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FORMULATION AND EVALUATION OF TOPICAL GEL OF ACELOFENAC

CONTAINING PIPARINE

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 Indo American Journal of Pharmaceutical Research, 2013 ISSN NO: 2231-6876

INDO AMERICAN
Journal home page: JOURNAL OF
http://www.iajpr.com/index.php/en/ PHARMACEUTICAL
RESEARCH

FORMULATION AND EVALUATION OF TOPICAL GEL OF ACELOFENAC CONTAINING

PIPARINE

Vijay Kumar Singh1*, Praveen Kumar Singh1, Purnendu Kumar Sharma2, Peeyush Kumar Srivastava2,
Ashutosh Mishra3
1
Kamla Nehru Institute of Management & Technology, Sultanpur, India
2
Technocrats Institute of Technology (Pharmacy), Bhopal, India
3
Acharya Narendra Dev College of Pharmacy, Babhnan Gonda, India

ARTICLE INFO ABSTRACT

Article history The present investigation involves formulation of Acelofenac topical gel having
Received 09/07/2013 linseed oil and piparine. Aceclofenac has been shown to have potent analgesic and
Available online anti-inflammatory activities, similar to indomethacin and diclofenac and due to its
31/07/2013 preferential cox-2 blockade, it has better safety than conventional NSAIDs with
respect to adverse effects on gastrointestinal and cardiovascular system.
Aceclofenac is used in treatment of osteoarthritis, rheumatoid arthritis, acute
lumbago, and dental pain condition. The formulation of Acelofenac topical gel was
Keywords prepared using HPMC, Carbopol 974 P and Sodium CMC in three different
Piparine, batches. Piperine was isolated from major alkaloids of the pepper fruits and was
HPMC, used as a penetration enhancer in formulation. Formulated gel was evaluated and
carbopol 974 P, compared with marketed Nusaid gel for pH, viscosity, spreadability, extrudability,
NSAIDs. drug content, in vitro drug diffusion, ex-vivo bio-adhesive test and skin irritation
test. Topical gel having best drug releasing profile was evaluated for anti-
inflammatory and analgesic potency by animal paradigms.

Corresponding author
Vijay Kumar Singh
Kamla Nehru Institute of Management & Technology, Sultanpur, India
Email id- vijaysinghs1@gmail.com
Contact No: 09452848617

Please cite this article in press as Vijay Kumar Singh et.al. Formulation and evaluation of topical gel of acelofenac containing
5266

piparine. Indo American Journal of Pharm Research.2013:3(7).

Copy right © 2013 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical
Page

Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Vol 3, Issue 7, 2013. Vijay Kumar Singh et al. ISSN NO: 2231-6876

INTRODUCTION
The I.P. defines Gels are homogeneous, semisolid preparations usually consisting of solutions or dispersions of
one or more medicaments in suitable hydrophilic or hydrophobic bases. As per the definition of U.S.P. Gels as a
semisolid system consisting of dispersion made up of either small inorganic particle or large organic molecule
enclosing and interpenetrated by liquid. The inorganic particles form a three-dimensional “house of cards”
structure.
As far as structure is concerned, Gels consist of two-phase system in which inorganic particles are not dissolved
but merely dispersed throughout the continuous phase and large organic particles are dissolved in the
continuous phase, randomly coiled in the flexible chains. Gels are typically formed from a liquid phase that has
been thickened with other components. They are normally prepared with the aid of suitable gelling agents like
HPMC, Carbopol and Sodium CMC etc. Substances such as antioxidants, stabilizers and antimicrobial
preservatives are used as additives in the formulation of gels.
Delivery of drugs to the skin is an effective and targeted therapy for local dermatological disorders. This route
of drug delivery has gained popularity because it avoids first pass effects, gastrointestinal irritation, and
metabolic degradation associated with oral administration. Due to the first past effect only 25-/45% of the orally
administered dose reaches the blood circulation. In order to bypass these disadvantages the gel formulations
have been proposed as topical application. Topical gel formulations provide suitable delivery system for drugs
because they are less greasy and can be easily removed from the skin. They are intended to be applied to the
skin or certain mucous membranes for protective, prophylactic or therapeutic purposes.
Aceclofenac is a Diclofenac derivative of the Non–Steroidal Anti-Inflammatory drug3,4,5 (NSAID), which is
chemically, (2-[2-[2-(2,6-dichlorophenyl) amino phenyl]acetyl] oxyacetic acid)6,7. Aceclofenac is used in
treatment of osteoarthritis, rheumatoid arthritis, acute lumbago, and dental pain condition. Like other NSAIDs,
oral administration of this drug is also associated with severe gastrointestinal side effects like- ulceration and
gastro intestinal bleeding liver and kidney trouble The solution of problems like side effects associated with
systemic delivery of NAIDs lies in the fact that, topically applied NSAIDs are safer than and as efficacious as
oral NSAIDs. Furthermore, the transdermal route of administration has a high patient compliance, which
derives from it being non-invasive and the long interval between applications. Transdermal administration also
provides a means to obtain constant systemic drug levels.
There are few dermatological inflammatory conditions and wounds where gel formation of NSAIDs like
aceclofenac could be helpful. Interestingly topical dermatological products are intended for localized action on
one or more layers of skin. Although some medication form this topical product may unintentionally reach
systemic circulation, it is usually in subtherapeutic concentrations, and does not produce effect of any major
concern except in special situations such as pregnant or nursing patients.
The major drawback of Aceclofenac is its poor aqueous solubility, carbomers are extensively used in non-
parenteral medicines particularly topical liquid and semi-solid preparations, HPMC is also widely used in
topical gels and ointments as an excipient it is non-toxic and non-irritant materials hence HPMC, Carbopol 940
and Sodium CMC were selected as an ideal gel base and are incorporated to gel bases by trituration.
Piperine is major alkaloids of the pepper fruits which belong to a piperaceae family. Piperine forms monoclinic
needles, slightly soluble in water and more soluble in alcohol, ether or chloroform. It is a weak base that is
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tasteless at first, but leaves a burning aftertaste. Its molecular formula is C17H19NO3, and its molecular weight is
285.34 daltons. Piperine is the trans-trans stereoisomer of 1-piperoylpiperidine. It is also known as (E, E)-1-
piperoylpiperidine and (E, E)-1-[5-(1, 3-benzodioxol-5-y1)-1-oxo-2, 4-pentdienyl] piperidine. 1,2,3
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Piperine may have bioavailability-enhancing activity for some nutritional substances and for some drugs.
Piperine induces modification in membrane dynamics and permeation characteristics of stratum corneum and

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Vol 3, Issue 7, 2013. Vijay Kumar Singh et al. ISSN NO: 2231-6876

act as a penetration enhancer. It has putative anti-inflammatory activity and may have activity in promoting
digestive processes. Piperine has been demonstrated to increase the serum levels and lengthen the serum half
lives of some nutritional substances, such as coenzyme Q10 and beta-carotene. It is speculated that piperine may
act as a so-called thermonutrient and increase the absorption of certain nutritional substances from the
gastrointestinal tract by producing a local thermogenic action.

MATERIALS
Chemicals
Acelofenac was a kind gift from Yag Mag Pvt. Ltd. and were supplied by Wardhman (Batch no.-
YM/AC/055/12-13). Nusaid gel from Molekule Pharmaceuticals Pvt. Ltd. was purchased from market. Methyl
Paraben was purchased from Hi Media labotatories Pvt. Ltd.,Mumbai. Propylene Glycol, Methyl salicylate were
purchased from Merk Limited Mumbai, Triethanolamine, Na lauryl sulphate, HPMC-E 50LVwere obtained
from LOBA CHEMIE PVT LTD, Mumbai. Menthol and Linseed oil were purchased from
Arora&Co.(Chemical Division)Delhi. Isopropyl Alcohol was obtained from Qualigens Fine chemicals GSK
Pharmaceutical Ltd. Mumbai.

Experimental Animal
Adult wistar albino rats, weighing (150-200g) were procured from Institutional of Health and Biologicals,
Mhow (Indore, India). The animals were feed a normal laboratory pellet diet and water adlibitum. They were
housed in colony cages under standard laboratory conditions (12:12h light and day cycle, temperature at
25±2°C and relative humidity at 55±10%). The ethical clearance was obtained from Institutional Animal Ethics
Committee (TIT/IAEC/831/PHARMACEUTICS/2013/07) at TIT, Bhopal, India for using animal in the present
study.

METHOD:
ISOLATION OF PIPARINE4, 5, 6:
150mL of 95% ethanol and 5 boiling chips was added in 15g black pepper and heat at reflux for 2hrs. Using
suction filtration, the mixture was filtered and then concentrated to a volume of 10-15mL by simple distillation.
10mL of 10% solution of KOH in 95% ethanol was added in a 125mL Erlenmeyer flask and concentrated
pepper extract was mixed and heated. The resulting mixture was diluted with water. A yellow precipitate was
formed. Water was added to yellow precipitate until no more solid appears and then the mixture was allowed to
stand at least overnight. The resulted solid extracts were collected by suction filtration and recrystallized with
10-20mL of acetone

PREPARATION OF ACECLOFENAC GEL:

Gel with Carbopol base:
Propylene glycol was heated at 65°C then methyl paraben and propyl paraben was added along with water and
carbopol, and kept for 8 hours for adequate swelling of polymer. Triethanolamine was added to neutralize the
carbopol and pH was adjusted to 6.7 – 6.9. Propylene glycol at 65°C was heated in another vessel, and drug
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aceclofenac was added. The above mixture was allowed to cool at room temperature and carbopol base was
added. Menthol was dissolved in IPA till the clear solution was obtained; this solution was added to above gel.
Oil phase was prepared by dissolving tween 60,chremophore Rh 40, methyl salicylate, linseed oil and peparine
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together, oil phase was slowly added in the above aqueous carbopol gel while constantly stirring to get emulgel

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Vol 3, Issue 7, 2013. Vijay Kumar Singh et al. ISSN NO: 2231-6876

and the pH was adjusted between 7.0 – 7.5. Gel was packed in aluminum collapsible tube. This formulation
was marked as batch no. Ac-1.

Gel with hydroxy propyl methyl cellulose:

Aceclofenac was dissolved in propylene glycol and menthol was dissolved in Isopropyl alcohol. The whole
amount of HPMC was sprinkled on drug solution with slow stirring then methyl paraben and propyl paraben
was added. The mixture of drug solution and polymer was kept aside for 6-7 hour, for adequate swelling of
polymer. The oil phase consisting of linseed oil, peparine and methyl salicylate was added slowly in above
aqueous gel with continuous stirring with overhead stirrer. The gel was packed in aluminum collapsible tube.
This formulation was marked as batch no. Ac-2.

Gel with Sodium CMC base:

Aqueous gel base was prepared by dissolving of sodium CMC in water and with continuous stirring propylene
glycol was added, and then Aceclofenac drug was added. The SLS was charged slowly and with continuous
stirring. Menthol was dissolved in IPA till the clear solution is obtained and was added to above gel. Oil phase
was prepared by dissolving, methyl salicylate, linseed oil and peparine and mixed till the clear solution is
obtained; oil phase is slowly added in the above aqueous gel while constantly stirring. Finally, methyl paraben
was added to the gel. Gel was packed in aluminum collapsible tube. This formulation was marked as batch no.
Ac-3.

EVALUATION OF ACECLOFENAC GEL:

Appearance - Colour is important for patient compliance. The prepared gels were inspected visually for clarity,
colour and presence of any particle.

pH -The pH of gel was determined using digital pH meter. 2 gm Aceclofenac gel was stirred in distilled water
till a uniform suspension is formed. The volume was made up to 40 ml and pH of the solution was measured.

Viscosity-Viscosity of the gel was determined by using (LV) Brookfield viscometer (Dial type). As the system
is non-Newtonian spindle no. 4 is used. Viscosity was measured for the fixed time 2 min for 0.3 rpm.

Skin irritation7 - Ten healthy male and female volunteers were selected for skin irritation testing. 100 mg gel
was applied on area of 2 cm for 6 hours, on the interior surface of upper arm and covered with cotton bandage.
After 6 hr the sites were cleaned with acetone and readings are made according to the scale given by Draize.
No irritation: 0
Slight irritation: 1
Irritation: 2

Spreadability8,9,10,11,12 - Spreadability of formulations was determined by an apparatus suggested by

Multimer45, which was fabricated itself in laboratory and used for slide fixed on wooded block and upper slide
with one end tide to glass slide and other end tied with other end tied to weight pan. An excess of gel (2 – 5 gm)
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was placed in between two glass slides and then 1000 gm weight was placed on slides for 5 min to compress the
sample to a uniform thickness. Weight (80 gm) was added to pan. The time (seconds) required to separate the
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two slides, was taken as a measure of spreadability.

It was calculated using formula,

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S = M. L / T
Where, S = spreadability
M = weight tied to upper slide
L = length of glass slide
T = time taken
Shorter time interval, to cover distance of 6.5 cm, indicates better spreadability.

Extrudability1, 13, 14 - In this test, sample is extruded from the tube by usual procedure. A closed collapsible
tube containing gel was passed firmly at crimpened end. When the cap was removed, gel extrudes until pressure
was dissipates. The weight in grams required to extrude 0.5 cm ribbon of gel in 10 seconds was determined. The
results for each formulation were recorded as extrusion pressure in grams.

Drug Content 15, 16 - 1gm gel was dissolved with little amount of methanol in a 100 ml volumetric flask and
mixture was shaken till solution was affected. The volume was made up to 100 ml with methanol. The solution
was filtered through Whatman filter paper (No. 41). Further dilute 5ml to 50 ml with methanol. The absorbance
of the solution was measured at 275 nm (systronics pc based double beam spectrophotometer 2202) against
reagent blank.

IN VITRO DRUG DIFFUSION STUDY

All formulations were subjected to in vitro diffusion through cellulose membrane by using Keshary- Chein type
cell. The receptor compartment was filled with saline phosphate buffer pH 7.4 and methanol (90:10) and kept at
32 + 0.5°C and stirred with the help of magnetic stirrer. Methanol (10%) was added to maintained sink
condition. About 200 – 300 mg of gel was placed on the cellulose membrane. One ml of sample was withdrawn
from the receptor compartment at 1, 2, 3, 4, 5, 6, 7, 8 hour and replaced with same volume of medium. All
samples were diluted up to 10 ml with medium and analyzed for aceclofenac content spectrophotometrically
(systronics pc based double beam spectrophotometer 2202) at wavelength 275 nm.

EX–VIVO BIOADHESIVE STRENGTH MEASUREMENT OF ACECLOFENAC GEL17, 18:

Fresh goat hairless skin was obtained from a local slaughter – house and used within 2 hours of slaughter. The
skin was separated by removing the underlying fat and loose tissues. The membrane was washed with distilled
water and then with 0.1 N NaoH.

The modified Patel et al (2007) method was used for the measurement of bioadhesive strength. The fresh skin
was cut into pieces and washed with 0.1 N NaoH. Two pieces of skin were tied to the two glass slide separately
from that one glass slide was fixed on the wooden piece and other piece was tied with the balance on right hand
side. The right and left pans were balanced by adding extra weight on the left – hand pan. 1 gm of topical gel
was placed between these two slides containing hairless skin pieces, and extra weight from the left pan was
removed to sandwich the two pieces of skin and some pressure was applied to remove the presence of air. The
balance was kept in this position for 5 minutes. Weight was added slowly at 200 mg/ min to the left – hand pan
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until the patch detached from the skin surface. The weight (gram force) required to detach the gel from the skin
surface gave the measure of bioadhesive strength. The bioadhesive strength was calculated by using following:
Bioadhesive Strength = Weight required (g) / Area (cm2)
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ASSESSMENT OF ANTI-INFLAMMATORY ACTIVITY 19,20,21,22:

Anti-inflammatory activity was evaluated by inducing paw oedema using carrageenan in rats. Rats were
deprived of food overnight and treated topically on the dorsal part of the hind paw with the 55 mg/kg
aceclofenac gel (Ac-2a, Ac-2b, Ac-2c, positive control (pure acelofenac 10mg/kg and control) 60 min before
0.1 mL 1% carrageenan in isotonic saline was injected sub-plantar into the left hind paw. The contralateral paw
was injected with 0.1 mL saline and used as a control. The volume difference between the carrageenan and
saline injected paws was used to evaluate the inflammatory response. Paw volume (V) was measured by water
plethysmometer immediately before and 1, 2, 3, 4, 5 and 6 h after the injection of carrageenan into the plantar
region of the left hind paw (n = 5 for each group). The percent inhibition of oedema induced by carrageenan
was calculated for each group using following equation:

Inhibition of oedema (%) = V control −V treated X 100 (Table 6)

V control
ASSESSMENT OF ANALGESIC ACTIVITY
Tail flick test 19,20,21,22
The tail flick test was performed by focusing radiant heat on the dorsal surface of the tail. Latency, or the time it
took the rats to withdraw their tails from a noxious thermal stimulus, was measured using a tail flick meter
(MAY-TF 0703, Ankara, Turkey). To minimize tissue damage, a maximum latency of 30 s was imposed. For
each set of experiments, 500 mg of Acelofenac gel formulations (containing 50 mg/kg) Ac-2a, Ac-2b and Ac-
2c, positive control (containing 10mg/kg pure acelofenac and control were applied to an area of approximately
4 cm on the dorsal skin. The nichrome wire was about 1/8 below the tail. Each rat was then tested before and
30, 45, 60, 75, 90, 105, 120 and 180 min after the topical administration of each formulation (n = 5 for each
group).The analgesic potency was calculated by the following Equation:

ME (maximum effect) (%) = TL−BL ×100

CL−BL
Where, TL was test latency, BL was baseline latency and CL was cutoff latency.

Hot plate test19, 20, 21, 22

Rats were placed on an aluminum hot plate kept at a temperature of 62 ±0.5 ◦C for a maximum time of 30 s.
The temperature of the plate was monitored at all times. For each set of experiments, 50 mg/kg of Aceclofenac
gel (Ac-2a, Ac-2b, and Ac-2c), positive control (containing 10mg/kg pure aceclofenac) and control were
applied to an area of approximately 4 cm on the dorsal skin. The reaction time was determined when animals
licked their fore and hind paws and jumped before and 15, 30, 45, 60, 90, 120 and 180 min after the topical
application of each formulation (n = 5 for each group). After each measurement, the plate was wiped with a
damp cloth to remove traces of urine and faeces.

STATISTICAL DATA ANALYSIS

Statistical analysis was performed using a one-way analysis of variance (ANOVA) to test the difference
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between the means of Aceclofenac gel formulation with control and positive control group. The mean and
standard error mean (SEM) of n = 5 were calculated. Data were considered statistically significant at P < 0.05.
For the in vivo studies, differences between drug treated and control groups were also evaluated using
Dennett‟s„t‟ test. The mean and SEM of n =5 were calculated. A probability level of P < 0.05 was considered
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statistically significant.

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RESULT AND DISCUSSION

All the aceclofenac gels formulations (Table -01) were good feel and showed no clogging and lumps which
indicate good texture of system. pH of hydrogels was around the neutral pH and in the range of 6.9-7.2. All the
formulations showed no significant skin irritation on intact skin. Thus, indicating skin acceptability of these
formulations for topical application. The description of appearance has been provided in Table-02. Viscosity is
an important parameter for characterizing the gels as it affects the spreadibility, extrudability and release of the
drug. Viscosities of formulations were ranges between 28600-32000 cps (Table -02). Easy spreadability is one
of the important characteristics of any topical preparation as far as patient compliance is concerned. Gel is
considered to be good if it takes minimum time to spread on the surface. Among the various gels studied Ac-2
aceclofenac gel has better spreadability and drug release. The values of spreadability indicate that the gel is
easily spreadable by small amount of shear.
Extrusion of gel from the tube is important during application and for the patient compliance. The values of
extrudability of different formulations were ranges in between 480-610 (Table-03). Drug content uniformity of
all formulations was observed and Ac-2b batch shows the 100.02% drug content (Table-03). Maximum drug
diffusion was observed from Ac-2 aceclofenac gel as compare to marketed Nusaid gel. Depending up on
different evaluation parameters made on all formulations, batch Ac-2 declared as an optimized batch.the
cumulative drug release profile of different formulation ranges from 49 to 83 percentage in 8 hours when
compared to marketed formulation ( Nusaid gel) having 75 percentage in 8 hours. However Ac-1c has shown
the best drug release profile as given in the Table-04. With consideration of all formulation characteristic and
parameter we selected Acelofenac Batch-2 formulation (Ac-2 Batch) for further study of anti-inflammatory and
analgesic activity.
Ac-2 Batch was used for the evaluation of anti-inflammatory and analgesic activity which was having 3
different concentrations of piperine. Ac-2 batch showed very significant anti-inflammatory and analgesic
potency (**p<0.01 by applying Dunnett‟s test) (Fig: 2, 3, and 4).The Ac-2b and Ac-2c formulation has shown
best anti-inflammatory activity when compared to Ac-2a and control, which show that this formulation has
potential against inflammation as shown in (Fig: 5, 6) Table-05 and Table-06 against paw oedema in rats.
The analgesic activity is measured by two methods which are Tail flick method and Hot plate method.

Anti-inflammatory Effect
The carrageenan-induced oedema test was used to examine the invivo effects of the developed Acelofenac gel
formulations. Intraplantar injection of carrageenan caused a time-dependent paw oedema in the rat, whereas the
saline injection did notcause swelling. The application of the formulations (Positive control, Ac-2a, Ac-2b, and
Ac-2c) inhibited paw swelling (Fig. 2).The maximum percent inhibition for Ac-2a was observed at 5 h which
was 61.42% , likewise, Ac-2b and Ac-2cwas observed at 4 h which was found to be 65.71% and 64.29%
respectively (Table 06).
Carrageenan administration into the rat hind paw produced a significant inflammation associated with
hyperalgesia, as shown by decreased rat paw withdrawal latency in response to a thermal stimulus.
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Previously it has been reported that the formulations having a better drug release profile will have the strongest
acute anti-inflammatory activity. Similarly, the results of this study demonstrated that the Ac-2a, Ac-2b and Ac-
2c having better cumulative % of drug release (99.3, 100.2 and98.1 respectively) possessed the strongest anti-
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inflammatory activity.

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In order to have a better comparison between the anti inflammatory activity of Aceclofenac pure (positive
control) and Aceclofenac gel (Ac-2a, Ac-2b and Ac-2c), evaluation was made on the basis of their ability to
inhibit the oedema produced in hind paw of rats after challenging with the carrageenan.
In control group, the increase in paw volume was (0.34±0.007) ml at first hour. With time, the increase in paw
volume increased up to 4th hour and then decreased, following administration of pure aceclofenac at a dose of
10mg/kg body weight.
On the other hand, the increase in paw volume following administration of aceclofenac gel (Ac-2a, Ac-2b, and
Ac-2c) was slightly higher than that produced by the pure drug. However, the change in paw volume with time
followed the same pattern. Pure Aceclofenac inhibits the paw oedema volume but to a lesser extent, on the other
hand the inhibitory or anti-inflammatory effect produced by aceclofenac loaded gel was found over a long
period of time.

From these observations it can be concluded that piperine is a bioavailability enhancing agent as it enhances
bioavailability of aceclofenac. It enhances local absorption and inhibits various metabolizing enzymes. This
provides scientific basis for use of Piperine to enhance the therapeutic efficacy of the concurrently administered
drugs.

Tail flick test and hot plate test

Ac-2a, Ac-2b, and Ac-2c administration increased tail flick and hotplate latency when compared with control
groups (Positive control and control group) (Fig. 4 and Table 7, 8). The latent period did not change in the tail
flick and hot plate tests in the control group animals.Other change in latent period was observed in Ac-2a, Ac-
2b, and Ac-2c along with positive control. The onset effect of the Ac-2a, Ac-2b, and Ac-2c was observed slight
less than positive control but then after 45 min, the latency period was found to be almost similar (Table 7, 8).
On comparison between hot plate and tail flick methods, the results were almost similar with p<.001 that means
very significant.

In the tail flick test, the maximum analgesic response of Ac-2a was observed at 45 and 90 min. Moreover, the
Ac-2a analgesic response peaked at 90 min. Likewise in Ac-2b the maximum response was observed at 45 min
and in Ac-2c was at 45 min (Table 07).
In the hot plate test, the maximum analgesic response of Ac-2a was determined to be 8.4 ± 0.25 at 90 min.
Likewise in Ac-2b the maximum response was observed at 45 and 60 min and in Ac-2c was at 45 min (Table
08). Statistically the difference was found to be very significant with p<0.001 with comparison to control with
treated group (Ac-2a, Ac-2b, and Ac-2c).

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CONCLUSSION
Table 1: Composition of Acelofenac gel formulation

Ingredients % w/w Gel 100 gm.

Batch Ac-1 Batch Ac-2 Batch Ac-3
Ac-1a Ac-1b Ac-1c Ac-2a Ac-2b Ac-2c Ac-3a Ac-3b Ac-3c
Acelofenac 1 1 1 1 1 1 1 1 1
Linseed oil 3 3 3 3 3 3 3 3 3
Piparine 0.5 1 1.5 0.5 1 1.5 0.5 1 1.5
Methyl salicylate 10 10 10 10 10 10 10 10 10
Carbopol 974 P 1 1.25 1.5 - - - - - -
HPMC - - - 1 1.5 2 - - -
Sodium CMC - - - - - - 3 4 5
Menthol 5 5 5 5 5 5 5 5 5
Triethanolamine 2 2 2 2 2 2 2 2 2
Methyl paraben 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
Propyleparaben 0.03 0.03 0.03 0.03 0.03 0.03
Chremophore Rh 40 4 4 4 - - - - - -
Sodium lauryl sulphate - - - - - - 0.03 0.03 0.03
Tween 60 1 1 1 - - - - - -
Isopropyl alcohol 6.5 6.5 6.5 6.5 6.5 6.5 6.5 6.5 6.5
Propylene glycol 15 15 15 5 5 5 5 5 5
Distilled Water (up to) 100 100 100 100 100 100 100 100 100

Table 2: Evaluation of appearance, pH, skin irritation, viscosity of different batch of acelofenac gel formulation

Batch Formulation Appearance pH Skin irritation Viscosity (Cp)

Ac-1a Cream 7.0 0 29000
Ac-1 Ac-1b Cream 7.0 0 29000
Ac-1c Cream 6.9 0 28980
Ac-2a White 7.0 0 31000
Ac-2 Ac-2b White 6.9 0 32000
Ac-2c White 7.0 0 32000
Ac-3a White 7.1 0 29400
Ac-3 Ac-3b White 7.1 0 28600
Ac-3c White 7.2 0 31000
Nusaid gel White 7.2 0 31000

The overall results of this study indicate that Acelofenac Topical gel having Piperine are superior to
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conventional dosage forms due to much better result of formulation, anti-inflammatory and anti-analgesic
profile. It can also play a vital role in efficient drug delivery of drug like various anti-inflammatory and anti-
analgesic agents.
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Table 3: Evaluation of spreadability, extrudability, bioadhesive strength and drug content of different batch of
acelofenac gel formulation
Formulations Time Spreadability Extrudability (Wt. Bioadhesive Drug Content
2
(sec) (g.cm/sec) required in g) strength (g/cm ) %
Ac-1a 10 52 554 1.17 96.3
Ac-1b 10 52 565 1.28 98.4.
Ac-1c 11 47.4 553 1.76 99.4
Ac-2a 12 43 610 1.02 99.3
Ac-2b 13 40 580 1.11 100.2
Ac-2c 14 38 560 1.14 98.1
Ac-3a 12 42 480 1.20 98.1
Ac-3b 14 37 496 1.19 97.3
Ac-3c 15 34 502 1.05 98.2
Nusaid gel 08 65 560 1.12 97

Table 4: Cumulative % Drug Release Profile of Aceclofenac Gels

Formulations
Time Ac-1a Ac-1b Ac-1c Ac-2a Ac-2b Ac-2c Ac-3a Ac-3b Ac-3c Nusaid Gel
(Hrs)
0 0.0 0.00 0.0 0.0 0.0 0.0 0.0 0.0 0.00 0.0
1 16.3 12.90 30.4 18.3 12.4 10.4 16.2 17.3 13.20 22.3
2 18.8 20.05 36.0 20.5 19.0 16.3 21.6 23.6 19.40 27.0
3 22.4 26.30 44.5 24.1 23.8 22.9 32.0 30.2 24.20 35.0
4 28.1 35.40 52.1 27.4 31.8 28.1 36.1 34.7 32.00 47.5
5 34.3 41.60 60.9 30.6 38.1 35.5 41.0 37.0 38.60 52.1
6 39.2 49.50 70.0 40.1 49.0 43.6 43.7 40.3 47.10 60.7
7 49.9 57.00 74.5 45.7 51.6 50.3 50.2 47.7 55.10 68.4
8 57.0 66.30 82.6 49.0 53.4 58.7 56.0 58.1 62.40 74.5

Table 5:Anti-Inflammatory Activity

0 1 Hr 2 Hr 3Hr 4 Hr 5 Hr 6 Hr

Control 00 0.34± 0.43± 0.64± 0.70± 0.65± 0.62±

0.003 0.003 0.004 0.008 0.005 0.004
Positive 00 0.21± 0.23± 0.24± 0.30± 0.28± 0.27±
Control 0.004** 0.003** 0.005** 0.003** 0.003** 0.007**
Ac-2a 00 0.23± 0.25± 0.28± 0.27± 0.25± 0.25±
0.003** 0.003** 0.009** 0.005** 0.003** 0.005**
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Ac-2b 00 0.22± 0.24± 0.25± 0.24± 0.24± 0.23±

0.004** 0.003** 0.004** 0.004** 0.005** 0.007**
Ac-2c 00 0.22± 0.24± 0.25± 0.25± 0.24± 0.23±
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0.004** 0.004** 0.003** 0.004** 0.002** 0.003**

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Vol 3, Issue 7, 2013. Vijay Kumar Singh et al. ISSN NO: 2231-6876

Table 6: Percentage Anti-inflammatory activity by inhibition of oedema

1 Hr 2 Hr 3 Hr 4 Hr 5 Hr 6 Hr
Positive Control 38.24% 46.51% 62.5% 57.14% 56.93% 56.45%
Ac-2a 32.35% 41.86% 56.25% 61.42% 61.54% 59.68%
Ac-2b 35.30% 44.18% 60.93% 65.71% 63.08% 62.90%
Ac-2c 35.30% 44.18% 60.93% 64.29% 63.08% 62.90%

Table 7: Analgesic Activity by Tail Flick Experiment

0 mins 15 mins 30 mins 45 mins 60 mins 90 mins 120 mins 180 mins
Control 4.2± 4.8± 4.2± 4.4± 4.8± 3.8± 4.4± 4±
0.20 0.20 0.37 0.24 0.20 0.37 0.40 0.32
Positive 4.6± 8.2± 9.4± 9.2± 7.4± 7.6± 5.8± 5.2±
Control 0.24 0.20** 0.24** 0.20** 0.24** 0.24** 0.20** 0.20*
Ac-2a 4.2± 5.2± 6.8± 8± 8.2± 8.6± 7± 6.2±
0.20 0.37 0.37** 0.31** 0.20** 0.24** 0.32** 0.37**
Ac2-b 5.2± 6± 7.8± 9± 8.2± 7.6± 6.4± 4.8±
0.49 0.32* 0.20** 0.32** 0.37** 0.24** 0.24** 0.20
Ac-2c 4.6± 6.2± 7.6± 8.4± 7.2± 6.6± 5.6± 5±
0.24 0.20** 0.24** 0.24** 0.20** 0.24** 0.24* 0.32

Table 8: Analgesic Activity by Hot plate experiment

0 15 mins 30 mins 45 mins 60 mins 90 mins 120 mins 180 mins

mins
Control 4.6± 5.0± 4.8± 4.6± 3.8± 3.4± 4.2± 4.8±
0.25 0.45 0.37 0.25 0.20 0.25 0.20 0.25
Positive 4.8± 8.4± 9.4± 9± 7.8± 7.4± 6± 5.2±
Control 0.200 0.25** 0.25** 0.32** 0.37** 0.25** 0.32** 0.20
Ac2-a 4.4± 6.2± 6.8± 8± 8.2± 8.4± 7.2± 6.4±
0.25 0.37 0.20** 0.32** 0.37** 0.25** 0.37** 0.25**
Ac-2b 4.6± 5.4± 7.6± 8.8± 8.8± 7.8± 6.6± 5.0±
0.40 0.40 0.25** 0.24** 0.37** 0.37** 0.24** 0.32
Ac-2c 4.8± 6.8± 7.4± 8.8± 7.4± 7.6± 6.0± 5.4±
0.20 0.37** 0.25** 0.37** 0.25** 0.40** 0.54** 0.25*
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Vol 3, Issue 7, 2013. Vijay Kumar Singh et al. ISSN NO: 2231-6876

Ac-1a
100
Ac-1b
Ac-1c
Ac-2a
80 Ac-2b
Ac-2c
Ac-3a

Cumulative Drug Relase

Ac-3b
60 Ac-3c
Nusaid Gell

40

20

0
0 2 4 6 8 10
Ti me (mi n.)

Figure 1: Drug release profile of acelofenac gel

Control
Posi ti ve Control
0.8 Ac-2a
Ac-2b
Ac-2c

0.6

0.4

0.2

0.0
0

Ti me (hr)

Figure 2: Inflammatory activity

80
% Inhibition of Oedema

60

40
Positive Control
Ac-2a
20 Ac-2b
Ac-2c

0
1

Ti me (hr.)

Figure 3: Percentage Inhibition of oedema

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Vol 3, Issue 7, 2013. Vijay Kumar Singh et al. ISSN NO: 2231-6876

15 Control
Posi ti ve Control
Ac-2a
Ac-2b
Ac-2c
**
10
** **
** ** ** **
** ****
** **
** ** **
**
** **
* ** **
**
**
*
5

0
15

30

45

60

90
0

0
12

18
Ti me (mi n.)

Figure 4: Tail flick method - assessment of analgesic activity

Control
15 Positive Control
Ac-2a
Ac-2b
Ac-2c

10 **
**
**** **
** ** ** **
** ****
**** ** **
** **
** **
** **
**
*
5

0
15

30

45

60

90
0

0
12

18

Ti me (mi n.)

Figure 5: Hot plate method - assessment of analgesic activity

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Vol 3, Issue 7, 2013. Vijay Kumar Singh et al. ISSN NO: 2231-6876

22 Mithun Dey 1, Saumen Karan1, Biswanath sa2 and Tapan Kumar Chatterjee1Comparative Study on
Ulcerogenicity and Anti-Inflammatory Activities of Pure Aceclofenac With The Aceclofenac Loaded
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