Formulation and in vitro evaluation of Cream containing Diclofenac Sodium and Curcuma Longa for the management of Rheumatoid Arthritis

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Vol. 4, No. 4 (2014): 654-660 Research Article

Open Access

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ISSSSNN:: 22332200--66881100

Formulation and in vitro evaluation of Cream

containing Diclofenac Sodium and Curcuma Longa for

the management of Rheumatoid Arthritis

Muhammad Razi Ullah Khan

1, 2

*, Shahaid Masood Raza

1

and Musaddiq Hussain

1

School of Pharmacy, The University of Faisalabad, Faisalabad, Pakistan

2

Faculty of Pharmacy, University of Sargodha, Sargodha, Pakistan

* Corresponding author: Muhammad Razi Ullah Khan; e-mail: razi_pharmacist@yahoo.com

ABSTRACT

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most frequently prescribed drug groups. These drugs are used dermal or systemically in treatment of various rheumatic diseases, including Rheumatoid arthritis (RA), as well as for Osteoarthritis, low back pain and some joint diseases. Diclofenac sodium is a well tolerated NSAID because of its limited numbers of adverse effects and topical formulation has excellent permeation and absorption into the skin and is frequently prescribed for the long term treatment of Rheumatoid arthritis, Osteoarthritis and Ankylosing spondylitis. The present investigation was to develop novel cream formulation containing Diclofenac sodium in combination with most effective and potent natural anti-inflammatory agent curcuma longa, which is reported to possess strong anti-inflammatory effects in Rheumatoid arthritis and Osteoarthritis, according to the study by University of Arizona researchers. Diclofenac sodium in combination with Curcuma longa is a good rational, where curcuma longa produces synergistic anti-inflammatory effects with Diclofenac sodium. Formulation containing fixed concentrations (1%) of Diclofenac sodium with curcuma longa was prepared. To access the efficacy of formulation different in-vitro tests including stability studies, tube extrude ability, spread ability, pH, skin irritation test, viscosity and rheological properties, drug diffusion study and anti- inflammatory studies were carried out. The results obtained were encouraging and formulation containing Diclofenac sodium (1%) with curcuma longa was found better than alone Diclofenac sodium cream formulation.

Keywords:

Diclofenac sodium, Curcuma longa, Rheumatoid arthritis, Cream.

INTRODUCTION

In the last few decades there has been an exponential

growth in the field of herbal medicine. It is getting

popularized in developing and developed countries

owing to its natural origin and lesser side effects

[1].The scientific evidence has brought about the possibility of utilization of herbal plant in the treatment of inflammatory diseases and the development of anti-inflammatory products [2]. Inflammation is a tissue reaction by the body to injury and involves a complex array of enzyme activation, mediator release, extravasations of fluid, cell migration, tissue breakdown and repair [3]. Rheumatoid arthritis

represents the commonest form of chronic

inflammatory joint disease [4]. Arthritis is one of the most distressing and disabling syndromes encountered in medical practice [5]. An estimated 1-2% of adult

population is affected [6]. Diclofenac Sodium a Non Steroidal Anti-inflammatory agent is frequently prescribed for the long term treatment of Rheumatoid Arthritis, Osteoarthritis and Ankylosing Spondylitis [7]. The drug undergoes substantial first pass effect and only 50% of drug is available systemically [8]. Further, the drug is known to induce ulceration and bleeding of the intestinal wall. To avoid the adverse effect, alternate routes of administration have been tried by investigators [9]. Delivery of Diclofenac Sodium via skin offers the potential advantage of by passing gastrointestinal first pass metabolism associated with oral administration [10]. Turmeric (the Common name for Curcuma longa) is a perennial member of the Zingiberaceae family, derived from the rhizomes of the plant and has a long history of use in Ayurvedic medicine as a treatment for inflammatory conditions [11]. The major pigment compound of Curcuma longa is

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Curcumin which has been shown to have including cytokines (TNF alpha and IL-1 beta) [12]. The present research has been undertaken with the aim to develop a topical Diclofenac Sodium cream formulation along with the Curcuma longa, which would attenuate the gastrointestinal related toxicities associated with oral dosage forms. Diclofenac Sodium having molecular weight of 296.1 and melting point of 157°C can be considered ideal to permeate through the skin.

MATERIALS AND METHODS

Chemicals:

Diclofenac Sodium was received as a gift from Sami Pharmaceuticals Pakistan , Stearic Acid (BDH labs, England), Stearyl Alcohol (BDH Labs, England), Bees Wax (Kukdong oils and chemicals, Korea), Methyl Parabens (BDH labs, England), Liquid Paraffin (Kukdong oil and chemicals, Korea), Polyoxyethylene (80) Sorbitan monooleate (Tween 80) (Merck, Germany), Potassium Hydroxide (Merck, Germany), , Sorbitol liquid USP (Merck, Germany) and De-ionized water (Medilines Diagnostic division)

Plant Material:

Plant material used for that study was collected from the surroundings of Kasur city of Pakistan in the month of March and identified by Department of Botony, Faculty of Biological Sciences, University of Sargodha Pakistan.

Apparatus:

Beaker 50ml, 100ml (Pyrex, England), Pipette 10ml (Preciclolor, Germany), Conical flask 50ml, 100ml (Pyrex, Germany), White colored jar, Amber colored glass jar, Aluminum foil and Aluminum collapsible tube.

Instruments:

Spectrophotometer U.V1700 (Shimdazu, Japan),

Magnetic stirrer/ Hot plate (Made in Germany), Weighing balance (Analytical grade), pH meter (Model No: 3510, England), Keshary-chien diffusion cell, Homogenizer (Euro-Star, IKA D 230, Germany), Brookfield digital viscometer (model DV-II+, Brookfield Engineering Laboratory, INC. USA), Refrigerator (Dawlance, Pakistan), Incubator (Sanyo MIR-162, Japan), Oven (Schutzartdin 40050 IP-20, Germany), Soxhlet Apparatus.

Animals:

Albino rats of either sex weighing between 200-250 g were used for the present investigation. The rats were housed at controlled temperature (25±2ºC) and 12hrs dark-light cycle and provided basal diet in the form of pellets, water and libitum.

Preparation of Turmeric Extract:

The rhizomes of curcuma longa (Turmeric) were cleaned, washed with de-ionized water, sliced and dried in the sun for one week. Dried rhizomes were cut in small pieces and powered by electronic mill. 200 gm of sample was taken into thimble and placed in a Soxhlet apparatus. The apparatus was setup with

various solvents ranging from non polar to polar. 1 lit of solvent was added and extracted according to their boiling point for seven hours. The solvents used were chloroform (B.P. =61˚C), ethyl acetate (B.P. =77˚C), methanol (B.P. =65˚C) and acetone (B.P. =56.53˚C). After completion of extraction the dark brown extract was then cooled, concentrated using rotary evaporator get a crude dried extract which was black orange in color.

Formulation of Diclofenac Sodium Cream

containing Curcuma longa:

1% by weight of Diclofenac Sodium cream containing curcuma longa was made according to the formulation given in Table I.

Table I: Formulation of Diclofenac Sodium cream containing Curcuma longa

S. No. Ingredients Percentage Composition 1 Diclofenac Sodium 1.0

2 Curcuma longa 3.0 3 Liquid Paraffin 5.0 4 Stearic Acid 0.30 5 Bees Wax 5.0 6 Stearyl Alcohol 10.0 7 Tween-80 8.0 8 Methyl Parabens 0.12 9 Sorbitol Solution 6.0 10 Potassium Hydroxide 1.50 11 De-ionized Water 60.08

Preparation of cream:

Diclofenac Sodium cream with turmeric extract was formulated by the method of Nazir et al [13]. The aqueous and oil phases were taken into bakers and heated to 75ºC over a water bath. The oil phase was comprised of Diclofenac Sodium, Liquid Paraffin, Bees Wax, Stearyl Alcohol, Tween-80 and Stearic Acid while the aqueous phase was composed of Extract of Curcuma longa, Methyl parabens, Sorbitol Solution and Potassium Hydroxide. Drop wise addition of the aqueous phase to the oil phase was done with constant stirring at 2000 rpm in a homogenizer for a period of 15 min. The homogenizer speed was then reduced to 1000 rpm and homogenization was continued for another 5 min. The speed was further reduced to 500 rpm and the homogenization extended for 5 min. Diclofenac cream containing the Curcuma longa extract was formulated.

Organoleptic Evaluation:

Changes in organoleptic properties of the cream were evaluated by visual inspection and the properties evaluated included the color of the creams, liquefaction and phase separation. These were evaluated over a period of 3 months at specific time intervals.

Physical Evaluation of Cream:

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Determination of pH:

The pH measurement was carried out by using a

calibrated digital type pH meter by dipping the glass

electrode and the reference electrodecompletely into

the cream so as to cover theelectrodes [14]. pH was

determined at the time of preparation and on monthly basis for the 3 months period.

Determination of Spread ability:

Spread ability of cream was determined by the apparatus which consists of a wooden block, which was provided by a pulley at one end [15]. By this method spread ability was measured on the basis of slip and drag characteristics of cream. An excess of cream (about 2g) under study was placed on this ground slide. The cream was then sandwiched between this slide and another glass slide having the dimension of fixed ground slide and provided with the hook. 1 kg weighted was placed on the top of the two slides for 5 minutes to expel air and to provide a uniform film of the cream between the slides. Excess of the cream was scrapped off from the edges. The top plate was then subjected to pull of 80 gm. With the help of string attached to the hook and the time (in seconds) required by the top slide to cover a distance of 7.5 cm be noted. A shorter interval Indicate better spread ability. Spread ability was calculated using the following formula:

S = M × L / T

Where,

S = Spread ability,

M = Weight in the pan (tied to the upper slide)

L = Length moved by the glass slide

T = Time (in sec.) taken to separate the slide

completely each other.

Determination of Tube Extrude ability:

Tube extrude ability was determined by filling the cream in clean, lacquered aluminum collapsible tube and pressed firmly at the crimped end. When the cap was removed, cream extruded until pressure dissipated. Weight in grams required to extrude 0.5 cm ribbon of cream in 10 seconds was determined [16].

Determination of Viscosity:

Viscosity of cream was determined by Brookfield

viscometer. The viscosity measurements were done

using Brookfield DV-II + viscometerusing LV-4 spindle.

The developed formulation was poured into the

adaptor of the viscometer and determined the viscosity

of the testsample as per standard operating procedure

of viscometer. The spindle was rotated at speeds of 0.5, 1, 5, 10 and 20 rpm. The reading near to 100% torque was noted [17].

In Vitro Diffusion Studies:

The diffusion studies were performed using a Keshary-chien diffusion cell. The cell was locally fabricated and had a 25 ml receptor compartment. The dialysis membrane was mounted between the donor and receptor compartments. The cream formulation was applied uniformly on the dialysis membrane and the

compartments were clamped together. The receptor compartment was filled with the phosphate buffer (pH 7.4) and the hydrodynamics in the receptor compartment were maintained by stirring with a magnetic bead. The study was carried out for 24 hrs with the interval of 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 hrs. 1ml of samples was withdrawn from the receptor compartment at pre-determined time intervals and an equal volume of buffer was replaced. The samples were analyzed after appropriate dilution for drug content Spectrophotometrically at 280 nm [18].

Skin Irritation Study:

In skin irritation test 0.5 gm of the cream formulation was used as the test substance was applied to an area

of approximately 6 cm2_{of skin and covered with a}

gauze patch. The patch was loosely held in contact with the skin by means of a semi-occlusive dressing for the duration of 1 hour and gauze was removed. At the end of the exposure period, i.e., 1 hour, residual test substance was removed, without altering the existing response or integrity of the epidermis. Observations have recorded after removal of the patch. The cream was applied to the skin once a day for 7 days and observed for any sensitivity and the reaction if any was graded [19].

Stability Studies:

Stability studies on the cream formulation were

conductedover a period of 6 months at three different

conditions: (a) At 25 ± 1ºC (b) at 40 ± 1ºC (c) at65 ±

1ºC. The cream was analyzed by UV- Visible Spectrophotometer, immediately after preparation (at zero time) and after every month until 6 months period [20].

The active contents in cream formulation were determined by measuring the absorbance of sample solution on UV Spectrophotometer at 280nm wavelength at above mentioned time intervals and by calculating the remaining %age of active content by following formula:

Remaining %age of active content in sample

solution = (Absorbance of Sample / Absorbance of

Standard) × (Conc. of Standard / Conc. of Sample) × % age purity of Standard [21].

Determination of Drug contents by

Spectrophotometric Method: Preparation of Standard Solution:

50 mg of Diclofenac sodium was carefully weighted on analytical balance and dissolved in ethanol (96%) and made the volume upto 100ml with ethanol. The solution was then filtered and 1ml was taken from that solution and made the volume of that solution up to 50ml with same solvent and it was taken to be the standard solution in UV Visible spectrophotometer.

Preparation of Sample Solution:

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the same solvent. The solution was then filtered and 1 ml was taken made up to 50 ml with ethanol. The absorbance was measured at 280nm using ethanol as blank solution.

RESULTS AND DISCUSSION

The purpose of the present investigation was to develop a topical Diclofenac sodium cream formulation along with a most effective and natural anti-inflammatory agent, curcuma longa, that conveniently deliver the drug to the localized area of the skin and is used for the management of Rheumatoid arthritis and does not produce any undesirable side effects. Diclofenac sodium cream along with potent anti-inflammatory agent Curcuma longa was prepared using

different concentration of excipients and active ingredient. Accelerated stability study was done at 25 ± 1°C, 40 ± 1°C and 65 ± 1°C. Stability testing was done

for the period of 6 months (180 days). It isevident from

the results that cream formulation is best suitable at (25 ± 1°C) as % age of drug remaining is not decreased by more than 10% [22]. It is also evident from the results of standard deviation at the end of 6 months that at 25 ± 1°C standard deviation was least and it fell into acceptable range, but at 40°C and 65°C the standard deviation is bit higher and away from normal and reasonable range. So it can be concluded, that at 25 ± 1°C, the cream formulation fulfils the criteria required for a pharmaceutical cream preparation to be acceptable concerning accelerated stability studies.

Table II percentage remaining of drug content at zero time

Absorption of Standard

Absorption of Sample Percentage of active

drug in the sample

Mean Standard Deviation

0.625 0.623 99.28% 0.624 0.001414

Table III percentage remaining of drug content after 1 month

Absorption of Sample Percentage of active drug in the sample

At 25 ± 1°C 0.617 98.32% 0.621 0.005656 At 40± 1°C 0.612 97.52% 0.618 0.009192 At 65 ± 1°C 0.610 97.20% 0.6175 0.01060

Table IV percentage remaining of drug content after 2 months

At 25 ± 1°C 0.604 96.25% 0.6145 0.014849 At 40± 1°C 0.597 95.13% 0.611 0.019798 At 65 ± 1°C 0.595 94.81% 0.61 0.021213

Table V percentage remaining of drug content after 3 months

At 25 ± 1°C 0.597 95.04% 0.611 0.019798 At 40± 1°C 0.585 93.22% 0.6105 0.028284 At 65 ± 1°C 0.581 92.58% 0.603 0.0311112

Table VI percentage remaining of drug content after 4 months

At 25 ± 1°C 0.590 94.92% 0.607 0.024748 At 40± 1°C 0.568 90.51% 0.596 0.040305 At 65 ± 1°C 0.563 89.71% 0.594 0.043840

Table VII percentage remaining of drug content after 5 months

At 25 ± 1°C 0.581 92.58% 0.603 0.031112 At 40± 1°C 0.547 87.16% 0.586 0.055154 At 65 ± 1°C 0.543 86.53% 0.584 0.057982

Table VIII percentage remaining of drug content after 6 months

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%

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Time in Months

Figure I. A graphical representation between percentage of drug concentration and time in months

The visual appearance of cream formulation was checked at the time of preparation and at the end of every month until 3 months period. The cream was light yellowish in colour and had a cool and smooth feeling on application and there was found no significant difference in visual appearance at the end of three months period from the time of preparation. pH evaluation is also important to check the stability of cream formulation. The pH of prepared cream with extract was found to be around 6 which is suitable for

topical application because the pH of skin is between 4.5-6. The result of spread ability varies from 12.78 to 17.01g/sec whereas the extrude ability of cream formulation from the collapsible tube varies from 180 to 190 g as shown in table IX. At 0.5 rpm to 20 rpm viscosity was decreased from 6896 to 671 cps. So, if we decrease the rate of shear it increases the viscosity of cream. Viscosity of creams is inversely proportional to rate of shear (rpm) and the results are showed in table no. X.

Table IX Evaluation data of Diclofenac cream with herbal extract

Parameters evaluated Study Period

At Zero time 1 Month 2 Months 3 Months

Visual appearance Light Yellowish No change No change No change pH 6.79 6.81 6.78 6.80 Spread ability (g/sec) 12.78 14.12 15 17.01 Tube extrude ability (g) 180 180 185 190

Table X Viscosity of cream formulation (cps) at different rpm

Speed in rpm At Zero time 1 Month 2 Months 3 Months

0.5 6710 6825 6883 6896 1.0 3147 3271 3291 3343 5.0 1614 1621 1591 1618 10 931 964 939 986 20 625 642 659 671

In Vitro Drug Diffusion study:

From the data we have found that the prepared topical Diclofenac cream formulation with herbal extract

releases 84.19% of drug over a period of 24 hours as

shown in Table XI. From the In – vitro drug diffusion

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prepared, controls the release of drug for longer period of time which will be helpful to avoid the more fluctuation and also reduces the cost of therapy.

Skin Irritation Test:

In skin irritation test, no signs of erythema and edema were found after 7 days of cream application in rats and are graded in table XII.

Table XI in Vitro Drug Diffusion Study over period of 24 Hours

Time (Hours) Percentage of drug release

0.0 0.0 0.5 7.78 1.0 15.77 2.0 23.57 4.0 34.98 6.0 43.03 8.0 51.17 10.0 66.82 12.0 74.45 24.0 84.19

Table XII Skin irritation study results over 7 days period

A – No reaction, B – Slight patchy erythematic, C –Slight but confluent or moderate but patchy erythematic, D – Moderate erythematic, E – Severe erythematic with or without edema.

CONCLUSION

The present cream formulation was developed by

taking into consideration that in cream formulations

there is present no direct contact of active drug with

stomach wall. This can be a reason to remove the

chances of gastric mucosal damage to a reasonable

level that is caused by the use of soliddosage forms of

NSAIDs. The cream formulation contains Diclofenac Sodium along with extract of Curcuma longa , a spice most often found in curry dishes may help prevent Rheumatoid arthritis and Osteoporosis. Diclofenac Sodium is an NSAID that is very effective to mimic the pain and inflammation in arthritis patients and

Curcuma longa performs a synergistic

anti-inflammatory effect.

Acknowledgements

The authors are thankful to The University of Faisalabad, Faisalabad, Pakistan for providing necessary laboratory facilities to carryout work with great ease and precision and also acknowledge with special thanks to Sami Pharmaceuticals, Pakistan for providing the Diclofenac sodium active material as a gift sample for present work.

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