INTRODUCTION:

Today vagina not only recognized as female genital organ but also it is well established as a route for drug administration due to some of its unique features like avoidance of first pass effect, large permeation area, permeability to large molecular weight drugs, rich vascularization and relatively low enzymatic activity ^[1],[2]. This anatomical area is very sensitive and frequently undergoes several bacterial or fungal infections. Among the several female health oriented problems, vaginal candidacies are most common gynecological problems ^[3].

It is reported that 30–50% of vaginitis episodes are cause due to Candida infection and two-third of world women population experience acute episodes of vaginal candidasis at least once during their life time ^[4]. In most of the cases it was found that an effective topical antifungal therapy is preferably required for the management of Candida infections. Some of the synthetic antifungal drugs are reported to serve the purpose effectively but at the same time it produce irritation in vaginal cavity and also disturbs vaginal flora when used at high concentration ^[5]. Again development of resistance against susceptible microbial stain is another major issue for those drugs ^[6].

It is a well established fact that phyctochemicals obtain from different plant sources have potent pharmacological activities ^{[7], [8]}. Among the several natural occouring phytochemicals like curcumin, aloin, aloe emodine, nimbidin, gingerol, allicin, coumarin etc. reported to have potent antimicrobial activities. These natural antimicrobial agents are used as alternate pharmaceutical APIs in combination with synthetic drugs not only to improve the effectiveness but also to reduce the chances of toxicity and microbial resistance^{[9], [10]}.

In consideration to the above fact this research work has been designed to develop a safe and effective antifungal gel formulation using combination of curcumin and fluconazole. The mucoadhesive gels were prepared using different ratios of polymers like HPMC K 4M, Carbopol 940. The prepared gels were submitted for in-vitro and in-vivo evaluation studies. Based on the results the final formulations were selected.

MATERIAL AND METHODS:

Materials used for the experimental work such as fluconazole was obtained as gift sample from cadila pharmaceutical ltd. India. Curcumin was purchase from Loba chemie ltd. India. Carbopol 940 and HPMC K4M were purchased from S D fine pvt.ltd. Triethanolamine and propylene glycol was purchased from loba Chemie Ltd. India.

Preparation of mucoadhesive Gel

Mucoadhesive gel was prepared, following the method as mentioned by (Basha N. B. et al) ^[11]. All the formulations were prepared using different ratios of Carbopol 940, Hydroxypropylmethyl cellulose as polymer system. Accurately weighted required quantities of polymers as well as APIs (Fluconazole along with Curcumin) were transferred to beaker. The hydro-alcoholic solvent system in required quantity was added to the beaker and allowed to soak for 24 hours. The hydrated mass was mixed properly using magnetic starrier. A few drops of triethanolamine as gelling agent, glycerin as a moistening agent along with propylene glycol was added slowly with continuous gentle stirring until the homogenous gel formed.

Visual and organoleptic examination

The prepared gel formulations were visually inspected for their color and appearance. It was found that gel formulations were slightly yellowish in color, free from any gritty particles and seems to be homogeneous. ^[12]

Spreadability Test

The test was performed as per Doaa A. H. et al. 2012, using parallel plate method for determining and quantifying the spreadability^[13]. The prepared formulations were placed in between a set of 20×20 cm glass slides. Then around 125 g weights were placed upon the upper slide so that the gel between the slides pressed uniformly to form a thin layer. The weight was removed and the excess of gel adhering to the slide was scrapped off. The set of slides were fixed in such a way that only upper slide may slip off freely due to the weight tied with it. The time taken for the upper slide to separate from the lower slide was noted. The experiment was carried out three times and the average of three reading was recorded. Following formula was used for calculation-

S = M.L/T

M = weight tied to upper slide

L = Length of glass slide

T = Time taken to separate the slide

Percentage Yield

In this study weight of empty container and weight of gel formulation along with container was measured accurately. Then the weight empty container was subtracted from the weight of container with gel formulation. It gives the practical yield. Then the percentage yield was calculated by the formula.[12]

Drug Content Determination

A specific quantity of prepared gel was weighted and dissolved in 100ml of buffer of pH 4.5. The volumetric flask containing gel solution was shaken for 2hr on mechanical rotary shaker in order to get complete release of drug from gel base. It was further kept aside for 24hrs. After that the content was filtered using Millipore filter (0.45μm). Absorbance was measured After suitable dilution using UV- visible spectrophotometer (UV – 1700, Shimadzu, Japan) at λ _max260 nm and 422 nm respectively using buffer (pH 4.5) as blank^[14].

Determination of pH

The pH of gels was determined using a digital Electronic pH meter. Initially the pH meter was calibrated using standard buffers of pH 4, 7 and 9. Accurately 5 gm of gel was weighed and dispersed in 50 ml of purified water ^[15]. The electrode of pH meter was dipped in dispersion and the numerical value displayed in PH meter was noted.

Viscosity and Rheological Studies

The viscosity of gels was determined by using Brookfield viscometer ^[16]. Formulations were placed in the sample holder and suitable spindle attached perpendicularly inside the sample. The spindle was attached to viscometer and allowed to rotate at a constant speed. The reading of viscometer was measured.

In-Vitro Drug Release Study

The apparatus consists of a glass cylinder with both the ends open, 10 cm in height, 3.8 cm in outer diameter and 3.2 cm in inner diameter was used as a permeation cell. A cellophane membrane previously soaked in distilled water for 24 hours was fixed to the one end of the cylinder. 10 mg of gel was taken in the cell (donor compartment) and the cell was immersed in a beaker containing 100 ml of buffer of pH 4.6 (receptor compartment). The whole assembly was fixed in such a way that the lower end of the cell containing gel was just touched (1-2 mm deep) to the diffusion medium, the medium in the compartment was agitated using a magnetic stirrer at the temperature 37±1ºC.^[17] Sink condition were maintain throughout the experiment and after suitable dilution; the sample was analyzed by using Shimadzu UV visible spectrophotometer at 260nm and 422 nm respectively.

Anti-fungal Studies

Antifungal study was performed following disk diffusion method. In this study sabouraud dextrose agar as media and fungal stain like Candida albicans (MTCC 227) were used.First sterile media plates were inoculated with Candida stain suspension aseptically. After that hole was created in each petridish with the help of sterile steel bore of 6 mm. Than required concentration of the standard drug Fluconazole and curcumin solution (individual as well as in combination) were prepare and placed in the bores and incubated the media plates for 72 h at 25°C in incubators ^[18]. Then the zone of inhibition was measured.

Kinetic Treatment of Dissolution Data

The kinetics of drug release was determined on the basis best fit to model. Data obtained from release study was analyzed according to Zero Order Kinetics, First order kinetics, Hixon -Crowell equation, Higuchi equation ^{[18], [20]}.

Vaginal irritation test

The primary vaginal irritation test was performed on Newzealand white female rabbit (1.5-2.5kg). All the animals were kept under standard laboratory condition. The total numbers of animals were divided into four batches, each batch containing three animals. 1ml of prepared gel was inserted daily, for 10 days, through a lubricated catheter into the vagina of rabbits. The external genitalia are observed regularly for any signs of oedema, erythema or discharge as a reaction to the exposure to the test materials ^{[21], [22]}. The experimental protocol of the study was approved by the Institutional Animal Ethics Committee (Regd. No. CIP / IAEC / 2013-14/044).

RESULT AND DISCUSSION:

As per visualization and organoleptic evaluation it was found that all the prepared gel formulations were smooth, free from any grittiness, transparent and homogeneous in nature. The gel formulations were off slightly yellowish in color and having very good spreadability. All the prepared formulations having good yield value and the pH of the prepared formulations were ranges within (5-6), that complies with vaginal pH hence can be consider suitable for vaginal application. The viscosity of prepared formulations were varied from (42000-70000), it is due to the difference in concentration of polymer present in the formulation. It has been found that viscosity of the prepared formulations were directly proportional to concentration of Carbapol 940 present in it. Again it is also a fact that higher the viscosity greater will be adhesion and better will be the retention time. Result of drug content study shows that all the prepared formulation contains around 97-99% of drugs, which consider as a sign of good formulation. Results of the all the essential evaluation parameters are shown in [Table no-02]

Fig 1. Cumulative % Drug release of curcumin from formulations

Fig 2. Cumulative % Drug release of Fluconazole from prepared formulations.

Table 1. Formula for preparation of gels.

Ingredients	F1	F2	F3	F4	F5
Curcumin%	0.1	0.1	0.1	0.1	0.1
Fluconazole%	0.4	0.4	0.4	0.4	0.4
Carbopol 940(mg)	250	167	125	100	333
HPMC(mg)	250	333	375	400	167
Water(ml)	50	50	50	50	50
Glycerin(ml)	2	2	2	2	2
Propylene glycol(ml)	4	4	4	4	4
Triethanolamine (drops)	3	3	3	3	3

Table 2. Study of Different in-vitro evaluation parameters of prepared gels.

Sr. no.	Formulation	Percentage yield%	pH of gel	Viscosity Cps	Spreadability gm.cm²	%Drug content
Sr. no.	Formulation	Percentage yield%	pH of gel	Viscosity Cps	Spreadability gm.cm²	Curcumin	Fluconazole
1	F1	96.5	5.66	42500	11.30	97.2 ±1.92	98 ±1.39
2	F2	97.62	5.55	42000	10.89	98 ± 1.68	98.5 ± 1.27
3	F3	98	6.62	66000	11.75	98.3 ± 1.97	99 ± 1.75
4	F4	97.2	6.6	54000	10.55	97 ± 1.16	98.3 ± 1.24
5	F5	98.2	5.12	70000	10.62	97.5 ± 1.59	97 ± 1.83

Table 3. Release kinetic best fit model for all formulations.

Formulation	Zero order R²		First order R²		Higuchi matrix R²		Peppas plot R²		Best fit model
Formulation	Cur	flu	Cur	Flu	cur	Flu	cur	flu	Best fit model
F1	0.9583	0.9928	0.9929	0.9976	0.8825	0.9684	0.8485	0.9259	First order
F2	0.9628	0.9743	0.9952	0.9914	0.8936	0.945	0.8706	0.968	First order
F3	0.92	0.9799	0.9747	0.9928	0.8286	0.9684	0.796	0.9259	First order
F4	0.9652	0.9539	0.9945	0.9899	0.8957	0.9181	0.8591	0.9404	First order
F5	0.9572	0.9754	0.9913	0.9955	0.8821	0.9079	0.843	0.9252	First order

Table 4. Antifungal screening based on Zone of inhibition.

Formula code	Curcumin mg	Area (cm²)	Fluconazole mg	Area (cm²)	Curcumin + Fluconazole mg	Area (cm²)
F1	0.5	0.28	0.5	0.50	0.5+4	17.34
F2	1.0	1.14	1	1.32	1+4	26.40
F3	1.5	1.76	2	5.30	1.5+4	22.89
F4	2.0	2.83	3	11.93	2+4	23.74
F5	2.5	3.46	4	19.63	2.5+4	24.61

On the basis of analysis of in-vitro release data and release kinetic study it was observed that almost all the formulation were showing 70-80% of drug release within 6-7 hrs following first order release kinetics. Based on the anti fungal study it has found that combination of curcumin and fluconazole shows better zone of inhibition as compare to their individual effect. Among the several formulations F2 formulation shows better result. The mark differences in in-vitro antifungal activities between single API and combination of APIs may be due to summation or addition or synergism effect. Clinical signs of irritation include the development of a rash, inflammation, swelling, scaling, and abnormal tissue growth in the affected area was not found after RVI test, that indicate the formulation was free from any short of irritation and will not produce any kind of discomfort to the patients during therapy. The data obtained after in-vitro evaluation represents that all the formulation gives satisfactory response. Among the several formulations F2 formulations that contain 1:4 ratios of curmumin and fluconazole shows better results. Further clinical studies are required to establish the formulation before commercialization.

ACKNOWLEDGEMENTS:

Author would like to acknowledge Cadila Pharmaceutical ltd. India for providing Fluconazole as drug sample and Columbia Institute of Pharmacy as research centre for providing the research facilities.

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Received on 04.04.2016 Modified on 11.05.2016

Res. J. Pharmacognosy and Phytochem. 2016; 8(3): 116-120.

DOI: 10.5958/0975-4385.2016.00021.2