Formulation and Evaluation of Herbal Emulgel of Pothos scandens

Mohammed Haneefa K.P et al /J. Pharm. Sci. & Res. Vol. 6(2), 2014, 63-67
Formulation and Evaluation of Herbal Emulgel of
Pothos scandens Linn for Burn Wound Healing Activity
Mohammed Haneefa K.P1*, Guru Prasad Mohanta2,Chandini Nayar3
1
Alshifa College of Pharmacy, Poonthavanam Post, Kerala, India
*Research Scholar, School of Chemical & Biotechnology, SASTRA University, Tamil Nadu, India
2
Department of Pharmacy, Annamalai University, Tamil Nadu, India
3
School of Chemical & Biotechnology, SASTRA University, Tamil Nadu, India
Abstract
The main aim of this work was to formulate the leaf extract of Pothos scandens in to an emulgel and investigate their burn
wound healing activity. Ethanolic extract of dried leaves of Pothos scandens were subjected to preliminary phytochemical
evaluation and wound healing activities studies. Emulgel formulations were prepared using three types of gelling agents:
Carbopol 934, Carbopol 940 and HPMC K4M. The influence of the type of the gelling agent on the drug release from the
prepared emulgel was investigated. The prepared emulgel were evaluated for their physical appearance, pH, viscosity,
spreadability, in vitro drug release, pharmacological activity and stability. It was finally concluded that the formulation F1 with
1%w/w Carbopol 940 was found to be more promising formulations as it shows better physicochemical characteristics and higher
pharmacological activity compared to other formulations. Herbal emulgel of ethanolic extract of Pothos scandens shows significant
improvement in burn wound contraction and hence this is a promising candidate in burn wound healing
Keywords: Pothos scandens, Burn wound healing, Emulgel , Carbopol 934, Carbopol 940, HPMC K4M.
INTRODUCTION:
Topical preparations are used for the localised effect at the
site of their application by virtue of drug penetration into
the underlying layer of skin or mucous membrane. Topical
drug administration is a localized drug delivery system
anywhere in the body through ophthalmic, rectal, vaginal
and skin as topical routes. The main advantage of topical
delivery system is to bypass first pass metabolism1, 2 .When
gels and emulsions are used in combined form the dosage
forms are referred as Emulgel. The presence of a gelling
agent in the water phase converts a classical emulsion into
an emulgel. Direct (oil-in-water) system is used to entrap
lipophilic drugs whereas hydrophilic drugs are
encapsulated in the reverse (water-in-oil) system3.
Emulsions possess a certain degree of elegance and are
easily washed off whenever desired. They also have a high
ability to penetrate the skin. Emulgels for dermatological
use have several favourable properties such as being
thixotropic, greaseless, easily spreadable, easily removable,
emollient, non-staining, water-soluble, longer shelf life,
bio-friendly, transparent & pleasing appearance4.
Pothos sc andens ( Family- Araceae ) is a climbing shrub
having adventitious aerial roots. The internodes of the plant
are 1.3-2.5 cm and its leaves are very variable. The leaves
are obovate, elliptic or lanceolate and coriaceous, having a
bright green colour. Pothos sca ndens has been used in
herbal medicine for the treatment of skin disease, boils,
swellings, wound ulcers, dropsy, menorrhagia, vomiting,
flatulence and burning sensations. The extract is also
reported regarding its usage in the treatment of cuts and
wounds5,6,7,8. The present study was conducted to formulate
herbal emulgel of Pothos scandens using gelling agents like
Carbopol 934, Carbopol 940 and HPMC K4M. The
prepared emulgels were evaluated for physicochemical as
well as for pharmacological activity.
MATERIALS AND METHODS
Materials
Carbopol 934, carbopol 940 and HPMC K4M were
obtained from Loba Chem. Pvt Ltd, Mumbai. Methyl
paraben sodium and propyl paraben sodium were obtained
from Hi Media laboratories. Glycerol and triethanolamine
were obtained from Nice chemicals Pvt. Ltd, Mumbai.
Plant materials and extract preparation
P.scandens Linn. (Family: Araceae) were collected from
local areas of Perinthalmanna, kerala and was authenticated
by Prof. M. Sabu, Head of Department of Botany,
University of Calicut. 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 herb was
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.
Animals
A total of 78, 12-week old healthy wistar strain rats
weighing 150-200g of either sex, bred locally in the animal
house of Al Shifa College of Pharmacy, Perinthalmanna,
were selected for the wound healing studies and 9 albino
rabbits (average wt 2.2 kg) were used for primary skin
irritation test. They were housed under controlled
conditions of temperature (23 ± 2 ) 0C, humidity (50 ±5) 0C
and 10-14 hours of light and dark cycles. The animals were
housed individually in polypropylene cages containing
sterile paddy husk bedding and free access to food and
water ad libitum. The study was conducted after obtaining
63
Mohammed Haneefa K.P et al /J. Pharm. Sci. & Res. Vol. 6(2), 2014, 63-67
the approval from Institutional Animal
Committee.(Reg No: 1195/ac/08/CPCSEA).
Ethical
Acute toxicity studies
Acute toxicity studies were performed for the extract as per
OECD guidelines (Class method). Three healthy albino
rates average weight 150 g were used for the study. A
maximum dose of 2g/kg body weight was administered
orally .Animals were observed individually after dosing
once during the first 30 minutes, periodically during the
first 24 hours, with special attention given during the first 4
hours, and daily thereafter, for a total of 14 days.
Wound healing studies of extract 9,10
Rats were anesthetized with ketamine + xylazine (50
mg/kg+5 mg/kg) and the hair on the back was clipped with
electric clippers. Burn wounds were created by using a
device with an iron piece and a wooden handle placed on
the back of the rat. It was heated to red hot over flame and
was placed in contact with the back of anaesthetized rat up
to 10 seconds without any pressure. After this, each animal
was placed in a separate cage for full recovery from
anaesthesia before being returned to holding rooms. The
wound of the test animal was applied with extract in
glycerol and the control group I was applied with glycerol
as the solvent control and control group 2 were left
untreated. The application was repeated daily for the next
20 post-operative days and the following parameters were
studied.
(1) Epithelization period: It was monitored by noting the
number of days required for scar to fall away, leaving no
raw wound behind;
(2) Wound contraction: To monitor this, progressive
changes in wound area were followed planimetrically.
Leaving the wounding day, wounds were traced on a
transparent paper on an alternate day. The animal was
restrained in proper position during tracing. The tracings
were then transferred to 1 mm2 graph sheet. From this,
wound areas were read and the percentage of wound
contraction was calculated taking the initial size of wound
(250 mm2) as 100%.
%Woundcontraction
initinalwoundsize − inalwoundsize
= × 100
initialwoundsize
Formulation of Emulgel11
Different formulations were prepared using varying amount
of gelling agent and penetration enhancer. 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 bases were prepared by
dispersing Carbopol 934 and Carbopol 940 in distilled
water separately with constant stirring at a moderate speed
using mechanical shaker. Formulations F1, F2 and F3 were
prepared by carbopol 934 and F4, F5 and F6 by carbopol
940 as gelling agent. In formulations F7, F8 and F9 the gel
were prepared by dispersing HPMC K4M in heated
distilled water (80°C), and the dispersion was cooled and
left overnight. The pH of all the formulations was adjusted
to 6 - 6.5 using tri ethanol amine (TEA). The oil phase of
the emulsion was prepared by dissolving Span 20 in light
liquid paraffin while the aqueous phase was prepared by
dissolving Tween 20 in purified water. Methyl and Propyl
parabens were dissolved in propylene glycol and mixed
with the extract dissolved in ethanol, being hydrophobic
was dissolved in oil phase. Clove oil was also mixed in oil
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 it got cooled to
room temperature. The obtained emulsion was mixed with
the gel in 1:1 ratio with gentle stirring to obtain the
emulgel. The composition of different formulations has
been discussed.
Evaluation of Emulgel
1. Physical Examination12
The prepared emulgel formulations were inspected visually
for their colour, homogeneity, consistency, grittiness and
phase separation
2. Estimation of drug content13
Preparation of standard solution
Take 100 mg of crude extract and dissolved in 100 ml of
ethanol (Stock solution 1).
From this stock solution 1ml was taken and diluted to 10
ml [stock solution - 2]. From stock solution - 2 take volume
2,4,6,8 and 10ml separately and made up to 10ml to get
concentration range 20, 40, 60, 80, 100mcg/ml
respectively. The absorbance was measured at 270 nm
against ethanol as blank.
Preparation of test solution of Pothos scandens Linn
emulgel
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 whatmann 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
270 nm
3. Measurement of pH14
The pH of emulgel formulations was determined by using
digital pH meter. 1gm of gel was dissolved in 100 ml of
distilled water and it was placed for 2 hr. The measurement
of pH of each formulation was done in triplicate and
average values were calculated.
4. Viscosity15
Viscosity of the emulgels was determined using Brookfield
viscometer. Spindle type, model LVDV-E at 10 rpm. 200
grams of the gel was taken in a beaker and the spindle was
dipped in it for about 5 minutes and then the reading was
taken.
5. Spreadability 16,17
Spreadability denotes the extent of area to which the
emulgel readily spreads on application to skin or the
affected part. The bioavailability efficiency of a emulgel
formulation also depends on its spreading value. The
64
Mohammed Haneefa K.P et al /J. Pharm. Sci. & Res. Vol. 6(2), 2014, 63-67
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 20 gm weight was tied to the upper slide
carefully. The time taken for the upper slide to travel the
distance of 7.5 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
taken for calculation.
Spreadability was calculated by using the following
formula:
=
×
Where,
S – Spreadability
m – Weight tied to the upper slide (20gm)
l - Length of the glass (7.5 cm)
t - Time taken in seconds
6. Permeability studies18
The in vitro diffusion studies of the emulgels were
performed using dialysis membrane. The membrane was
soaked in phosphate buffer pH 6.8 for 6 – 8 hr & was
clamped carefully to one end of the hollow glass tube.
Phosphate buffer of pH 6.8 was used for in vitro release as
a receptor medium. The emulgel sample was applied on the
membrane and then fixed in between donor and receptor
compartment of glass tube. The receptor compartment
contained phosphate buffer (100ml) of pH 6.8. The
temperature of diffusion medium was thermostatically
controlled at 370 C ± 10C by surrounding water in jacket
and the medium was stirred by magnetic stirrer at 500rpm .
The sample were withdrawn at predetermined intervals and
was replaced by equal volume of fresh fluid . The samples
withdrawn were spectrophotometrically estimated at
270nm using phosphate buffer as blank.
Pharmacological studies of formulation
Primary skin irritation test19,20
This study employed nine rabbits (white, average weight of
2.2 kg) to test for the skin irritation. They were kept
carefully following an acclimation period of 7 days to
ensure their suitability for the study. Test animals were kept
within a limited-access rodent facility with environmental
conditions set to a temperature of 25 ±2 ºC, a humidity of
60-90% RH and a 12-h light / 12-h dark cycle. Animals
were provided ad libitum access to a commercial rabbit-diet
and drinking water was supplied to each cage. The area on
the back of each rabbits was shaved prior to the
experiment. The back was divided into five marked areas
for the topical application of the gel containing various
concentrations of extract. Test product was placed on each
area for 24 hours using adhesive tape. Scoring of the
erythema and edema was performed at 24 and 72 hours
with Draize technique. The positive control of this
experiment is 98% lactic acid.
Wound healing studies of the formulation 9,10
Wound healing studies of formulation were prepared
similar to wound healing studies of the extract in which the
control groups of rats were applied with dummy gel. The
application was repeated daily for the next 20 post
operative days and the following parameters were studied,
epithelisation period and wound contraction.
Stability studies of formulation 18
Stability studies were carried out on formulations according
to International Conference on Harmonization (ICH)
guidelines.Short term accelerated stability study was
carried out for the period of 3 months for the formulations.
The samples were stored at different storage conditions of
room temperature (25 ± 0.50 C), elevated temperature such
as 40°C at 75% RH. Samples was withdrawn on weakly
interval and analyzed for visual appearance, clarity, pH,
spreadability and viscosity. All the emulgel formulations
were stored for a period of 12 weeks. Samples were
withdrawn at 1,3,6,9 and 12 week and assessed for drug
content. At the end of 12th week they were evaluated for
physical parameters and integrity of the product.
RESULTS AND DISCUSSION
Acute toxicity studies
Study revealed that there was no mortality and hence the
extract was found to be safe for administration. After
administration there was no significant change in the body
weight of the animals.
Wound healing studies of extract
Table 1 shows the effect of P. sca ndens leaf extract
administered topically on wound healing in rats with burn
wound. Wound contraction studies in Pothos sc andens
Alcoholic extract revealed that 4 % of Alcoholic extract
have maximum wound healing activity.
In this model, P. scand ens treated animals were found to
epithelise in 22 days while the solvent control and the
untreated rat’s epithelise with 35 and 40 days respectively.
On the 20th day, the percentage wound area reduction in
solvent controls, untreated rats and the extract treated rats
were 64 %, 54% and 95% respectively.
Formulation and evaluation of gel
Herbal emulgels of P.scandens Linn were prepared
successfully by using different concentration of Carbopol
934, Carbopol 940 and HPMC K4M. It was found that all
the formulations were light green in colour and the physical
evaluations were given in Table 2. The P. sc andens
at 270 nm. All the
alcoholic extract shows λmax
formulations were found to be neutral (pH 6.2 to 7.2) and
drug content was found to be in the range of 89%-100 %
w/w (Table 2). Viscosities of all the formulations are given
in Table 3.
65
Mohammed Haneefa K.P et al /J. Pharm. Sci. & Res. Vol. 6(2), 2014, 63-67
Table 1- Wound healing effect of Pothos scandens extract in burn wound model.
Conc. of the
alcoholic
extract
(%w/v)
Solvent
control
Day 4
Day 8
Day 12
Day 16
Day 20
Epithelization time
(In days)
3.10±0.72
24.25±1.24
32.56±2.01
48.1±1.59
64.43±2.70
35 ± 1.69
2%
8.61±2.22
24.03±2.53
39.11±1.46
46.21±2.47
71.30±2.18
33 ± 2.15
4%
5.25±1.36
28.6±2.13
58.1±3.69
87.16±1.88
95.56±2.37
22 ± 2.43
6%
7.46±0.60
24.60±1.69
42.65±1.57
52.50±1.6
76.10±2.32
30 ± 1.22
8%
5.00±1.82
25.25±2.80
52.34±3.23
69.64±2.07
77.28±2.79
29 ± 2.25
10%
5.98±1.38
24.36±2.48
35.61±2.22
65.06±2.65
84.55±2.60
26 ± 2.4
untreated
control
2.90±1.21
12.40±0.92
24.20±0.81
34.25±1.29
54.50±2.02
40 ± 1.06
Values are mean ±SD of six readings
Table 2-Characteristics of P. scandens gel formulations.
Formulation
pH
F1
F2
F3
F4
F5
F6
F7
F8
F9
6.8
6.9
6.8
6.8
6.8
7.0
6.9
6.8
6.8
+++ Excellent
Viscosity
(cps)
8377
9770
14288
11370
16882
17638
3173
3582
4361
Spreadability gm
cm/sec
34.6±0.66
21.4±0.45
15.8±0.38
29.4±0.55
14.6±0.4
13.4±0.29
33.3±0.61
31.3±0.53
28.9±0.49
Drug content
(%w/w)
92.93±0.510
0.32±0.23
95.55±0.25
98.68±0.30
97.86±0.20
98.35±0.24
93.75±0.18
91.61±0.12
89.47±0.40
Extrudability
Nature of gel
+++
+++
+++
+++
+++
+++
+++
+++
+++
Light green, Homogenous
Light green, Homogenous
Light green, Homogenous
Light green, Homogenous
Light green, Homogenous
Light green, Homogenous
Light green, Homogenous
Light green, Homogenous
Light green, Homogenous
++ Good
Table-3- Regression co-efficient (R2) values of kinetic models for formulation F1and F4
Formulation code
F1
F4
Zero Order
0.984
0.920
First Order
0.979
0.920
Higuchi Model
0.992
0.982
Peppas Model
0.920
0.944
Table-4 Wound healing effect of formulations in burn wound model.
Day 4
Day 8
Day 12
Day 16
Day 20
Control of Carbopol 934
4.60±1.05
22.89±1.58
43.09±1.08
61.2±2.60
71.2±1.28
Epithelization
time
28±1.05
F1
7.96±1.17
32.03±1.61
61.26±1.50
89.05±0.65
97.66±1.32
21±1.25
F2
8.92±1.03
24.1±1.60
49.6±1.51
89.2±3.21
94.61±1.20
23±1.02
F3
6.25±1.02
23.03±1.26
49.76±1.26
73.91±1.66
90.12±1.42
24±1.54
Control of Carbopol 940
2.69±1.13
9.25±1.38
24.46±0.65
58.13±2..14
70.83±1.25
30±1.28
F4
9.14±0.32
30.1±1.23
59.25±1.26
81.21±0.27
99.91±1.62
20±0.84
F4
6.8±0.24
28.12±1.12
47.12±0.89
75.3±1.23
92.32±2.12
24±0.32
F6
6.82±0.23
27.18±1.69
46.16±1.02
76.02±0.62
90.21±2.31
27±1.03
Spreadability denotes the extent of area to which the gel
readily spreads on application to skin or the affected part.
The bioavailability efficiency of a emulgel formulation also
depends on its spreading value. Maximum spreading value
was found for formulation with 1% carbopol 934. The
extrudability reflects the capacity of the gel, to get ejected
in uniform and desired quantity when the tube is squeezed.
The results of extrudability are shown in Table 2.
Permeability studies
The results obtained in in vitro release studies were plotted
in different kinetic models. Regression coefficient (R2)
values of different kinetic models are shown in Table 3.
This indicated that the release data was best fit with
Higuchi model kinetics because the value of R2 is greater in
this model. Higuchi equation explains the diffusion release
mechanism, so formulations follow the diffusion
mechanism of drug release.
66
Mohammed Haneefa K.P et al /J. Pharm. Sci. & Res. Vol. 6(2), 2014, 63-67
Stability studies
The formulated emulgels were subjected for stability
studies. No colour fading was observed for all prepared
gels. The pH of all formulations were not affected and
found to be within the range of 6.2-7.2. The viscosity and
spreadability of all emulgels was found to be same
especially at ambient, 4-5OC and 370C temperature. The
drug content was found to be in the limit 90-101 % for all
emulgel formulation at all temperature conditions.
Pharmacological studies of formulation
Primary skin irritation test
The skin irritation studies revealed that all formulations
were non-sensitizing and safe for use.
Wound healing activity studies of the formulation
In this model, formulation with carbopol 940 treated
animals shows better healing compared to carbopol 934
treated rats. Formulations with 1% carbopol 940 treated
rats were found to epithelise in 20 days while the control
group epithelise with 30 days. Results are shown in
Table 4.
Statistical analysis
Data obtained from pharmacological experiments are
expressed as mean ± SD. Differences between the control
and the treatments in these experiments were tested for
significance using student t test. Differences were
considered significant if P > 0.05.
CONCLUSION
The aim of the study was to formulate and evaluate Pothos
scandens Linn leaf extract in to a emulgel for burn wound
healing activity. The leaves were successfully extracted
with ethanol and water. 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 Pothos scandens Linn
leaf possess maximum wound healing effect in rats and the
effects produced was maximum in 4% alcoholic extract and
this concentration was used for the formulation.
The extract was formulated in to 9 different emulgel
formulations by changing the concentration of Carbopol
934, Carbopol 940 and HPMC K4M. All the formulations
were found to be neutral (pH 6.8 to 7.0) and drug content was
found to be in the range of 89 -100%w/w. On physical
evaluation of all the formulations, F1 (1% Carbopol 934) and F4
(1% Carbopol 940) was found to be optimum in terms of gel
consistency, viscosity, spreadability and extrudability. The
invitro release studies were conducted and results are
shown in Tables 23-26 & Graphs-4-12. Maximum release
was shown by F1 and F4, 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 six weeks.
Pharmacological evaluation of gels revealed that all
formulations are non sensitizing and safe for use.
Pharmacological studies of the formulations showed that
F1 (1% Carbopol 934) has greatest pharmacological
activity. It was finally concluded that the formulations F1 was
found to be more promising formulation as it shows better
physicochemical characteristics and higher pharmacological
activity compared to other formulations.
REFERENCES
1. C. Surver and F.A. Davis, Bioavailability and Bioequivaance, In: K.A
Walter (eds.), Dermatological and Transdermal Formulation, Marcal
Dekker, New York, 2002, pp. 323- 327,403.
2. Sharma S. Topical drug delivery system. Available from:
http://www.pharmainfo.net/ Section/science-news/. [Cited in 2011
Aug 9].
3. Kuller R, Saini S, Seth N, Rana AC, Emulgel: A surrogate approach for
topical used hydrophobic drugs. Int J Pharm Bio Sci, 1(3):117-128,
(2011).
4. Jain A, Gautam SP, Gupta, Jain S, Development and characterization of
Ketoconazole emulgel for topical drug delivery. Der Pharmacia
Sinica, 1(3):221- 231, (2010).
5. Ogner J. A revised classification of Araceae with
dichotomouskeys.Willdenowia 1991; 21:35-50.
6. Boyce PC, Poulsen AD. Notes on Pothos insignis (Araceae:
Pothoideae).KewBulletin 1994; 49(3): 523-528.
7. View Abstract: Plant Resources of South-East Asia No.12 (3):
Medicinal and poisonous plants 3. 2003.
8.Udayan P.S and Indira Balachandran, Medicinal plants of
Aryavaidyasala herb garden, page no: 292
9. Nayak B S; Investigation of wound healing activity of Lantana camara
L. in Sprague dawley rats using a burn wound model; International
Journal of Applied Research in Natural Products; Vol. 1(1),
March/April 2008), pp. 15-19
10.Farnood Shokuhi; The efficacy of alcoholic extract of garlic on the
healing process of experimental burn wound in the rabbit, Journal of
Animal and Veterinary advances 8 (4), 2009, Page no: 655-659
11. Vikas S, Seema S, Rana AC, Gurpreet S. Development and evaluation
of topical emulgel of lornoxicam using different polymer bases.
Internationale Pharmaceutica Sciencia 2012; 2 (3): 36 - 44.
12. Jain A, Gautam SP, Gupta Y, Khambete H, Jain S. Development and
characterization of ketoconazole emulgel for topical drug delivery.
Der Pharmacia Sinica 2010; 1(3):221-231.
13.Nandgude T, Rahul , Jaiswal N, Deshmukh P, ChatapV, Nitin .
“Formulation and evaluation of pH induced insitu nasal gel of
salbutamol sulphate”, International journal of pharmaceutical
sciences and nano technology, 2008; vol 1(2):177-183.
14. Kaur LP, Garg R, Gupta GD. Development and evaluation of topical
gel of minoxidil from different Polymer bases in application of
alopecia. Int J Pharmacy and Pharm Sci 2010; 2(3): 43-47.
15. Bhowmik BB, Nayak BS, Arkendu. “Formulation Development and
Characterization Of Metronidazole Microencapsulated Bioadhesive
Vaginal gel”, International Journal Of Pharmacy And Pharmacy
Practise. 1(1): 240.
16. Panigrahi L, Ghosal SK, Pattnaik S, Maharana L, Barik BB. “Effect of
Permeation Enhancers on the Release and Permeation Kinetics of
Lincomycin Hydrochloride Gel Formulations through Mouse Skin”.
Indian J Pharm Sci 2006; 68: 205-211.
17. Bhaskar G, Arshia S, Priyadarshini SRB, Bhaskar G, Arshia S,
Priyadarshini SRB. “Formulation and Evaluation of Topical
Polyherbal Antiacne Gels Containing Garcinia mangostana and Aloe
vera”, Pharmacognosy magazine,vol 5, Issue 19(suppl), Jul- Aug
2009, page 93-99.
18. Panigrahi L; “Effect of Permeation Enhancers on the Release and
Permeation Kinetics of Lincomycin Hydrochloride Gel Formulations
through Mouse Skin”; Indian J Pharm Sci. 68: (2006), Page no:
205-11.
19. Nawanopparatsakul et al. “Skin Irritation Test of Curcuminoids Facial
Mask Containing Chitosan as a Binder”.
20. Kirwin C.J. “Eye and skin local toxicity testing In Toxicology:
Principles and practice” Vol 2 edited by Sperling F.a WileyInterscience publication 1984; 169-175.
67