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FORMULATION AND EVALUATION OF HERBAL EMULGEL OF LANTANA

CAMARA LEAVES EXTRACT FOR WOUND HEALING ACTIVITY IN DIABETIC RATS

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 Indo American Journal of Pharmaceutical Research, 2016 ISSN NO: 2231-6876

FORMULATION AND EVALUATION OF HERBAL EMULGEL OF LANTANA CAMARA

LEAVES EXTRACT FOR WOUND HEALING ACTIVITY IN DIABETIC RATS
Sk. Shaheda Sultana*1, G.Swapna1, G. Sai Sri Lakshmi1, S. Swathi1, G. Nirmala Jyothi1, A. Seetha Devi2
1
Nirmala College of Pharmacy, Atmakuru(V), Mangalagiri-522503, Andhra Pradesh.
2
Hindu College of Pharmacy, Amaravathi Road, Guntur-522002, Andhra Pradesh, India.
ARTICLE INFO ABSTRACT
Article history The main aim of this work was to formulate the leaf extract of Lantana camara in to an
Received 06/08/2016 emulgel and investigate their excision wound healing activity in Diabetic rats. Ethanolic
Available online extract of dried leaves of Lantana camara were subjected to preliminary phytochemical
30/08/2016 evaluation. Emulgel formulations were prepared using different types of gelling agents:
Carbopol 934, Na CMC, HPMC, HPMC K15M, and HEC. The influence of the type of the
Keywords gelling agent on the drug release from the prepared emulgel was investigated. The prepared
Emulgel, emulgel were evaluated for their physical appearance, pH, Viscosity, Spreadability, in-vitro
Lantana Camara, drug release, pharmacological activity and stability. From the results it was found that the
Wound Healing, formulation EGF2 with 1%w/w Sodium CMC shows better drug release (92.8% at 8 h) and
Diabetic Rats. higher pharmacological activity compared to other formulations. Herbal emulgel of ethanolic
extract of Lantana camara shows significant improvement in excision wound contraction and
hence this is a promising candidate for wound healing in diabetic rats.

Corresponding author
Shaik Shaheda Sultana
Department of Pharmaceutics
Assistant professor
Nirmala College of pharmacy
MangalagiriGuntur dist.
Phone: 9493133208
sshahedasms@gmail.com

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Please cite this article in press as Shaik Shaheda Sultana et al. Formulation and Evaluation of Herbal Emulgel of Lantana
Camara Leaves Extract for Wound Healing Activity in Diabetic Rats .Indo American Journal of Pharmaceutical
Research.2016:6(08).
Page

Copy right © 2016 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical
Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Vol 6, Issue 08, 2016. Shaik Shaheda Sultana et al. ISSN NO: 2231-6876

INTRODUCTION
80% of the world population relies on medicinal plants for their primary health care. Such herbal medicines that are easily
available, cheaper, time tested and considered safer than most of modern synthetic drugs. Over 50% of the best selling
pharmaceuticals in use today were derived from natural products. Plants provide a bank of rich, complex and highly varied structures,
which are unlikely to be synthesized in laboratories. Lantana camara (Family- Verbanaceae) is a low, erect shrub which can grow to 2
- 4 meters in height. The leaf is ovate or oblong, 2 - 10 cm long and 2 - 6 cm wide, arranged in opposite pairs. Leaves are bright green,
rough, finely hairy with serrate margins and emit a pungent odor when crushed. [1] Since very long time Lantana camara.L reported to
be used in traditional medicine system for itches ,cuts , ulcers, swelling, [2,3] bronchitis and arterial hypertension.[4, 5]
Most of the topical preparations are used for the localized effects by virtue of drug penetration into the underlying layers of
skin or mucous membranes. Gels are a relatively newer class of dosage form created by entrapment of large amounts of aqueous or
hydro alcoholic liquid in a network of colloidal solid particles, which may consist of inorganic substances, such as aluminum salts or
organic polymers of natural or synthetic origin. [6] They have a higher aqueous component that permits greater dissolution of drugs, and
also permit easy migration of the drug through a vehicle that is essentially a liquid, compared with the ointment or cream base.[7]
These are superior in terms of use and patient acceptability. In spite of many advantages of gels a major limitation is the delivery of
hydrophobic drugs. So to overcome this limitation, emulgels are prepared and used so that even a hydrophobic therapeutic moiety can
enjoy the unique properties of gels [8]. When gels and emulsions are used in combined form the dosage forms are referred as
EMULGELS.[9] Emulgels for dermatological use have several favorable properties such as thixotropic, greaseless, easily spreadable,
easily removable, emollient, nonstaining, long shelf life, bio-friendly, transparent and pleasing appearance.[10]
The present study was conducted to formulate herbal emulgel of Lantana camara using gelling agents like Carbopol 934, Na
CMC, HPMC, HPMC K15M, and HEC. The prepared emulgels were evaluated for physicochemical as well as for pharmacological
activity.

MATERIALS
Carbopol 934, Na CMC, HPMC, HPMC K15M, HEC, Span 80, and Tween 80 were obtained from Loba Chem. Pvt Ltd,
Mumbai, Liquid Paraffin and Tri ethanolamine from Qualigens, and Propylene glycol from SD Fine Chemicals.

Plant materials and extract preparation: [11]

Lantana camara linn (Verbanaceae) were collected from Hindu College of Pharmacy Garden, Guntur, Andhra Pradesh and
was authenticated by Prof. Z Vishnuvardhan Ph.D. Dean, faculty of natural Sciences, ANU, Guntur. The foreign, earthy matter and
residual materials were removed carefully from the leaves and then cleaned and dried in the shade. It was then powdered and used for
extraction. Powdered leaves were placed inside a thimble made from thick filter paper, which was loaded into the main chamber of the
soxhlet extractor. The soxhlet extractor was placed onto a flask containing the extraction solvent until it get exhausted. The extract is
filtered and concentrated under reduced pressure. It was stored at 4- 80 C until use.

FORMULATION DEVELOPMENT: [12]

The composition of Lantana camara emulgel formulations was shown in Table 1. Different formulations were prepared
using varying amount of gelling agent. The method only differed in the process of making gel in different formulations. The
preparation of emulsion was same in all the formulations. The gel in formulations F1, F6, and F11, were prepared by dispersing
Carbopol 934 in purified water with constant stirring at a moderate speed then the pH was adjusted to 6 to 6.5 using triethanolamine.
The gel bases were prepared by dispersing Na CMC, HPMC, HPMC K15M and HEC in heated purified water (80°C) separately, and
the dispersion was cooled and left overnight. Formulations F2, F7, and F12 were prepared by Sodium CMC as a gelling agent; F3, F8
and F13 were prepared by HPMC K15M as a gelling agent; F4, F9, and F13 were prepared by HPMC as a gelling agent; F5, F10, and
F15 were prepared by HEC as a gelling agent. The oil phase of the emulsion was prepared by dissolving Span 80 in light liquid
paraffin while the aqueous phase was prepared by dissolving Tween 80 in purified water. Propyl paraben was dissolved in propylene
glycol whereas Lantana camara extract was dissolved in ethanol, and both the solutions were mixed with the aqueous phase. Both the
oily and aqueous phases were separately heated to 70° to 80°C; then the oily phase was added to the aqueous phase with continuous
stirring until cooled to room temperature. The obtained emulsion was mixed with the gel in 1:1 ratio with gentle stirring to obtain the
emulgel.
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Table 1: Composition of emulgel formulations containing ethanolic extract of Lantana camara.

Ingredients FORMULATION CODE

EG EG EG EG EG EG EG EG EG EG EG EG EG EG EG
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F 13 F 14 F 15
Lc extract (g) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Carbapol 934 (g) 0.1 - - - - 0.15 - - - - 0.2 - - - -
Na CMC (g) - 0.1 - - - - 0.15 - - - - 0.2 - - -
HPMC K 15M(g) - - 0.1 - - - - 0.15 - - - - 0.2 - -
HPMC (g) - - - 0.1 - -- - - 0.15 - - - - 0.2 -
HEC (g) - - - 0.1 - - - 0.15 - - - - 0.2
Light liquid paraffin 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4
(ml)
Tween 80(ml) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
Span 80 (ml) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05
Prop. glycol (ml) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Alcohol (ml) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Water suf. for 10 g qs qs qs qs qs qs qs qs qs qs qs qs qs qs qs

Characterization of Emulgel:
Physical Examination:
The prepared emulgel formulations are inspected visually for their color, appearance, extrudability and phase separation.

pH Evaluation:
pH evaluation is the important criteria especially for the topical formulation. The pH of the emulgel should be between 5-7 to
mimic the skin condition. If the pH of the prepared emulgel is acidic or basic, it may cause irritation to the patient. pH of the prepared
emulgel was measured using digital pH meter (ELICO LI 613). 1gm of gel was dissolved in 100 ml of distilled water and it was
placed for 2 hr and then dip the glass electrode into an emulgel. The measurement of pH of each formulation was done in triplicate
and average values were calculated.

Compatibility studies by FT-IR:

FTIR Spectroscopy was carried out to check the compatibility between drug and polymer .The IR spectra of Lantana camara,
Carbopol 934, Sodium CMC, HPMC,HPMC K 15,HEC and physical mixtures of drug and polymer was carried out by using FTIR
,Bruker. The scanning range was 400 to 4000 cm-1 and the resolution was 1cm-1. The wave numbers of characteristic peaks of physical
mixtures were compared with the pure samples and interpreted.

Rheological Studies:
The viscosity of the emulgel during handling, transport and storage is an important criterion. Viscosity of the emulgels was
determined using Brookfield viscometer, Spindle (no: 42) type, model LVDV-E at 0.5 and 1 r/min. Small amount of the emulgel was
taken in the cup and the spindle was dipped in it for about 5 minutes and then the readings were taken.

Spreadability:
Spreadability denotes the extent of area to which the emulgel readily spreads on application to skin or the affected part. The
bioavailability efficiency of an emulgel formulation also depends on its spreading value. The Spreadability was expressed in terms of
time in seconds taken by two slides to slip off from the emulgel which was placed in between the slides, under certain load. Lesser the
time taken for separation of the two slides, better the spreadability. Two sets of glass slides of standard dimensions were taken. The
herbal emulgel formulation was placed over one of the slides. The other slide was placed on the top of the emulgel, such that the
emulgel was sandwiched between the two slides in an area occupied by a distance of 7.5 cm along the slide.
100gm weight was placed upon the upper slides so that the emulgel between the two slides was pressed uniformly to form a thin layer.
The weight was removed and the excess of emulgel adhering to the slides was scrapped off. The two slides in position were fixed to a
stand without slightest disturbance and in such a way that only the upper slide to slip off freely by the force of weight tied to it. A 10
gm weight was tied to the upper slide carefully. The time taken for the upper slide to travel the distance of 6.8 cm and separated away
from the lower slide under the influence of the weight was noted. The experiment was repeated by three times and the mean time was
taken for calculation. Spreadability was calculated using formula:
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S = M. L / T

Where M= weight tied to upper slide (10g)

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L = length of glass slide (6.8cm)

T = Time taken to separate the slides

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Extrudability Study of Topical Emulgel (Tube Test) [13]:

It is an usual empirical test to measure the force required to extrude the material from tube. The method applied for
determination of applied shear in the region of the rheogram corresponding to a shear rate exceeding the yield value and exhibiting
consequent plug flow. In the present study, the method adopted for evaluating emulgel formulation for extrudability is based upon the
quantity in percentage of emulgel and emulgel extruded from lacquered aluminum collapsible tube on application of weight in grams
required to extrude at least 0.5 cm ribbon of emulgel in 10 seconds. More quantity extruded better is the extrudability. The
measurement of extrudability of each formulation is in triplicate and the average values are presented. The extrudability is then
calculated by using the following formula:

Extrudability = Applied weight to extrude emulgel from tube (in gm) / Area (in cm 2)

Drug Content Determination: [12]

1 gm each formulation containing approximately 40 mg of drug was taken in a 50 ml volumetric flask and diluted with
ethanol and shaken to dissolve the drug in ethanol. The solution was filtered through whattmann filter paper, 0.1 ml of the filtrate was
pipette out and diluted to 10 ml with ethanol. The content of the drug was estimated spectrophotometrically by using standard curve
plotted at 332 nm.

Drug Content = (Concentration × Dilution Factor × Volume taken) × Conversion Factor

In-vitro Release/Permeation Studies: [12]

In vitro release studies were carried out using Franz diffusion cell of 10ml capacity. Egg membrane was isolated and used for
the study. Pre-weighed emulgel was spread evenly on the egg membrane. The egg membrane was clamped between donor and
receptor compartment. The receptor compartment was filled with 10 ml of pH 6.8 phosphate buffer maintained at 37 oC and stirred by
using magnetic stirrer. 1 ml sample was collected at suitable time intervals (i.e., for every 30mins until complete drug was released)
and replaced with fresh buffer. The collected samples were analyzed for drug content by UV –Visible Spectrophotometer at 332nm.

Skin Irritation Test: [12]

0.5 g of the optimized formulation was applied on the hair – free skin of rabbits by uniform spreading over an area of 4 cm2.
The skin surface is observed for any visible change such as erythema (redness) after 24, 48 and 72 hours of the formulation
application. The mean erythmal sores are recorded depending on the degree of erythema.
No erythema =0
Slight
Moderate erythema (dark pink) =2
Moderate to severe erythema (light red) =3
Severe erythema (extreme redness) =4

Ex-vivo Studies of Optimized formulation: [14]

In the experiment, a total of 24 rats were used. The rats were divided into 4 groups comprising of 6 animals in each group as follows:
Group I : Control group (wounded rats)
Group II : Diabetes was induced by Alloxan in wounded rats (150mg / kg)
Group III : Standard drug (soframycin ointment) was given to the diabetes induced wounded rats.
Group IV : Test drug of optimized formulation (EGF2 i.e., Sodium CMC (0.1%) emulgel of Ethanolic extract of L.camara)
was given to the diabetes induced wounded rats.

Induction of diabetes mellitus in rats:

After overnight fasting, 24 rats were induced diabetes mellitus by giving a single injection of Alloxan (150mg/kg, I.P.)
prepared by dissolving in Normal saline solution. Fasting blood glucose level was estimated using Glucometer (Accuchek meter) after
24 h of the injection by taking blood from tail vein. Normal control group was not induced diabetes mellitus. Rats showing blood
glucose level of 250-300mg/dl were included for the wound healing study.

Excision wound and treatment:

The experimental animals were anaesthetized with Di-ethyl ether, over the local selected region and under anesthesia, the hair
of rats was shaved from the dorsal thoracic central region. One excision wound was inflicted by cutting away 500 mm 2 full thickness
of skin from the predetermined area. Excision wound was induced in diabetic as well as normal rats. Each animal was maintained in a
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separate cage till the end of the study. The Emulgel was applied topically twice daily from the day of creation of excision wound, till
the wound was completely healed. Normal control and diabetic control groups received vehicle (simple Emulgel base) topically.
Group III were treated with the standard drug which is available in market and Group IV were treated with optimized formulation
twice a day. The wound areas were measured on 0th, 3rd, 6th, 9th and 12th day, until the wound was healed for the experimental rats.
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Measuring wound healing activity:

Wound healing activity was measured by assessing two parameters such as % wound contraction rate and epithelization
period. Wound area was traced using butter paper and was retracted on a millimeter scale graph paper. Percent wound contraction was
calculated using the following formula;

Initial wound size- specific day wound size x 100

% wound contraction =
Initial wound size

Epithelization period was monitored by noting the number of days required for each to fall away, leaving no raw wound behind.

Stability Studies: [15]

Stability studies were carried out for the optimized formulation (EGF2) according to International Conference on
Harmonization (ICH) guidelines. Short term accelerated stability studies were carried out for the period of 3 months for the
formulations. The samples were stored at different temperature conditions i.e., refrigeration temperature (4-8oC), room temperature
(25 ±2oC) and oven maintained at (45°C ± 2oC). Sample was withdrawn on weakly interval and analyzed for visual appearance,
clarity, pH, spreadability, viscosity, and drug content. Sample was withdrawn at 1,3,6,9 and 12 week and assessed for various
parameters. At the end of 12th week they were evaluated for physical parameters and integrity of the product.

RESULTS AND DISCUSSIONS

Compatibility studies:
From the (Fig 1-2) FT-IR spectra of physical mixture of the drug, polymer, and other ingredients, it was revealed that there
was no chemical interaction of plant extract with other excipients.

Fig 1: FT –IR spectrum of ethanolic extract of Lantana camara.

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Fig 2: FT –IR spectrum of optimized formulation(EGF2).

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Calibration curve of ethanolic extract of Lc in phosphate buffer of pH 6.8:

Standard plot of Lantana camara was plotted as per the procedure in experimental methods and its linearity was shown in
Table 2 and Fig 3. The standard graph of Lantana Camara shows good linearity with R2 value of 0.999, which indicates that it obeys
Beer’s-Lambert’s Law in the concentration range of 0-100 µg/ml.

Table 2: Calibration curve of ethanolic Lantana camara in phosphate buffer of pH 6.8.

S.No Concentration (µg/ml) Absorbance (332nm)

1. 0 0
2. 20 0.112 ±0.002
3. 40 0.213 ±0.008
4. 60 0.318 ±0.006
5. 80 0.426 ±0.004
6. 100 0.524 ± 0.006

Fig 3: Calibration curve of ethanolic Lantana camara in phosphate buffer of pH 6.8.

Physical appearance:
All the formulations were evaluated for their color and appearance. The physical appearances of all the formulations were found to be
greenish, clear and transparent. (Table 3)

Extrudability studies:
Extrudability studies of all formulation were carried out as per procedure stated in methodology section. The results were shown in
table 6.6. It was found that formulations EGF2, EGF4 and EGF5 are having excellent extrudability; Formulations EGF1, EGF3,
EGF7, EGF9, EGF10 and EGF12 were having good extrudability; Formulations EGF6, EGF8, EGF14 and EGF15 were having poor
extrudability while formulations EGF11 and EGF13 were having very poor extrudability.

Table 3: Characterization of Lc Emulgel Formulations for Color, Appearance and Extrudability.

Formulations Colour Appearance Extrudability

EG F1 Greenish Clear & Transparent Good
EG F2 Greenish Clear & Transparent Excellent
EG F3 Greenish Clear & Transparent Good
EG F4 Greenish Clear & Transparent Excellent
EG F5 Greenish Clear & Transparent Excellent
EG F6 Greenish Clear & Transparent Poor
EG F 7 Greenish Clear & Transparent Good
EG F8 Greenish Clear & Transparent Poor
EG F9 Greenish Clear & Transparent Good
EG F10 Greenish Clear & Transparent Good
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EG F11 Greenish Clear & Transparent Very poor

EG F12 Greenish Clear & Transparent Good
EG F13 Greenish Clear & Transparent Very poor
EG F14 Greenish Clear & Transparent Poor
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EG F15 Greenish Clear & Transparent Poor

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pH determination
Skin compatibility is the primary requirement for a good topical formulation. It was found that the pH of all formulations
were in the range of pH 6.4 to 7.0 (Table 4) which indicates skin capatibility i.e., Lc emulgels can be applied to the skin without any
discomfort or irritation.

Spreadability studies
Spreadability study is one of the criteria for an emulgel to meet the ideal qualities that it should possess good spreadability. If
spreadability value is more, it would be properly spread over the skin which is more beneficial as per patient compliance concern.
All the formulations were checked for the spreadability and the data was given in the table 5. The values of the spreadability indicated
that the emulgels were easily spreadable by small amount of shear. The order of increasing spreadability was EGF2> EGF4> EGF7>
EGF5> EGF9> EGF3> EGF1> GF10 >EGF12> EGF6> EGF14> EGF15> EGF13> EGF11. By taking the data into consideration, it
was observed that concentration of polymers makes the difference in the spreadability. Among all the formulations EGF2 emulgel
formulation containing Sodium CMC (1%) has shown highest spreadability value (17 cm/sec).

Rheological study
The viscosities of all the formulations were measured using Brookfield viscometer, spindle 42 (DV++) at 0.5 and 1 rpm and
the viscosities for all the formulations were given in the table 5. It was found that all the formulations followed shear thinning effect
with thixotropic property. It was observed that the viscosity of the formulation increased with the increase in polymer concentration.
Among all the formulations EGF2 emulgel formulation containing Sodium CMC in minimum concentration (1%) has shown low
viscosity (80.4 cps) while EGF11 emulgel formulation containing Carbopol 934 in maximum concentration (2%) has shown high
viscosity (720 cps).

Drug content determination:

Drug content of all the formulations was carried out as per procedure stated in the methodology section. Drug content of all
the formulations were found to be in the range of 95-100% as indicated in the table 5. The result indicates that uniform amount of drug
is present in all the emulgel formulations.

Table 4: Characterization of Lc Emulgel Formulations For pH, Spreadability, Viscosity, Drug content.

Formulation pH* Spreadability Viscosity (cps) %Drug content*

(cm/sec)* 0.5 rpm 1 rpm
EG F1 6.8±0.01 9.06±0.11 589 308.3 98 ±0.12
EG F2 6.82±0.4 17±0.56 80.4 67.8 100 ±0.5
EG F3 6.52±0.8 9.7±0.68 205.2 175.2 98 ±1
EG F4 6.9±0.3 15.11±0.36 106.8 93 100 ±0.1
EG F5 6.8±0.11 11.3±1.09 113 111.6 99 ±0.6
EG F6 6.9±0.56 6.8±0.54 601 556 96±0.7
EG F7 6.8±0.1 11.33±0.12 228 195 100±0.01
EG F8 6.6±0.15 7.15±0.32 328.7 319.1 97 ±0.09
EG F9 6.8±0.1 10.46±0.67 250 195 98±0.1
EG F10 7±0.11 8.5±0.76 291.5 222 89 ±0.6
EG F11 6.9±0.2 5.23±0.25 720 638 95 ±0.3
EG F12 6.45±0.3 8.5±0.12 263.9 225 98 ±0.02
EG F13 6.39±0.1 6.18±0.85 650.9 589.1 96 ±0.6
EG F14 6.8±0.01 6.8±0.76 417.5 359.3 98 ±0.5
EG F15 6.9±0.05 6.47±0.13 436.7 379.1 97±0.13
*All values represent mean ±standard deviations (SD), n=3

In-vitro drug permeation for Lc emulgel formulations:

The emulgel formulations of Lantana camara (EGF1-EGF15) were characterized for their drug diffusion study using Franz
diffusion cell through a membrane. The release of Lc from the prepared emulgels was performed in order to study the effect of
different polymer types and concentration on the release of Lc aiming to select the best formula.
The percentage of Lantana camara released from emulgel formulations containing 1% of different polymers were reported in table 5
and fig 4. The percentage of Lantana camara released from emulgel formulations containing 1.5% of different polymers were
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reported in table 6 and fig 5. The formulations containing Carbopol 934 1.5%
The percentage of Lantana camara released from emulgel formulations containing 2% of different polymers were reported in
table 7 and fig 6.
The release of the drug from its emulgel formulations can be ranked in the following descending order: EGF2> EGF4>
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EGF5> EGF7> EGF9> EGF12> EGF10> EGF14> EGF15> EGF3> EGF8> EGF13> EGF1> EGF6> EGF11.
Among the five polymers used the drug release is higher for Na CMC then followed by HPMC, HEC, HPMC K15M and Carbopol
934.

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These results suggested that EGF2 is effective for topical application as highest percentage of drug released after 8hrs
(92.5%). It was observed that the most influenced factor in the Lantana camara release is the polymer type followed by the
concentration of the polymer.
Finally the results conclude that as the polymer concentration increases the spreadability and drug release decreases, whereas
viscosity of the formulations increases.
EGF2 containing Sodium CMC 1% gelling agent has shown high spreadability (17 cm/sec), less viscosity (80.4 cps),
excellent extrudability, more drug content and highest drug release (92.5% in 8hrs) than all other emulgel formulations. So, it was
selected as the optimized formulation.

Table 5: In-vitro drug release profiles of Lc emulgel formulations (EGF1-EGF5).

Percentage release of ethanolic Lantana camara plant drug*

Time (min)
EG F1 EG F2 EG F3 EG F4 EG F5
0 0 0 0 0 0
30 9±0.11 23.75±0.25 14.5±0.32 21.5±0.30 21.5±0.45
60 19±0.56 34.75±1.05 25±0.82 32.75±0.33 32±0.22
90 29.25±0.68 46±1.22 36±0.32 44.25±0.63 43.75±0.92
120 31.75±0.36 48.25±0.40 39±0.74 46.25±0.38 45.75±0.72
150 34.5±0.9 50.5±0.40 41±0.45 48.25±0.35 47.75±0.82
180 40.5±1.09 56±1.01 47±0.86 53.75±0.54 52.75±0.12
210 46.5±0.54 61.5±0.18 52±0.29 59.5±0.678 58±0.37
240 52.25±0.32 68.25±0.67 58±0.39 66.25±0.17 64.5±0.77
270 58±0.67 75±0.47 65±0.33 73±0.78 71±0.98
300 61±1.11 77.25±0.85 67±0.34 75.25±0.19 73.5±0.45
330 64.25±0.35 79.5±0.22 70±0.55 77.75±0.26 76.25±0.55
360 66.5±0.12 82.25±0.99 72±0.78 80±0.76 78.75±0.88
390 69±0.76 85±0.34 73±0.32 82.50.17 81.5±0.41
420 69.5±0.25 86.75±0.12 75±0.49 84.5±0.03 82.75±0.56
450 70±1.05 88.75 76.5±0.50 86.5±0.31 84.25±0.8
480 73.25±0.18 92.5±0.3 80±0.26 90.25±0.76 88.25±0.5
*All values represent mean ±standard deviations (SD), n=3

Table 6: In-vitro drug diffusion profiles of Lc emulgel formulations (EGF6-EGF10).

Time (min) Percentage release of ethanolic Lantana camara plant drug*

EG F6 EG F7 EG F8 EG F9 EG F10
0 0 0 0 0 0
30 7±0.45 20±0.30 13±0.44 19.5±0.89 19±0.13
60 15.5±0.22 29.75±0.40 22±0.54 29±0.52 28.5±0.25
90 24.25±0.92 39.5±0.51 30±0.64 38.75±0.32 38.25±0.21
120 26.25±0.72 41.5±0.74 33±0.53 40.5±0.34 39.75±0.6
150 28.5±0.82 43.75±0.41 35.5±0.09 42.25±0.47 41.5±0.94
180 36.75±0.12 51.5±0.05 42±0.51 50.25±0.62 49.25±0.33
210 45±0.37 59.5±0.98 48±0.13 58.25±0.77 57±0.33
240 49±0.77 64.5±0.74 55±0.21 63±0.30 61.75±0.30
270 53.25±0.98 69.5±0.41 57±0.43 68±0.45 66.5±0.29
300 57.25±0.45 72.5±0.56 62±0.42 71.25±0.32 70.25±0.45
330 61.25±0.55 75.75±0.76 68±0.23 74.5±0.34 74.25±0.18
360 62.75±0.88 78.25±0.51 70±0.53 76.75±0.67 76.5±0.45
390 64.25±0.56 80.75±0.4 72±0.13 79.25±0.21 78.75±0.87
420 66.5±0.8 82.25±0.56 73.2±0.23 80.75±0.07 80±0.66
450 69±0.56 84±0.32 75±0.55 82.25±0.09 81.5±0.77
480 70±0.88 87±0.8 78±0.37 86±0.9 83.75±0.69
*All values represent mean ±standard deviations (SD), n=3.
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Table 7: In-vitro drug diffusion profiles of Lc emulgel formulations (EGF11-EGF15).

Time (min) Percentage release of ethanolic Lantana camara plant drug*

EG F11 E F12 E F13 E F14 EG F15
0 0 0 0 0 0
30 4.5±0.32 17.75±0.25 10.75±0.22 17.25±0.45 16.5±0.03
60 13±0.89 27.75±0.36 15±0.45 26.75±0.80 25.5±0.09
90 21.75±0.50 38±0.92 25±0.34 36.5±0.09 34.5±0.2
120 23±0.54 38.75±0.35 28±0.23 37.25±0.07 35.25±0.60
150 24.5±0.72 39.75±0.98 30±0.76 38±0.56 36.25±0.80
180 31.75±0.29 47±0.34 38±0.32 45.25±0.11 44±0.45
210 39.25±0.52 54.5±0.22 46±0.53 52.75±0.12 51.75±0.34
240 44.5±0.45 59.75±0.21 50±0.23 58±0.59 56.5±0.56
270 50±0.22 65.25±0.90 55±0.12 63.25±0.60 61.5±0.34
300 54±0.78 69.5±0.45 59±0.02 68±1 66±0.32
330 58±0.09 74±0.76 64±0.09 73±0.90 70.75±0.56
360 61±0.76 76.25±0.23 67±0.12 75±0.61 73.5±0.54
390 64±0.45 78.5±0.12 69.3±0.67 77.25±0.63 76.25±0.45
420 65.5±0.12 80.25±0.23 71±0.70 79±0.55 78.25±0.34
450 67±0.23 82.25±0.56 74±0.15 80.75±0.31 80.25±0.23
480 68.5±0.32 85.5±0.46 76.1±0.34 83.75±0.07 81.5±0.34
*All values represent mean ± standard deviations (SD), n=3

Fig 4: Drug release profiles of Lc emulgel formulations (EGF1-EGF5).

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Fig 5: Drug release profiles of Lc emulgel formulations (EGF6-EGF10).

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Vol 6, Issue 08, 2016. Shaik Shaheda Sultana et al. ISSN NO: 2231-6876

Fig 6: Drug release profiles of Lc emulgel formulations (EGF11-EGF15).

In vitro drug release kinetics:

The kinetics were estimated by fitting the data in various kinetic models like zero order, first order and the the results were
shown in Table 8. When the R2 values of regression plots for first order and zero order were considered, R2 values of first order plots
were found to be higher than zero order plots. Hence it is evident that the drug release from all emulgel formulations followed zero
order kinetics.

Release mechanisms
By incorporating release data in Higuchi and erosion models, the R 2 values of all the formulations were found to be greater
for Higuchi model. So, all the formulations in this study were best expressed by Higuchi’s classical diffusion equation. The linearity
of plot indicated that the release process was diffusion controlled.
To further confirm the exact mechanism of drug release, the data was incorporated into koresmeyer Peppas model and the
mechanism of drug release was indicated according to the value of exponent ‘n’. For all the emulgel formulations the release exponent
‘n’ value found to be between 0.696 to 0.7. This indicates the drug released from all the emulgel formulations followed non – fickian
diffusion mechanism.

Table 8: Correlation Coefficient values of Lc emulgel formulations (EGF1-EGF15)./

2 2 2 Erosion Korseymeyer peppas

Formulations Zero order (R ) First order (R ) Higuchi (R ) 2 2
(R ) R n
EG F2 0.985 0.906 0.993 0.480 0.966 0.7

Skin Irritation Test:

The optimized formulation EGF2 (Na CMC 1%) was applied on the hair free skin of the rabbit. The applied area is observed
for 3 days and the result shows that the primary irritation index of the sample was 0.00 i.e., no erythema, edema and no irritation were
observed on the skin of the rabbit. The formulation is safe for topical use.

Ex-vivo wound healing activity assessment

The animal activity was started after getting IAEC permission (HCOP/IAEC/PR-7/2014).The glucose levels of wister albino
rats were measured before and after the alloxan induction. The glucose levels are shown in the Table 9. The effect of ethanolic extract
of leaves of Lantana camara emulgel on excision wound model was evaluated, the wound healing contracting ability of standard and
test were significantly greater than that of control and diabetic control (Table 10 and fig 7, 8). In excision wound model the percentage
of wound closure or closure rate were studied by recording the changes in wound area at fixed intervals of time.
Test rats (which received optimized formulation) showed comparable wound healing activity to that of standard rats (which
received soframycin ointment). Control group showed better significant wound healing activity compared to diabetic control rats.
It was further found that all the four groups showed decreasing wound area from day to day. However on 12 th day, group I
and II showed 54 and 49 % protection, which may be due to self immunity of animals; whereas standard showed 97.67%. Test showed
94% closure of wound which was comparable to that of standard rats.
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DISCUSSION
In this study the topical application of the optimized formulation on the infected wound of the rats caused a significant
(P<0.001) and faster wound closure and reduced the epithelization period. Although wound treatment with emulgel containing the
Page

extract of Lc showed a wound healing activity, the exact step and mechanism in wound repair process affected by the extract was not
established.

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Higher percentage of wound closure was observed in the group of animals treated with test on the 12th day of the experiment
which was comparable to that of standard treated animals.
Period of epithelization of test treated animals is similar to that of standard treated animals.
Further phytochemical studies are needed to isolate the active compounds responsible for wound healing activity. Further
studies with purified constituents are needed to understand the complete mechanism of wound healing activity of Lc leaves extract.
Thus, this investigation confirms the use of Sodium CMC emulgels containing Lc leaf extract as a wound healing emulgel preparation
in diabetic rats.

Table 9: Blood glucose values in rats before and after Alloxan induction.

Treatment group Alloxan induction – Blood glucose values in rats (mg/dl)

Before Alloxan induction After one week of induction
Group I 100±1.23 101±1.33
Group II 99±1.13 295±2.23
Standard 103±0.97 289±1.35
Test 98±1.11 290±0.35
n=6, all the values are mean ± SD.

Table 10: Ex-vivo wound healing activity assessment.

Percentage of closure of excision wound area (original wound area 500 mm2 Epithelization
Group Treatment
Day 3 Day 6 Day 9 Day 12 in days
1 Control 14.13±0.46 22.0±1.53 39.67±2.90 54.67±2.90 19.0±0.32
2 Diabetic Control 10.0±0.53 16.0±0.66 33.7±0.67 49.13±1.74 21.0±0.86
3 Standard 28.0±0.57* 46.0±1.15* 64.00±5.03* 97.67±0.33** 13±0.26**
(Soframycin)
4 Test (EGF2) 24.27±0.81* 38.00±1.15** 60.00±1.15** 94.00±2.08** 14.2±0.31**
All values are mean ± SEM where (n==6). One way ANNOVA followed by Dunnet’s test.*P<0.005, **P<0.001 when compared to
diabetic control animals.

Fig 7: Ex-vivo Wound Healing Activity for Optimized Formulation (EGF2)

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Diabetic control Control Standard Test

1st Day

3rd Day

6th Day

9th Day

12th Day

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15th Day

Fig 8: Wound Healing Images.

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Stability studies
Stability study was performed on optimized batch EGF2 at ambient conditions. The results obtained after 3 month time
period are shown in table 11. The result revealed that there was no significant change in the colour, pH, and drug content in optimized
formulation. The results conclude that the optimized formulation is stable for 3 months and can be applied topically.

Table 11: Short term stability data of optimized formulation.

Initial After 3 month

Appearance pH Drug content (%) Appearance pH Drug content (%)
Greenish 6.82 100±0.01 Greenish 6.82 100±0.0
All values represent mean ± standard deviations (SD), n=3

Abbrevations:
Sodium CMC - Sodium Carboxy methyl cellulose; HPMC- Hydroxy Propyl Methyl Cellulose
HEC- Hydroxy Ethyl Cellulose; EGF- Emulgel Formulation

CONCLUSION
The phytochemicals present in the extract were identified by qualitative phytochemical screening, which reveals the presence
of alkaloids in chloroform extract, alkaloids, saponins, and carbohydrates in aqueous extract and alkaloids, proteins and flavonoids in
the alcoholic extract. The preliminary pharmacological study showed that the ethanolic extract of Lantana amara Linn leaf possess
maximum wound healing effect in rats and the effects produced was maximum in 10% alcoholic extract and this concentration was
used for the formulation.
All the formulations were found to be neutral (pH 6.4 to 7.0) and drug content was found to be in the range of 95 -100%. On
physical evaluation EGF2 (SCMC) and EGF4 (HPMC) were found to be optimum in terms of viscosity, spreadability and
extrudability. Maximum release was shown by EGF2 which follows Higuchi diffusion model. Stability studies revealed that there was
no significant difference in the physical and chemical parameters. Thus the formulations were found to be stable for 3 months.
Pharmacological evaluation of emulgels revealed that all formulations are non sensitizing and safe for use. Pharmacological studies of
the formulations showed that EGF2 (SCMC) has greatest pharmacological activity. It was finally concluded that the formulations
EGF2 was found to be more promising formulation as it shows better physicochemical characteristics and higher pharmacological
activity compared to other formulations. Future Research works can be done to get better results

ACKNOWLEDGEMENT
Authors would like to thank Nirmala & Hindu college of pharmacy, Guntur, for providing chemicals and required
infrastructure to carry out this research work.

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