Vs time

Figure 6: Zero order plot for drug release kinetics of formulation in PBS at pH 7.4.


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First Order Plot

Graph was plotted between log % cumulative drug remaining VS time.

Figure 7: First order plot for drug release kinetics of formulation in PBS at pH 7.4.

Higuchi’s Model.

Graph was plotted between % cumulative drug release vs square root of time.

Figure 8: Higuchi plot for drug release kinetics of formulation in PBS at pH 7.4.

Korsmeyer-Peppas Model

Graph was plotted between log % cumulative drug release vs log time.


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Figure 9: Peppas plot for drug release kinetics of formulation in PBS at pH 7.4 Table 6:

Kinetics of drug release of formulation in PBS at pH 7.4.

Plot K0 R2

Zero order 0.078 0.7712

First order 0.001 0.9826

Higuchi 4.82 0.9446

Peppas 1.44 0.912

The data obtained for in vitro release were fitted into equations for zero order, first order,

Higuchi and Korsmeyer Peppas release models. The interpretation of data was based on the

value of the resulting regression coefficients.

From these values, it was observed that the First order model was found to be best suited with

R2 value of 0.9826. First order model be used to describe the drug dissolution from several

types of modified release pharmaceutical dosage forms.

Stability Studies

The Optimized formulation was stored at 40ºC/75% relative humidity (RH) in closed glass

vials for 6weeks. Gel was analyzed at specified time intervals (0, 2, 4, 6 weeks) for the %

drug content and in vitro study.

Table 7: Antimicrobial activity by cup-plate method.

S.No. Time (in weeks) % Drug Content

1 0 92.80

2 2 91.81

3 4 91.75

4 6 90.98


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Physical appearance

It was observed that optimized gel kept for 3 months under 40C±1

0C as well as 25

0C±1

0C at

temperature conditions showed no change in their physical appearance.

Phase separation

No phase separation was observed in the optimized mupirocin gel.

Antimicrobial activity

Antimicrobial activity was determined by cup plate method on S. aureus.

Table 8: Antimicrobial activity by cup-plate method.

Concentration (µg/ml) Pure Mupirocin Formulation

0.5 9 ± 1 mm 6± 2 mm

1 11 ± 1 mm 9± 3 mm

4 15 ± 2 mm 13 ± 3 mm

20 16± 3 mm 15 ± 2 mm

30 17± 1 mm 16 ± 2mm

40 18± 2 mm 18 ± 1 mm

MIC 4µg 20µg

Figure 9: Antibacterial activity of pure Figure 10: Antibacterial activity of

gel drug mupirocin. containing mupirocin.

CONCLUSION

The present work on the preparation of carbopol gel containing mupirocin in microbial

strains with minimum side effects by formulating the topical form of drug. In vitro release

study show 97.4% release of drug, stability is good with effective antibacterial activity. it was


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observed that the first order model was found to be best suited with R2 values of 0.9826. First

order model be used to describe the drug dissolution fro, several types of modified release

pharmaceutical dosage forms. Mupirocin containing carbapol based gel displayed superior

efficacy against S. aureus owning to prolonged release as compared to pure drug.

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