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Poonam Jadhav. / International Journal of Research in Pharmaceutical and Nano Sciences. 7(5), 2018, 175-199.

Available online: www.uptodateresearchpublication.com September – October 175

Research Article CODEN: IJRPJK ISSN: 2319 – 9563

FORMULATION AND EVALUATION OF NANOSPONGES CONTAINING

MURRAYA KOENIGII EXTRACT FOR BURN WOUND HEALING

Poonam Jadhav*1

1*Satara College of Pharmacy, Satara, Near MIDC, Maharashtra, India.

INTRODUCTION

The ideal delivery systems solubilize the drug; lead

the drug to the target site to fulfill the individual

need of the patient and disease stage. Nanosponges

are versatile drug delivery system as it can load

both hydrophilic and hydrophobic drugs.

Nanosponges are solid, porous, biocompatible, tiny

in size with 3 dimensional structures and nanomeric

cavity size which have unique ability to entrap wide

variety of drugs.

ABSTRACT

Murraya koenigii is a medium sized tree belonging to the family Rutaceae. The major constituents present in the

Murraya koenigii leaves treat burns, bruises. Ayurveda is a traditional Indian medicinal system practiced for

thousands of years. Natural remedies are more acceptable in the faith that they are safer with less side effects than the

synthetic ones. The novel formulations are reported to have remarkable advantages over conventional formulations of

plant actives and extracts which include enhancement of solubility, bioavailability, protection from toxicity,

enhancement of pharmacological activity, enhancement of stability, improved tissue macrophages distribution,

sustained delivery and protection from physical and chemical degradation. Our main purpose is to treat/heal wound at

faster rate with minimum side effects. Nanosponges alone are difficult to use on local tissues because they diffuse

away to other parts of the body very quickly so to overcome the drawback we combined nanosponges with the herbal

extract of Murraya Koenigii. According to literature survey it was found that etanolic and aqueous extract shows

good wound healing results. Research on curry leaves revealed that they are also effective in fighting bacterial and

fungal infections. The leaf extracts from the plant have been comparable to popular main stream antibiotic drugs.

During this research work, we prepared ethanolic extract of Murraya Koenigii leaves and prepared nansponges from

it for treating burn wound. The nanosponges are prepared by quasi emulsion solvent diffusion method. The prepared

nanosponges were evaluated for various In-vitro parameters and the results obtained were satisfactory.

KEYWORDS

Nanosponges, Murraya koenigii, Wound and Solubility.

Author for Correspondence:

Poonam Jadhav,

Satara College of Pharmacy,

Satara, Near MIDC, Maharashtra, India.

Email: [email protected]

International Journal of Research

in

Pharmaceutical and Nano Sciences Journal homepage: www.ijrpns.com



Nanosponges are a novel class of nanoparticles with

nanostructured hyper branched polymers and few

nanometers wide cavities in which a large variety of

substances can be encapsulated. The nanosponges

drug delivery platform is a network of specific

polymers that slowly degrades and thus releases the

chosen drug. The nanosponges can be synthesized

to be of specific size and to release drugs over time

by varying the proportion of cross-linker to

polymer.

Now regarding its mechanism of drug release, the

sponge particles contain an open structure and it

contains the active ingredients which are free to

move in and out from the particles and into the

medium until equilibrium is reached. In case of

topical drug delivery, once the finished product is

applied to the target tissue, the active ingredient

which is already present in the vehicle will be

absorbed into it, depleting the vehicle, which will

become unsaturated, hence disturbing the

equilibrium.

This will lead to flow of the active drug ingredient

from the sponge particles into the vehicle and from

it to the target tissue until the vehicle is either

dried/absorbed.

The term gel was introduced in late 1800. The USP

defines gel as a semisolid system consisting of

dispersion made up of either small inorganic

particles or large organic molecules enclosed and

interpenetrated by liquid.

A burn is a type of injury to skin caused by heat,

electricity, light, radiation or friction. The wound

healing process is a series of independent and

overlapping stages. In these stages both cellular and

matrix compounds work to reestablish the integrity

of damaged tissues and replacement of lost tissues.

These overlapping series are classified into 5 stages

– Haemostasis, Inflammation, Migration,

Proliferation and maturation.

MATERIAL AND EQUIPMENTS

The raw materials like drug, polymers, excipients

and chemicals required for the present work were

procured from different sources. Following

materials were used for the formulation and

evaluation of nanosponges and gel.

Preformulation studies

Characterization of Murraya koenigii extract

Organoleptic properties Murraya Koenigii leaves extract was evaluated for

its organoleptic properties such as colour, odour,

and taste.

Determination of pH

The crude powder of Murraya Koenigii was

dissolved in distilled water and was kept in water

bath for 20 min, filtered and then pH of the filtrate

was noted down with the help of pH meter.

Phytochemical tests performed

Determination of total ash

The total ash value of crude powder of Murraya

Koenigii was determined by incinerating 2 g of

accurately weighed crude powder in a silica

crucible. It was incinerated in a muffle furnace at a

temperature not exceeding 450º C until free from

carbon, then cooled and weighed.

Determination of water soluble ash

The total ash obtained was boiled with 25 ml of

distilled water for 5 min. The insoluble matter was

collected on an ash less filter paper, washed with

hot water and ignited to constant weight at allowed

temperature. The weight of insoluble matter was

subtracted from the weight of total ash. The

difference in weight represents the water soluble

ash. The percentage of water soluble ash with

reference to the air dried drug was calculated.

Determination of acid insoluble ash

The ash obtained in the above method was boiled

with 25 ml of dilute hydrochloric acid for 5 min.

The residue was collected on ash less filter paper

and washed with hot water, ignited, cooled and

weighed. The percent of acid insoluble ash with

reference to air dried drug was calculated.

Determination of loss on drying

The loss on drying was determined by weighing 2 g

of crude powder of Murraya Koenigii in an

evaporating dish and then dried in an oven at 105ºC

till constant weight was obtained and loss on drying

was calculated. The percent loss on drying was

calculated on the basis of sample taken initially.



Determination of melting point

Melting point of Murraya Koenigii extract was

determined by micro controlled based melting point

apparatus. The sample was inserted in capillary

having one end closed. Then the capillary was

inserted in bath of silicone oil which was heated in

controlled manner with the help of electric heating

coil. The temperature at which bubble formation

occurs was noted as melting temperature.

Calibration curve of Murraya Koenigii extract in

Phosphate buffer pH 5.5

Accurately weighed 100 mg (0.1 gm) Murraya

Koenigii extract was taken and transferred to 100

ml volumetric flask and volume was made to 100

ml with petroleum ether (Stock I). The 10ml

solution from above stock I solution was again

diluted with methanol and volume was made to 100

ml (Stock II). The final solutions of stock II were

then prepared in methanol. From Stock II solution

aliquots of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 ml were

transferred to 10 ml volumetric flasks and final

volume was made to 10ml with methanol in the

concentration range of 10-100 µg/ml. The

absorbance values of these solutions were measured

at 239.5 nm using double beam UV

spectrophotometer (Shimadzu, Pharmaspec UV-

1700, Japan) against blank of methanol.

Drug – excipient compatibility studies

Visual observations

The samples subjected to drug-excipient

compatibility studies were assessed for any visual

changes. The samples were observed for change in

colour and nature.

Infrared Spectroscopy

IR was determined by Fourier Transform infrared

spectrophotometer (FTIR – 4100, Jasco, Japan).

Preparation of Blank Nanosponges

Blank nanosponges were prepared by Quasi –

emulsion solvent diffusion method. The inner phase

was prepared by dissolving Eudragit RS 100 in a

suitable solvent i.e dichloromethane. The inner

phase was then poured into the polyvinyl alcohol

solution in water (Outer Phase). Following 60 min

of stirring (rpm 800-900), the mixture was filtered

to separate the nanosponges. The nanosponges were

dried in an air heated oven at 40ºC for 12 h.

Preparation of drug loaded Nanosponges

Drug loaded nanosponges were prepared by Quasi -

emulsion solvent diffusion method. The inner phase

was prepared by dissolving Eudragit RS 100 in a

suitable solvent i.e. dichloromethane. Then drug

was added to solution and dissolved under

ultrasonication at 35ºC. The inner phase was then

poured into the polyvinyl alcohol solution in water

(Outer Phase). Following 60 min of stirring (rpm

800-900), the mixture was filtered to separate the

Nanosponges. The Nanosponges were dried in an

air heated oven at 40ºC for 12 h.

Evaluation of Nanosponges

Visual inspection

The visual inspection of nanosponges was

determined by optical or binocular microscopy.

Determination of production yield

The production yield of the nanosponges was

determined by calculating accurately the initial

weight of the raw materials and the final weight of

the nanosponges obtained. Production Yield (PY) = Practical mass of nanosponges × 100 (Eq-1)

Theoretical Mass (Polymer + Drug)

Actual drug content and Entrapment Efficiency

The actual drug content was determined by the

amount of drug which was entrapped in

nanosponges. The weighed amount of drug loaded

nanosponges (50mg) was kept in 10 ml ethanol and

soaked for 3 h. The samples were filtered and

analyzed at 239.5nm against blank using UV

spectrophotometer (Shimadzu, PharmaSpecUV-

1700, Japan). Encapsulation efficiency was

calculated by following formula: Entrapment efficiency (%) = Total amount of drug ─ Free unentrapped drug × 100 (Eq-2)

Total amount of drug

Differential Scanning Calorimetry (DSC)

Thermal analysis is an important evaluation

technique to find any possible interaction between

the drug and excipient. Such interaction can be

identified by any change in thermogram.

Thermogram of pure Murraya koenigii leaves

extract and finished nanosponges formulations were

obtained using DSC instrument (Mettler Toledo

DSC 821Ce, Switzerland) equipped with an



intercooler. Indium standard was used to calibrate

the DSC temperature and enthalpy scale. The

powder sample of nanosponges was kept in the

aluminium pan and heated at constant rate of

5°C/min up to 300°C. Inert atmosphere was

maintained by purging nitrogen at the flow rate of

10ml/min.

Scanning Electron microscopy

Scanning electron microscopy was used to study the

microscopic aspects of the nanosponges. The

morphology of nanosponges was carried out by

using zeta sizer.

Particle size analysis

Particle size analysis of prepared nanosponges was

carried out by using zeta sizer (Particulate system

nano plus).

Zeta potential

Zeta potential of optimized nanosponges was

measured by using zeta sizer at 25°C (Particulate

system nano plus).

Formulation and evaluation of Murraya koenigii

extract nanosponges gel

Preparation of Murraya koenigii extracts

nanosponges gel:

1% Carbopol 934 was allowed to soak for 24 h in

distilled water. On next day accurately weighed

Murraya koenigii extract nanosponges were added

to the gel base. Triethanolamine was added drop

wise to the formulation for adjustment of required

pH (5.5-5.6) and to obtain the gel in required

consistency. Finally preservatives were added in the

carbopol solution

Physical appearance

The physical appearance of the formulation was

checked visually.

Color

The color of the formulation was checked out

against white and black background.

Consistency

The consistency was checked by applying on skin.

Odor

The odor of gel was checked by mixing the gel in

water and taking the smell.

Determination of pH

The pH of gel was determined using digital pH

meter by dipping the glass electrode completely into

the gel system.

Determination of Spread ability

Spread ability was determined by modified wooden

block and glass slide apparatus. The apparatus

consisted of a wooden block with fixed glass and a

pulley. A pan was attached to another glass slide

(movable) with the help of a string. For the

determination of Spread ability measured amount of

gel was placed on the fixed glass slide. The

movable glass slide with a pan attached to it, was

placed on other fixed glass slide such that the gel

was sandwiched between the two slides for 5 min.

About 50gm of weight was added to the pan. Time

taken for the slides to separate was noted. Spread

ability was determined using formula:

S=M.L/T ------------- (Eq-3)

Determination of drug content

The drug content of gel formulation was determined

by dissolving an accurately weighed quantity (1g)

of gel in 100 ml of solvent (pH 5.5-5.6). The

solutions were kept for stirring up to complete

dissolution of the formulations. Solutions were

filtered and were subjected to spectrophotometric

analysis. The drug content was calculated from

calibration data. Drug Content = Actual conc. × 100 ------ (Eq-4)

Theoretical conc.

In vitro drug release studies

Release of the Murraya koenigii extract from

nanosponges incorporated in various gel

formulations was measured through standard

cellophane membrane using a Franz diffusion cell.

Prior to study, cellophane membrane was soaked in

diffusion medium for overnight, and then placed on

the support screen of the diffusion cell assembly.

Phosphate buffer pH 5.5 was used as the receptor

medium and 1gm of the gel was placed on the donor

side. All batches of Murraya koenigii extract (F1-

F9) were used for diffusion study. At predetermined

time intervals, 2ml of sample was withdrawn from

the receptor compartment and replaced with same

volume of phosphate buffer pH 5.5. The aliquots



were analyzed by UV spectrophotometer at 239.5

nm against PBS (pH 5.5).

In-vivo studies for burn wound healing activity

by using suitable animal model:

Burn wound healing activity

• Animals: Wistar rats Male/Female (n=18)

• Weight: 200-300 g.

• Group I: Nanosponges gel containing

Murraya koenigii extract.

• Group II: Standard (Silver sulphadiazine

cream)

• Group III: Control (No treatment)

Procedure

Albino Wistar rats of either sex weighing 200- 250

gm (3 months old) were randomly selected marked

to permit individual identification and kept in their

cages for at least 7 days prior to dosing to allow for

acclimatization to the laboratory condition. Animals

were not being deprived of food and water. The

animals were divided into 3 groups of 6 animals

each then dorsum of each rat was shaved.

Immediately after the procedure anesthesia was

given to animal. The burn was made by exposing

the back skin to red hot beaker. The wounding day

was considered as day 0. Prepared formulations

were placed on the wound site. Average wound

contraction was measured by a tracing paper on the

wounded margin and calculated as percentage

reduction in wound area. The wound contraction is

calculated as percentage of the original wound size

for each animal of a group. The percent of wound

size induced was calculated by: Percentage Wound Contraction (%) = Initial Wound

size-specific wound size / initial wound size ×100

RESULTS AND DISCUSSION

Collection of plant material

Authentication of plant material

The authentication of seeds was done from

department of Botany, Yashwantrao Chavan

institute of Sciences Satara (Maharashtra) and it

was confirmed that the procured leaves were of

Murraya koenigii plant.

Extraction of Murraya koenigii leaves

Extract was obtained in sufficient quantity from the

leaves of Murraya koenigii by soxhlet extraction

method.

Preformulation study

Characterization of Murraya koenigii extract:

Organoleptic properties

Determination of pH

pH of the extract was found to be 6.7, while as per

literature standard it is reported to be 6.3-6.4. As

experimental values were in good agreement with

official values, it could be concluded that procured

extract was in pure form.

Phytochemical tests performed

Test for alkaloids

Dragendroff’s test

This test was performed which showed formation of

orange brown precipitate that indicated the presence

of alkaloids.

Hager’s test


yellow precipitate that indicated the presence of

alkaloids.

Test for glycoside

Molisch’s test


violet ring at the junction of 2 liquids that indicated

the presence of glycosides.

Test for Saponin

Foam test

This test was performed which showed foam

formation that indicated the presence of saponin.

Test for proteins and amino acids

Millions test


white precipitate. Warm the obtained precipitate

which converted it into brick red colour that

indicated the presence of proteins and amino acids.

Test for Triterpenoids

Lieberman bur chard test


first red, and then blue and finally green colour

indicated presence of Triterpenoids.



Test for flavonoids

Shinoda test


red coloration that indicated presence of Flavinoids.

Determination of total ash

The total ash value of Murraya koenigii was found

to be 12.5%, while as per literature standard it is

11.33%.

Determination of water soluble ash

The water soluble ash value was found to be 2%,

while as per literature standard is 1.97%.

Determination of acid insoluble ash

The acid insoluble ash value was found to be 5%,

while as per literature standard it is 5.33%.

Determination of loss on drying

The loss on drying was found to be 10.43 gm, while

as per literature standard it is 10.06±0.15gm.

Determination of refractive index

The refractive index was found to be 1.5010, while

as per literature standard it is 1.5021.

Determination of melting point

The melting point of Murraya koenigii was found to

be 99ºC- 100ºC, while as per literature standard it is

98°C-100°C.

Determination of extractive values

Alcohol soluble extractives

The alcohol soluble extractive was found to be

7.2%, while as per literature standard it is reported

to be 7.75%.

Water soluble extractive

The water soluble extractive was found to be

9.52%, while as per literature standard it is reported

to be 9.56%.

Identification of Murraya koenigii extract

Ultraviolet spectroscopy

The λmax value of Murraya koenigii extract was

found to be 239.5nm in phosphate buffer pH

5.5.This was in well compliance with the λmax

value of Murraya koenigii extract in literature.

Calibration curve of Murraya koenigii extract in

phosphate buffer pH 5.5

The calibration curve for Murraya Koenigii extract

in phosphate buffer pH 5.5 was plotted by using

following results of absorbance at various

concentrations.

Drug - excipient compatibility studies

Visual observations

No notable change was observed in the sample on

visual observation. There was no observable color

change.

Infrared spectroscopy

FTIR spectrum of the mixture of Murraya koenigii

extract and excipients was compared with spectra of

individual components. An FTIR spectrum of

physical mixture shows significant peaks of

Murraya koenigii extract and respective excipient

indicating no chemical interaction between Murraya

koenigii extract and excipient.

Evaluations of Murraya koenigii extract

nanosponges

Visual inspection

In this figure, the nanosponges were observed in the

optical microscope. The nanosponges were spread

on neubauer’s chamber; small cube contains two or

three nanosponges. From the figure it could be

concluded that the obtained product was in nano

range. The binocular image of nanosponges also

exhibited the small and spherical nanosponges.

Determination of Production Yield, Entrapment

Efficiency and Actual Drug Content:

Production Yield, Entrapment Efficiency and

Actual Drug Content was calculated

The % production yield of all batches was ranged

from 19.38% to 88.78%, it was found that the

production yield was greatly affected by polymer

concentration as well as by concentration of

polyvinyl alcohol.

Use of higher amount of PVA while preparing

nanosponges at higher amount of polymers caused

slightly an increased viscosity of the dispersed

phase. When solvents from inner phase diffused

out, nearly all of the dispersed phase was converted

to the form of solid nanosponges and separated

particles appeared and also it has given higher

percent entrapment efficiency and % actual drug

content.

Scanning electron microscopy

From SEM photographs, it was clear that the

nanosponges so obtained were having the spherical

shape and the spongy nature of the nanosponges



was clearly evident, thus it could be concluded that

the adopted method for the preparation of

nanosponges was useful.

Particle size analysis

Particle size of nanosponges should be in the range

of 5-500 nm. The visual inspection of all batches for

particle size using optical and binocular microscope

revealed that the particle size was increased with the

increase in the Eudragit RS100 amount. This might

be due to increasing polymer wall thickness which

led to the larger size of nanosponges. The F6 batch

possessed more percent of intact, uniform, spherical

particles in optical microscopy; so the batch F6 was

chosen for further analysis. A mean particle size of

formulation F6 was found to be 55.2 nm.

Zeta potential

Highly negatively or highly positive zeta potential

value indicates good physical stability of the

formulation. The optimised nanosponge

formulation of murraya koenigii was measured by

using zetasizer at 25°C, it was found to be 27.11

and it indicate the formulation to be stable.

In vitro drug release studies In vitro drug release study was carried out using

phosphate buffer pH 5.5 using Franz diffusion cell,

from the results it could be revealed that % CADD

was founded and ranged between 62.14% to

82.16%. Formulation F6 gave better drug release

than other formulations. The cumulative percent

drug release (% CADD) for all formulations was

calculated. It is given in table below

Evaluations of Murraya koenigii extract

nanosponges gel

Physical appearance

The prepared gel formulations of Murraya koenigii

leaves extract nanosponges were visually inspected

for their colour, consistency and odour.

Colour: Pale green

Consistency: Good

Odour: Aromatic

The values of spred ability of all prepared

formulation were found to be in the range of

12.33±1.08 to 29.03±2.62 g.cm/sec and drug

content ranges from 81.1 to 92.5. Hence, it indicates

that spred ability and drug content of gel was good.

In vitro drug release studies

In vitro drug release study of gel was carried out

using phosphate buffer pH 5.5 using Franz diffusion

cell. From the results it could be concluded that %

CADD of gel formulations gave better release. At

the end of 8 h, the total amount of drug release from

the formulation was found to be 82.16%.

Stability study

The stability studies of formulated gel were carried

out at room temperature for one month. The effect

of temperature, humidity and time on the physical

characteristics of the gel was for assessing the

stability of the prepared formulations. The stability

studies were carried out when the room temperature

was 25º C. The Results were shown in Table No.32.

Therefore no evidence of degradation of drug was

observed.

DSC of formulated gel

In DSC thermogram of Murraya koenigii extract

containing gel endothermic peak was observed at

105.26ºC. The DSC thermogram of drug and

formulation compared with each other both shows

endothermic peak. The study indicated that drug has

not under go any chemical interaction with the

polymer.

Data analysis of formulations

Traditional design of the pharmaceutical

formulations are based on time consuming approach

of changing one variable at a time which does not

take into consideration the joint effect of

independent variables. Thus, factorial design can

serve as an essential tool to understand the

complexity of the pharmaceutical formulations.

A 32 full factorial design was selected and the 2

factors were evaluated at 3 levels. The amount of

Eudragit RS100 (A) and polyvinyl alcohol (B) were

selected as independent variables and the dependent

variables were % production yield, %CADD and %

Entrapment efficiency. The data obtained was

treated using Stat-Ease Design Expert software.



Table No.1: List of drug, Excipients, Polymer and Solvent

S.No Drug/Polymer/Excipients/Solvent Manufacturer

1 Leaves of Murraya Koenigii Botanical garden of Satara College of Pharmacy,

Satara

2 Eudragit RS100 Loba Chemie Pvt. Ltd., Mumbai

3 Polyvinyl alcohol Loba Chemie Pvt. Ltd., Mumbai

4 Dichloromethane S.D. Lab Chem. Mumbai

5 Ethanol S.D. Lab Chem. Mumbai

6 Carbopol 934 Research –Lab Fine Chem. Industries, Mumbai

7 Methyl paraben Loba Chemie Pvt. Ltd., Mumbai

8 Propyl paraben Loba Chemie Pvt. Ltd., Mumbai

9 Triethanolamine Research- Lab Fine Chem. Industries, Mumbai.

Table No.2: Samples used in drug-excipient compatibility studies

S.No Pure sample Drug + Excipient

1 Murraya Koenigii extract -----

2 Eudragit RS 100 Eudragit RS 100 + Extract

3 Polyvinyl alcohol Polyvinyl alcohol + Extract

4 Carbopol 934 Carbopol 934 + Extract

Formulation of Nanosponges

Table No.3: Ingredients used for formulation of nanosponges of Murraya koenigii leaves extract

S.No Ingredients Use

1 Murraya Koenigii extract API

2 Eudragit RS 100 Polymer

3 Polyvinyl alcohol Stabilizer

4 Dichloromethane Solvent

5 Distilled water Vehicle

Full Factorial design

Table No.4: Full factorial experimental design layout

Formulation code Coded values

A B

F1 +1 0

F2 0 +1

F3 -1 -1

F4 +1 -1

F5 -1 0

F6 -1 +1

F7 +1 +1

F8 0 0

F9 0 -1



Table No.5: Design Summary

S.No Factor Name Unit Type Coded level Actual level

Low Medium High Low Medium High

1 A Eudragit

RS100 gm

Numer

ical -1 0 +1 0.5 1 1.5

2 B Polyvinyl

alcohol gm

Numer

ical -1 0 +1 0.25 0.5 0.75

Table No.6: Composition of blank nanosponges

Table No.7: Composition of drug loaded Nanosponges

Table No.8: Composition of Gel containing Nanosponges of Murraya koenigii extract

S.No Batches F1 F2 F3 F4 F5 F6 F7 F8 F9

1 Nanosponges eq. to

100mg drug 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

2 Methyl paraben(g) 0.015 0.020 0.025 0.015 0.020 0.025 0.015 0.020 0.025

3 Propyl paraben(g) 0.05 0.010 0.015 0.05 0.010 0.015 0.05 0.010 0.015

4 Triethanolamine(ml) q.s q.s q.s q.s q.s q.s q.s q.s q.s

5 Carbopol 934(gm) 0.1 0.1 0.1 0.2 0.2 0.2 0.3 0.3 0.3

6 Distilled water (ml) q.s q.s q.s q.s q.s q.s q.s q.s q.s

Table No.9: Phytochemical tests

S.No Constituents Tests Observations

1 Alkaloids 1. Dragendroff’s test

2. Hager’s test

Present

Present

2 Protein Millions test Present

3 Saponin test Foam test Present

4 Glycoside Molisch’s test Present

5 Protein Millions test Present

6 Triterpenoids Liberman burchard test Present

7 Flavinoids Shinoda test Present

8 Terpenoids Noller’s test Present

S.No Batch no F1 F2 F3 F4 F5 F6 F7 F8 F9

1 Eudragit Rs 100(gm) 0.5 1 1.5 0.5 1 1.5 0.5 1 1.5

2 Dichloromethane (ml) 20 20 20 20 20 20 20 20 20

3 Polyvinyl alcohol (gm) 0.25 0.25 0.25 0.50 0.50 0.50 0.75 0.75 0.75

4 Distilled water (ml) 100 100 100 100 100 100 100 100 100

S.No Batch no F1 F2 F3 F4 F5 F6 F7 F8 F9

1 Drug (gm) 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2

2 Eudragit Rs 100(gm) 0.5 1 1.5 0.5 1 1.5 0.5 1 1.5

3 Dichloromethane (ml) 20 20 20 20 20 20 20 20 20

4 Polyvinyl alcohol (gm) 0.25 0.25 0.25 0.50 0.50 0.50 0.75 0.75 0.75

5 Distilled water (ml) 100 100 100 100 100 100 100 100 100



Table No.10: Absorbance of Murraya koenigii extract in phosphate buffer pH 5.5

S.No Concentrations (µg/ml) Absorbance

1 0 0

2 10 0.0794

3 20 0.176

4 30 0.330

5 40 0.472

6 50 0.5721

7 60 0.7021

8 70 0.7237

9 80 0.9212

10 90 1.0419

Table No.11: Production Yield, Entrapment Efficiency and Actual Drug Content of Murraya koenigii

extract nanosponges

Batches

Amount

of Eu

RS 100

(g)

Amount

of PVA

(g)

Theoretical

yield

(g)

Practical

yield

(g)

Production

yield

(%)

Entrapment

efficiency

(%)

Actual

drug

content

(%)

F1 0.5 0.25 0.95 0.4122 43.38 67.7 67.7

F2 1 0.25 1.45 0.7393 50.98 77.8 77.8

F3 1.5 0.25 1.95 1.7314 29.65 77.3 77.3

F4 0.5 0.5 1.2 0.3662 30.51 66.8 66.8

F5 1 0.5 1.7 0.7512 44.18 53 53

F6 1.5 0.5 2.2 0.6523 88.78 86.6 86.6

F7 0.5 0.75 1.45 0.7465 51.48 52 52

F8 1 0.75 1.95 0.3780 19.38 45.1 45.1

F9 1.5 0.75 2.45 1.1010 44.93 77.5 77.5

Table No.12: The cumulative percent drug release of all formulations

Time

(hr)

% CADD

F1 F2 F3 F4 F5 F6 F7 F8 F9

0 0 0 0 0 0 0 0 0 0

1 15.38 42.18 29.5 37.24 32.85 27.42 27.69 37.81 20.15

2 28.35 47.29 36.96 42.29 39.15 37.35 35.78 44.68 28.52

3 32.54 49.65 39.16 49.73 40.72 41.47 40.87 48.71 32.54

4 42.30 51.7 42.18 55.64 42.12 59.14 52.67 65.3 36.65

5 45.76 57.12 49.72 59.16 51.79 62.42 54.35 69.73 42.39

6 52.37 65.99 52.34 63.1 56.12 69.55 56.19 73.15 49.79

7 62.55 72.31 65.16 70.12 62.14 75.21 58.78 75.79 58.92

8 68.63 81.46 80.16 72.32 69.16 82.16 62.14 79.12 68.32



Determination of pH

Table No.13: pH of all formulations

S.No Batches pH Std. Deviation

1 F1 5.22 5.20 5.22 5.21±0.07234

2 F2 6.22 6.22 6.22 6.22±0

3 F3 5.29 5.10 5.32 5.23±0.1193

4 F4 5.89 5.90 5.92 5.90±0.01528

5 F5 5.62 5.58 5.60 5.59±0.02

6 F6 5.28 5.25 5.27 5.26±0.01528

7 F7 6.00 6.00 6.00 6.00±0

8 F8 6.67 6.69 6.67 6.67±0.01155

9 F9 5.76 5.75 5.74 5.75±0.01

10 5.28 5.25 5.27 5.26±0.01528 5.28

Spread ability and Drug Content

Table No.14: Spread ability and % drug content of all formulations

Batches Spread ability Std. Deviation Drug content (%)

F1 20 20.65 19 19.88±0.83 85.54

F2 29.16 25 27.77 27.31±2.11 89.6

F3 12.5 13.33 11.17 12.33±1.08 86.6

F4 22.36 22 19.31 21.22±1.66 81.1

F5 24.82 18.75 13.88 19.15±5.48 87.2

F6 13.87 16.30 14.81 14.99±1.22 92.5

F7 31.45 29.41 26.25 29.03±2.62 89.9

F8 18.4 21.18 17.96 19.18±1.74 82.1

F9 27.77 25 21.59 24.78±3.09 84.4

Table No.15: % CADD of optimized formulation

Time (hr) CADD (%)

0 0

1 27.42

2 37.35

3 41.47

4 59.14

5 62.42

6 69.55

7 75.21

8 82.16

Table No.16: Appearance, pH and homogeneity of formulation

Time interval

(days)

Formulation

Appearance pH Homogeneity

0 Pale green 5.20 Very good

7 Pale green 5.20 Very good

15 Pale green 5.36 Good

30 Pale green 5.38 Good



Table No.17: Design Summary

S.No Factor Name Unit Type Coded level Actual level

Low Medium High Low Medium High

1 A Eudragit

RS100 gm Numerical -1 0 +1 0.5 1 1.5

2 B Polyvinyl

alcohol gm Numerical -1 0 +1 0.25 0.5 0.75

Table No.18: Response Summary

S.No Response Name Unit Observations Analysis Minimum Maximum

1 M1 Entrapment

efficiency % 9 Polynomial 45.1 86.6

2 M2 CADD % 9 Polynomial 27.42 82.10

3 M3 PY % 9 Polynomial 19.38 88.78

Table No.19: The responses of all formulations (F1-F9)

S.No Responses A B CADD (%) EE (%) %PY

1 F1 +1 0 68.63 67.7 43.38

2 F2 0 +1 81.46 77.8 50.98

3 F3 -1 -1 80.16 77.3 29.65

4 F4 +1 -1 72.32 66.8 30.51

5 F5 -1 0 69.16 53 44.18

6 F6 -1 +1 82.16 86.6 88.78

7 F7 +1 +1 62.14 52 51.48

8 F8 0 0 79.12 75.1 19.38

9 F9 0 -1 68.32 77.5 44.93

In-vivo burn wound healing

Weight of animals after creating burn wound

Table No.20: Weight of animals after creating burn wound

S.No Time (days) Group 1 (Test) Group 2 (Standard) Group 3 (Control)

1 0 262.5±26.22 225±22.36 220.83±36.79

2 7 266.66±30.27 212.5±26.22 237.5±26.22

3 14 287.5±26.22 250±15.81 212.5±26.22

4 21 241.66±20.41 212.5±26.22 220.83±36.79

5 28 250±15.81 225±22.36 233.33±20.41

Surface area of burn wound

Table No.21: Surface area of burn wound

S.No Time (days) Group 1 Group 2 Group 3

1 0 10.89±2.088 9.216±1.114 11.75±1.665

2 7 9.142±1.579 7.738±0.999 10.14±1.659

3 14 7.411±1.019 5.878±1.987 8.588±1.424

4 21 5.79±1.289 3.615±1.717 5.015±2.245

5 28 0.859±0.499 1.000±0.839 1.734±1.172



% of burn wound contraction

Table No.22: % of burn wound contraction

S.No Time (days) Group 1 (Test) Group 2(Std) Group 3 (Control)

1 0 - - -

2 7 15.611±7.203 15.72±8.292 13.86±4.140

3 14 30.56± 19.11 32.42±20.506 26.96±5.303

4 21 47.87±19.105 46.235±10.848 35.57±2.106

5 28 89.56±8.434 85.106±9.196 78.24±9.916

a. Shaved animal b. 0th day wound 1st day wound

Figure No.1: Burn Wound

Figure No.2: Soxhlet extraction of leaves of plant and extract of leaves

Figure No.3: UV spectrum of Murraya koenigii extract in phosphate buffer pH 5.5



Infrared spectroscopy

3795.65

3763.74

3733.34

3554.06

2914.84

2751.40

2701.88

2587.62

2467.22

2355.50

2230.01

2204.85

2139.39

2009.89

1727.70

1404.52

1315.53

1024.84

500100015002000250030003500

Wavenumber cm-1

020

40

60

80

100

Transmittance [%]

MK KADIPATTA.0 R. L. Jadhav Satara College of Pharmacy, Satara 2/22/2017 4:26:43 PM

Figure No.4: FTIR spectrum of Murraya koenigii extract

DSC

Figure No.5: DSC of Murraya koenigii dried powder

In DSC thermogram of Murraya koenigii extract endothermic peak was observed at 75.82ºC

Figure No.6: DSC Thermogram of Murraya koenigii extract

In DSC thermogram of Murraya koenigii extract endothermic peak was observed at 130.40ºC

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3795.65

3763.74

3733.34

3554.06

2914.84

2751.40

2701.88

2587.62

2467.22

2355.50

2230.01

2204.85

2139.39

2009.89

1727.70

1404.52

1315.53

1024.84

500

1000

1500

2000

2500

3000

3500

Wavenum

ber cm

-1

0 20 40 60 80 100

Transmittance [%]

MK

K

AD

IP

AT

TA

.0 R

. L

. Jad

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: FT

IR sp

ectru

m o

f Mu

rray

a k

oen

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3931.37

3898.25

3865.99

3823.73

3747.70

3669.89

3642.47

3598.34

3555.86

3493.80

3430.58

3398.80

3355.11

3305.82

3226.43

3194.31

3115.87

3069.44

2983.55

2953.78

2918.55

2835.48

2760.89

2699.88

2634.16

2549.46

2475.41

2426.34

2361.81

2330.63

2283.25

2199.67

2133.04

2104.86

2072.29

2040.51

1992.46

1869.73

1833.02

1721.01

1646.02

1516.24

1451.10

1344.44

1233.29

1048.86

985.38

907.77

854.01

803.09

754.16

685.64

618.45

573.77

511.19

465.10

423.42500

1000

1500

2000

2500

3000

3500

Wavenum

ber cm

-1

0 20 40 60 80 100 120

Transmittance [%]

F

igu

re No

.9: F

TIR

spec

trum

of E

ud

rag

it RS

100

3925.70

3867.65

3807.24

3674.51

3616.27

3564.80

3426.33

3382.48

3307.77

3282.60

3253.85

3188.07

3139.71

3112.26

3001.15

2924.61

2855.02

2734.32

2704.07

2591.86

2412.44

2381.43

2336.60

2303.65

2252.32

2213.49

2072.88

1997.43

1926.13

1847.01

1747.13

1583.91

1469.13

1434.39

1383.96

1272.06

1229.04

1144.20

1097.23

1022.27

849.49

724.44

656.57

575.75

534.55

489.65

500

1000

1500

2000

2500

3000

3500

Wavenum

ber cm

-1

0 20 40 60 80 100 120 140

Transmittance [%]

MK

K

AD

IP

AT

TA

+ E

RS

100.0 R

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spec

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100

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3947.11

3887.54

3850.39

3784.38

3743.17

3683.66

3617.19

3560.71

3501.82

3407.41

3351.65

3289.11

3195.39

3128.83

3035.71

3000.84

2921.37

2846.18

2792.64

2760.73

2688.85

2645.89

2562.86

2489.44

2416.84

2365.15

2328.75

2244.08

2181.88

2148.28

2116.96

2060.83

2002.92

1920.35

1872.82

1833.62

1732.64

1706.70

1649.00

1522.71

1455.94

1424.99

1373.04

1314.64

1132.46

1072.00

1029.02

915.23

831.27

741.10

691.59

665.97

604.74

563.06

518.27

486.55

430.52

500

1000

1500

2000

2500

3000

3500

Wavenum

ber cm

-1

0 10 20 30 40 50 60

Transmittance [%]

PO

LY

VIN

YL

A

LC

OH

OL

.0 R

. L

. Jad

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Fig

ure N

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1: F

TIR

spectru

m o

f po

lyv

iny

l alco

ho

l

3931.62

3898.49

3852.75

3742.64

3677.69

3647.02

3616.73

3565.39

3414.70

3360.93

3313.30

3291.15

3245.04

3172.82

3119.49

3069.13

3001.29

2943.91

2893.11

2835.12

2788.99

2765.11

2689.44

2616.62

2584.04

2520.10

2425.30

2361.56

2327.06

2235.87

2181.24

2150.97

2066.16

1991.41

1917.87

1868.84

1832.89

1793.01

1742.68

1699.30

1649.19

1518.38

1458.77

1424.23

1395.97

1317.22

1274.40

1196.41

1133.82

1036.24

984.40

922.09

846.00

748.30

671.89

608.14

519.31

465.55

448.20

500

1000

1500

2000

2500

3000

3500

Wavenum

ber cm

-1

0 20 40 60 80 100 120 140

Transmittance [%]

PO

LY

VIN

YL

A

LC

OH

OL

+

MU

RR

AY

A K

OE

NIG

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XT

RA

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spec

tra

of p

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ture o

f Mu

rray

a k

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tra

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3937.73

3897.35

3860.84

3829.01

3770.20

3720.68

3678.04

3624.03

3591.32

3561.98

3513.97

3489.39

3445.89

3413.88

3367.24

3336.12

3304.00

3265.58

3232.63

3155.76

3126.28

3065.72

3048.74

2989.68

2957.67

2889.51

2840.04

2807.43

2763.47

2721.26

2667.30

2628.97

2564.48

2470.27

2408.75

2355.91

2267.75

2198.01

2170.04

2124.62

2039.94

1996.81

1914.91

1832.75

1706.86

1563.62

1514.39

1449.37

1411.97

1240.31

1170.95

1112.26

1031.72

891.03

802.58

636.49

587.51

532.51

496.69

460.47

444.15

427.58

500

1000

1500

2000

2500

3000

3500

Wavenum

ber cm

-1

20 40 60 80 100

Transmittance [%]

CA

RB

OP

OL

934.1 R

. L

. Jad

hav

Satara C

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12/31/2016 2:41:56 P

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Fig

ure N

o.1

3: IR

spec

tra

of C

arb

op

ol 9

34

3939.69

3907.89

3881.89

3851.57

3744.92

3707.91

3611.12

3564.46

3530.11

3450.52

3405.32

3099.93

3043.58

2881.52

2821.19

2792.67

2731.48

2671.90

2580.23

2539.62

2359.92

2316.99

2176.13

2142.44

2114.20

2036.02

1957.14

1890.51

1704.57

1446.83

1413.68

1231.55

1169.35

1063.85

1019.70

896.40

798.48

735.27

615.70

542.30

519.42

458.32

500

1000

1500

2000

2500

3000

3500

Wavenum

ber cm

-1

0 20 40 60 80 100 120 140 160

Transmittance [%]

MK

K

AD

IP

AT

TA

+C

AP

934.0 R

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2/22/2017 4:36:19 P

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Fig

ure N

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4: IR

spec

tra

of P

hy

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f Mu

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a k

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34



Figure No.15: Optical and Binocular microscope images of nanosponges

Figure No.16: Percent production yield of nanosponges

Figure No.17: Percent Entrapment efficiency of nanosponges

Figure No.18: Percent Actual drug content of nanosponges



Figure No.19: SEM of Nanosponges of Murraya Koenigii extract

Figure No.20: Particle size of nanosponges

Figure No.21: Zeta Potential of nanosponges



Figure No.22: In vitro diffusion study of formulations F1-F3





Infra red spectroscopy

Blank nanosponges

3736.77

3617.23

3531.91

3483.34

3412.59

3270.57

2990.53

2947.52

1724.76

1441.89

1383.45

1234.44

1141.86

1020.58

986.83

908.02

845.92

752.37

671.57

659.99

580.18

554.38

500100015002000250030003500

Wavenumber cm-1

50

100

150

200

250

Transmittance [%]

Figure No.25: The IR Spectrum of blank nanosponge

Drug containing nanosponges

3939.36

3740.32

3647.73

3559.41

2988.74

2947.72

2794.02

2689.73

2551.41

2329.78

2240.18

2067.22

1895.80

1725.69

1442.80

1383.90

849.03

809.19

753.65

662.74

626.17

500100015002000250030003500

Wavenumber cm-1

020

40

60

80

100

Tra

nsmittance [%]

Figure No.26: The IR spectrum of drug containing nanosponges

Differential scanning Calorimetry (DSC)

Figure No.27: DSC of drug loaded nanosponges



Figure No.28: DSC of Murraya koenigii containing gel

Figure No.29: Weight of animals after creating burn wound

Figure No.30: Surface area of burn wound



Figure No.31: % of burn wound contraction

Day ‘0’

Group 1 Group 2 Group 3

Day ‘7’


Day ‘14’


Day ‘21’




Day ‘28’

Group 1 Group 2 Group3

Figure No.32: Images of burn wound healing in rats

CONCLUSION

Burn wound injuries to the skin result in loss of its

protective function as a barrier to the

microorganism leading to the high risk of infection.

Thus burn wound patient face high morbidity than

mortality because of the large uncovered burns

getting infected; healing of wound takes place long

period of dressing, leading to deformities and

contractures. Managing burn injuries properly is

important because they are painful and can cause

disabling scarring, exclusion of affected parts or

even death in severe cases. Problems such as

infection, electrolyte imbalance, shock and

respiratory suffering may occur. Herbal drugs have

gained more popularity during last decade

throughout the world. It had been used by 80% of

population in developing countries. Herbal drug

contains many phytoconstituents and some of them

are important active phytoconstituents. These

herbals when formulated should contain similar

quantity of these constituents as that of original

herb. Nanosponges are mesh like structure and due

to their small size and porous nature. They can

easily bind poorly water soluble drug. The F6 batch

showed very fine, spherical and free flowing

nanosponges. By formulating nanosponges we got

free flowing drug loaded nanosponges. Further

Nanosponges having Murraya koenigii

implemented in gel and In-vivo activity was

checked. It was concluded that prepared

formulation F6 showed significant burn wound

healing activity.

ACKNOWLEDGEMENT

We, the authors, very much greatful to Dr. N.H.

Aloorkar (Principal, Satara College of Pharmacy,

Satara), Dr. A.S. Kulkarni (Vice- Principal, Satara

College of Pharmacy, Satara), Dr. S. H. Mujumdar

(H.O.D., Dept. of Pharmaceutics, Satara College of

Pharmacy, Satara), Dr. S.S. Gilda (H.O.D.,

Department of QAT, Satara College of Pharmacy,

Satara) for their valuable guidance and technical

suggestion and also for their support to make this

article with full of information in every aspect.

CONFLICT OF INTEREST

We declare that we have no conflict of interest.

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Please cite this article in press as: Poonam Jadhav. Formulation and evaluation of nanosponges containing murraya

koenigii extract for burn wound healing, International Journal of Research in Pharmaceutical and Nano Sciences,

7(5), 2018, 175-199.

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