Asian Journal of Biomaterial Research 2015; 1(1):23-26 23

Research Article

Comparative studies on pure curcumin ointment and curcumin loaded

transferosomes for wound healing potential
*
Preeti Sharma, Fedelic Ashish Toppo, R. S. Pawar

Pharmacognosy and Phytochemistry Laboratory, VNS Institute of Pharmacy, Neelbud, Bhopal, MP,
462044, India

Received: 29 September 2015 Revised: 18 October 2015 Accepted: 21 October 2015

Abstract
Objective: The aim of present research is to improve bioavailability of curcumin by tranferosome. Materials and
methods: The transferosomes of pure curcumin were prepared and characterized for optical study and entrapment
efciency. Incision wound model was performed and the parameters like tensile strength and hydroxyproline content
was estimated. Results: In control group, hydroxyproline content observed was 9.19 μg/500mg of tissue. It was higher
in group treated with transferosomes that is 23.44 μg/500mg of tissue. In standard and curcumin ointment treated
groups it was 16.36 and 18.99 μg/500mg of tissue. The tensile strength in control was only 328g and in standard it was
649g. In groups treated with transferosomes and curcumin ointment it was 665g and 654g respectively. Conclusion:
Thus, the results showed that transferosomes can be better option for skin treatments than ointments.
Keywords: Wound healing, Curcuma amada, Transferosomes, Curcumin, Hydroxyproline

Introduction variety of clinical indications (Barry, 2002). The flexible or

Wounds are physical injuries that outcome in an opening or
rupture of the skin. Conventional or synthetic drugs used as
wound healing agents are often inadequate, so peoples shifted
their choice to naturals from synthetics. Thus, there is the
demand to find additional information regarding the wound
healing potential of plant species (Vassilopoulos, 2002).
Plants and their extracts have immense potential for
the management of different types of wounds. The advantage of
an in-vivo model in wound healing research is that the wounded
tissue is similar to wound found in clinical practice and in the
case of skin wounds, can be made in human subjects.
Transdermal delivery of drugs through the skin to the systemic
circulation provides a convenient route of administration for a
*Address for Corresponding Author:
* Corresponding Author
Dr. R. S. Pawar
Pharmacognosy and Phytochemistry Laboratory,
VNS Institute of Pharmacy,VNS Campus, Vidya Vihar, Neelbud,
Bhopal-462044, India
E-mail:dr_pawar14@rediffmail.com
Tel.: 09826219429
deformable vesicles are called elastic vesicles or
transferosomes. A transferosome carrier is an artificial
vesicle and resembles the natural cell vesicle. Thus it is
suitable for targeted and controlled drug delivery (Barry,
2004; Honeywell et al., 2005).
Transferosomes are composed of phospholipids like
phosphatidylcholine which self assembles into lipid bilayer
in aqueous environment and closes to form a vesicle (Dubey
et al., 2006. Saraf, et. al., 2006). An edge activator consists
usually of single chain surfactant that causes destabilization
of the lipid bilayer thereby increasing its fluidity and
elasticity [Pirvu et al., 2010].
Curcumin is the product obtained by solvent extraction of
turmeric i.e., the ground rhizomes of Curcuma longa L.
(Curcuma domestica Valeton) and purification of the extract
by crystallization. The principal colouring components of
curcumin exhibit a keto-enol tautomerism and antioxidative
properties. Curcumin is an orange-yellow crystalline
powder (Jonathan et al., 2009).
Curcumin is an herbal drug hence devoid of any side effect
like bleeding in upper GIT, hepatotoxicity etc. but main

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 Asian Journal of Biomaterial Research 2015; 1(1):23-26
problem with curcumin when given orally is its poor
bioavailability due to less GI absorption. Nearly 25 to 85% of
orally administered curcumin is eliminated unabsorbed by
faeces. Difficulty is also associated with its topical application
due to less permeability through skin (Jonathan et. al., 2009). To
alleviate this problem vesicular drug delivery system
transferosomes is formulated to deliver curcumin across skin.
Materials and methods
Animals
Healthy albino rats of either sex (100-150g) with no prior drug
treatment were selected to carry out all the present in vivo
studies. The animals were used after an acclimatization period of
10 days to the laboratory environment. They were housed in
standard metal cages and provided with food and water ad
libitum. Animal study was performed in division of
pharmacology, VNS Institute of Pharmacy, Bhopal (M.P.) with
due permission from institutional ethical committee
(Registration No. 778/03/C/CPCSEA).
Formulation
A simple ointment base as per Indian pharmacopeia was
prepared by fusion method. Simple ointment base was served as
control. Formulation of pure curcumin drug, 10% of ethanolic
extract of Curcuma amada was prepared by fusion method
(Bhandarkar et al., 2015;Akanksha et. al., 2009).
Transferosomes of pure curcumin
Formula
Drug (pure curcumin): 50 mg
Soya lecithin : 100 mg
Tween 80 : 250 μl
Chloroform : 10 ml
Ethanol : 10 ml
Method: Drug, Lecithin and Tween 80 were dissolved in 20 ml
of ethanol: chloroform (1:1) mixture. The above solution was
mixed uniformly with the help of bath sonicator (Dolphin). The
solution was then placed in a rotary flask and attached to the
Rota evaporator (IKA HB10 Digital). The Rota evaporator was
o
set at 60 rpm at 43 C.Athin lipid film was formed inside the flask
wall with rotation. The thin film was kept overnight for complete
evaporation of solvent. The film was then hydrated with
phosphate buffer (pH 7.4). The transferosome suspension was
then collected. Transferosome was then centrifuged in Cooling
o
Centrifuge (Remi C-24B1) for 90 min at 2 C, 14000 rpm. The
pellets of transferosomes were obtained and suspended in a very
little amount of phosphate buffer just to suspend the pellets. The
transferosomes were now ready to apply on the wounds.
Incision wound model
For incision wound model, 24 animals of either sex weighed
between 100-150 g were divided into four groups consist six rats
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in each as follows:
Group I: Control (untreated)
Group II: Standard (0.1 % Silver sulfadiazine ointment)
Group III: Transferosome of pure curcumin
Group IV: 10% pure curcumin ointment
All animals were anaesthetized before wound creation and
two full thickness paravertebral long incisions were made
through the skin at the distance of about 1 cm from midline
on each side of the depilated back of rat. The both edges
kept together and stitched with black silk surgical thread
(no. 000) and a curved needle (no.11) was used for stitching.
The continuous threads on both wound edges were
tightened for good closure of the wound. After stitching,
wound was left undressed then transferosome of pure
curcumin and pure curcumin ointment were applied daily
up to 9 days. Tensile strength of the wound skin was
measured using tensiometer. From the healed wound, a
specimen sample of tissue was also collected from each rat
for histopathological examination. Hydroxyproline content
in healed tissues was also determined (Ehrlich and Hunt,
1968).
Characterization of Transferosome
Optical study of curcumin-loaded transferosomes
The optical study of the curcumin-loaded transferosomes
was studied with the help of Labomed CXR3 Optical
microscope at 10X.
Determination of percentage entrapment efficiency
Transferosome entrapped curcumin was estimated by
centrifugation method. The prepared transferosomes were
placed in centrifugation tube and centrifuged at 14000 rpm
for 90 minutes. The pellets obtained from the formulation
were then dissolved in the ethanol and absorbance was
measured. The standard curve of pure curcumin was also
prepared in ethanol.
Calculations for entrapment efficiency
Let the concentration of curcumin in 10 ml solution of pellet
in ethanol be 'x' μg/ml.
Then the amount of curcumin present in 10 ml of solution of
pellet in ethanol will be 10x ug/ml, which is equal to amount
present in pellet obtained from 5 ml formulation equal to
amount entrapped in transferosomes pellet obtained from 5
ml formulation.
Theoretical amount of drug present in 5 ml formulation=
[drug entrapped + drug unentrapped].
Theoretical amount of drug present in 40 ml formulation
is = 50 mg
 Asian Journal of Biomaterial Research 2015; 1(1):23-26
Theoretical amount of drug present in 5 ml formulation is
= 50 x 5 mg = 6.25 mg or 6250μg/5ml 40
Now, Calibration curve equation for curcumin in ethanol is ABS
3 2
= K3C + K2C + K1C + K0
K3 = 0.0000, K2 = 0.0000, K1 = 0.1527, K0 = 0.0000
2
r = 0.9934
Thus,ABS = 0.1527 x C
Where, ABS is the absorbance of curcumin in pellets 5 ml
formulation = 0.671
Amount of drug present in 5ml formulation = 4.394 x 10 x 5 =
219.711 ug/5 ml.
Wound healing evaluation parameters
Epithelialization period
Epithelialization period was monitored by noting the number of
days required for the Escher to fall off from the wound surface
without leaving a raw wound behind. The epithelialization period
was measured from initial day (Ehrlich and Hunt, 1968).
Tensile strength
Tensile strength is the resistance to breaking under tension. It
indicates how much the repaired tissue resists to breaking under
tension and may designate in part of repaired tissue. Tensile
strength of wound also represents the effectiveness of wound
healing. Usually wound-healing agents promote the gaining of
tensile strength. For this the newly repaired tissue including scar
was excised to measure the tensile strength. The instrument used
for measurement is called tensiometer (Rashed, et al., 2003).
Hydroxyproline estimation
Increase in hydroxyproline content indicates increased collagen
synthesis, which in turn leads to enhanced, wound healing. Its
estimation helps clinically to understand progress rate at which
the healing process is going on the connective tissue of the wound
(Kuwano et. al., 1994).
For the determination of hydroxyproline content, the
animals from each group were anaesthetized by using diethyl
ether. The wound tissues were excised and dried in a hot air oven
o and 18.99 μg/500mg of tissue.
at 60-70 c to constant weight and were hydrolysate was
neutralized to pH 7.0 and was subjected to Chloramine-T
oxidation for 20 min. the reaction was terminated by addition of
0.4 M perchloric acid and colour was developed with the help of
o
Ehrlich reagent at 60 C. The absorbance was measured at 557 nm
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using a spectrophotometer. The amount of hydroxyproline
in the samples was calculated using a standard curve
prepared with pure L-hydroxyproline in the same time.
StatisticalAnalysis
All wound area measurements were expressed as
percentage initial wound size. The values are expressed as
mean ± SEM of six animals in each group. The data were
statistically analysed by Dunnett using the program Instat
Graph pad. Data are significant, P<0.001 compared with
control.
Result and Discussion
In wound healing activity, 10% ointment of Curcuma
amada extract (ethanolic) pure curcumin transferosomes
and its ointment were evaluated in incision wound model.
Effect of pure curcumin transferosomes, curcumin
ointment and standard ointment on epithelialization
period of incision wound models in rats.
The epithelialization time was measured from initial day. It
was significantly reduced in all the groups. The time taken
for complete epithelialization of the incision wound was
18.533 days in control group. The epithelization time was
15, 16.13 and 17.33 for standard, transferosomes of pure
curcumin and 10% pure curcumin ointment.
Table 1. Effect of pure curcumin transferosomes, curcumin
ointment and standard ointment on epithelialization period
in incision wound model in rats
Groups Epithelialization period
(Mean time in days)
Group I (Control) 18.53 ± 0.4282*
Group II (Standard) 15.0 ± 0.3651*
Group III (Transferosomes of pure curcumin) 16.13 ± 0.4773*
Group IV (10% pure curcumin ointment) 17.33 ± 0.4944*
n = 6 albino rats per groups; values are represents mean ± SEM. *P<0.001
(Comparison of I with II, III and IV)
Effect of pure curcumin transferosomes, curcumin
ointment and standard ointment on hydroxyproline
content and tensile strength of incision wound model in
rats.
In group I, hydroxyproline content observed was 9.19
μg/500mg of tissue. It was highest in group III treated with
transferosomes that is 23.44 μg/500mg of tissue. In
standard and curcumin ointment treated groups it was 16.36
The tensile strength in control was only 328g and in
standard it was 649 g. While it was 665 g and 654 g in groups
treated with transferosomes and curcumin ointment.
 Asian Journal of Biomaterial Research 2015; 1(1):23-26
Table 2 Effect of topical application of curcumin transferosomes
and curcumin ointment on hydroxyproline content and tensile
strength of incision wound model in rats
Groups Hydroxyproline content Tensile strength
(µg/500mg tissue)
2
(g/mm )
Group I (Control) 9.19 ± 0.55* 328 ± 3.64*
Group II (Standard) 16.36 ± 0.62* 649 ± 4.91*
Group III Transferosomes
23.44 ± 0.33* 665 ± 2.63*
of Pure curcumin
Group IV Pure curcumin
18.99 ± 0.37* 654 ± 3.29*
Ointment
n = 6 albino rats per groups; values are represents mean ± SEM. *P<0.01
(Comparison of I with II III and IV)
Some flavonoids (e.g. curcumin) possess wound healing
potential by increasing cellular proliferation and collagen
synthesis at the wound site, as evidenced by increase in DNA,
total protein and type III collagen content of wound tissues.
Curcumin treatment was shown to decrease the levels of lipid
peroxides (LPs), while the levels of superoxide dismutase
(SOD), catalase (CAT), glutathione peroxidase (GPx), activities
were significantly increased exhibiting the antioxidant
properties of curcumin in accelerating wound healing. Since,
flavonoid was found positive in ethanolic extract; the wound
healing activity may be due to flavonoids (curcumin). All the
values were expressed as mean ± SEM of six animals in each
group. All the results were significant (p< 0.001) when compared
to control (Woessner, et al., 1961). The result obtained showed
that pure curcumin transferosomes and pure curcumin ointment
of Curcuma amada possesses wound healing potential. The
transferosomes of curcumin shows better healing rate, tensile
strength and hydroxyproline content when compared to other
groups. The results indicate that transferosomes which is a novel
preparation have faster as well as better results in skin diseases
for those phytoconstituents in which tissue penetration is poor.
Acknowledgement
We would like to thank VNS Group of Institutions, Bhopal
(M.P.), for giving us the freedom to pursue interesting avenues of
inquiry and different ideas of work.
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