1. INTRODUCTION:

Periodontal disease refers to a group of problems that arise in the sulcus, the gap between the gums and the tooth. The term periodontal literally means “around the tooth” Periodontal disease are generally divided in to two groups (1) gingivitis which causes lesions (wounds )that affect the gums and (2) periodontitis, which damages the bone and the connective tissue that supports the teeth. The term periodontitis is derived from three words: ‘Peri’ (around), ‘odont’ (tooth) and ‘itis’ (inflammation).

Gingivitis is the mild form of periodontal disease. It causes the gums to become red swollen and bleed easily. There is usually little or no discomfort at this stage. Gingivitis is often caused by inadequate hygiene. Gingivitis is reversible with professional treatment and good oral home care. Untreated gingivitis can advance to periodontitis and eventually lead to tooth loss and other health problems^.1

Periodontal disease is a chronic bacterial infection that affects the gums and bone supporting the teeth, it can affect one tooth or many, and it begins when the bacteria in the plague (the sticky, colorless film) that constantly forms on your teeth causes the gums to become inflamed. With time, plaque can spread and grow below the gum line. Toxins produced by the bacteria in plaque irritate the gums. The toxins stimulate a chronic inflammatory response in which the body in its presence turns on itself its defense mechanism, and the tissues and bone that support the teeth are broken down and destroyed. Gums separate from the teeth, forming pockets (spaces between the teeth and gums) that become infected. As the disease progresses, the pockets deepen and more gum tissue and bone are destroyed. Often, this destructive process has very mild symptoms. Eventually, teeth can become loose and may have to be removed.

Periodontitis is very common, and is widely regarded as the second most common disease worldwide, after dental decay, and in the United States has a prevalence of 30–50% of the population, but only about 10% have severe forms. The local delivery of antimicrobial therapy to periodontal pockets has the benefit of putting more drugs at the target site while minimizing exposure of the total body to the drug. Pocket irrigation has been found to reduce microbial levels and provide some improvement in the clinical parameters, but the response to this therapy has been mixed and the therapy requires daily professional or patient administration for best results. The lack of drug retention in the periodontal pocket is probably the chief reason for these mixed results. The attractiveness of treating periodontal disease using the sustained release of antimicrobial agents in the periodontal pocket is based on the prospects of maintaining effectively high levels of the drug in the gingival crevicular fluid for a sustained period of time to produce the desirable clinical benefits of attachment level gain, pocket depth reduction, and reduction in bleeding on probing. In addition, a local delivery device should have high patient acceptance and a method of application acceptable to the dentist's practice^.2

2. METHODS AND MATERIALS:

Metronidazole (Batch No. 20100655) was from Maxil labs, Surat (Gujarat, India). Carbopol 934 was taken from Khanderwal labs (Mumbai, India). All other materials were of analytical grade and they were used without any further purification.

2.1 Preparation of Standard Materials:

2.1.1 Preparation of phosphate buffer pH 6.8.

2.1.2 λ- max Determination

2.1.3 Determination of calibration curve of Metronidazole

2.1.1 Preparation of Phosphate Buffer pH 6.8.:

Place 50 ml of 0.2 M potassium dihydrogen phosphate in a 200 ml volumetric flask, add 22.4 ml of 0.2 M NaOH and then make up the volume with water.^[^3]

2.1.2 λ- max Determination:

Dissolve 100mg of the Metronidazole in 3 ml of rectified spirit (solubilizing solvent) then make up the volume upto100ml with phosphate buffer of pH 6.8(1mg/ml). Pipetted out10 ml of it and further diluted up to 100ml with same buffer to produce 0.1mg/1ml. Now from this 1ml was pipetted and diluted up to 10 ml to get final dilution of 10µg/ml. This solution was scanned in UV-visible range of UV-visible spectrophotometer and maximum absorbance is determined. Figure No 1 is the Scanned Spectrum of Metronidazole which gives the maximum peak of Metronidazole at 277nm.

Fig.1: Scan Spectrum of Metronidazole

2.1.3 Determination of Calibration Curve of Metronidazole:

The dilution of 0.1mg/ml was prepared as discussed above. Now from this solution dilution of 10,20,30,40,50,60µgm/ml were prepared by withdrawing 1,2,3,4,5,6ml of the solution of concentration 0.1mg/ml and the volume was made up to 10ml by phosphate buffer of pH 6.8.The prepared solutions were measured sprectrophotometrically by UV at 277nm.The results given in the Table No 1 are graphically represented in the figure No 2 which shows the calibration curve of Metronidazole.

TABLE 1: CALIBERATION CURVE OF METRONIDAZOLE

Sr. No	Concentration (µg/ml)	Absorbance
1	10	0.153
2	20	0.291
3	30	0.456
4	40	0.597
5	50	0.730
6	60	0.888

Fig.2: Calibration Curve of Metronidazole

2.2 Preparation And Formulation:

2.2.1 Preparation of plain Carbopol 934 gel bases

2.2.2 Preparation of Carbopol 934 gel base containing metronidazole

2.2.1 Preparation of Plain Carbopol 934 Gel Bases:

Carbopol 934 gel base were prepared by homogenizing 0.5% and 1% (w/v) Carbopol in sufficient distilled water using a magnetic stirrer for 30 min and kept it to equilibrate for 24 h. After that, pH was adjusted to 5-7 with triethanolamine.^[4]

2.2.2 Preparation of Carbopol 934 Gel Base Containing Metronidazole:

It was prepared by addition of 0.1% Metronidazole solution into gel base during the stirring process and the steps were completed as mentioned for Carbopol 934 gel bases. ^[4]

2.3 Evaluation of Formulation:

2.3.1 Appearance

2.3.2 Syringybility

2.3.3 pH measurements

2.3.4 Metronidazole drug content study

2.3.5 In –vitro release of Metronidazole from gel base

2.3.1 Appearance:

The prepared Metronidazole gels were evaluated for its physical appearance.

2.3.2 Syringybility:

The prepared Metronidazole gels were passed through the 5ml syringe of grade 21 and 18. Time was recorded for the formulation to eject out completely from the syringe.

2.3.3 pH Measurement:

The pH was measured in each base using a pH meter that was accurately calibrated. A defined amount of each base was taken and diluted with distilled water and mixed well. The electrode of the pH meter was immersed in the prepared base solution for the pH determination.^[^5]

2.3.4 Metronidazole Drug Content Study:

An accurately weighted quantity of each gel base (1mg) was dissolved in about 50 ml of phosphate buffer (pH 6.8) to prepare 20µg/ml solution. These solutions were then filtered through 0.45μm membrane filters and analyzed sprectrophotometrically by U.V. ^[^6,
7]

2.3.5 In–Vitro Release of Metronidazole from Gel Base:

A piece of gelatin sheet of appropriate size previously soaked in distilled water for 24 hrs was placed in between the donor and the recipient compartment maintained at constant temperature of 37±2^oC. Lower compartment was filled up with 50 ml phosphate buffer of pH 6.8 .3g of Metronidazole gel was applied on the paper. The paper was kept between the donor and the recipient compartment in inverted position. 5 ml sample was withdrawn from the recipient compartment at the time interval of 5, 10, 15, 30, 45, 60, 120, 180 min. The withdrawn sample was replaced with phosphate buffer of pH 6.8 each time to maintain the constant volume for the receiving medium. The amount of the drug released from the bases was determined sprectrophotometrically at 277nm.^[^{8, 9]}

Fig. 3: (A) Metronidazole Gel With 0.5% Gel Base Concentraion ; (B) Metronidazole Gel With 1% Gel Base Concentration

3. RESULTAND DISCUSSION:

3.1. Appearance:

Both the prepared Metronidazole gel was found to be of buff white color and opaque in appearance with smooth texture and overall good appearance. Figure No 3 gives the picture of prepared Metronidazole gel with 0.5% and 1% polymer concentration and 0.1% Metronidazole.

3.2. Syringybility:

The syringybility of Metronidazole gel prepared by using different Carbopol 934 concentration was done and the results are given in Table No 2.The results show that the syringybility in seconds of the prepared gel with 0.5% polymer from the needle of gauze 24 and 18 was found to be 79 and 45 seconds respectively and that with 1% polymer was found to be 125 and 50 seconds respectively which clearly represent that the syringybility of the gel decreases with the increase in the polymer concentration.

TABLE 2: SYRINGYBILITY STUDY OF PREPARED METRONIDAZOLE GEL

Gauze of needle	Eject time for 0.5% w/v polymer(sec)	Eject time for 1% w/v polymer(sec)
24	79	125
18	45	50

3.1.

3.2.

3.3. pH Measurement:

pH measurement studies shown that the pH of the prepared gels were between 5-7. The readings are shown in the Table No 3.The results show that the pH of the prepared gel with 0.5% and 1% polymer concentration has been found to be between 5-7 which is suitable for it to exhibit thixotropic property.

TABLE 3: pH MEASUREMENT OF PREPARED METRONIDAZOLE GEL

Concentration of polymer (%)	Observed pH
0.5	6.15
1	6.2

3.4. Drug Content Study:

In the drug content analyses the given sample of 20µg/ml of prepared gel was studied and it gave the concentration as depicted in the Table No 4.The results show that absorbance value of 20µg/ml solution of prepared gel with 0.5% and 1% polymer fits the calibration curve of Metronidazole.

The deviation of the drug concentration in the gel solution from the standard calibration curve of Metronidazole can be attributed to the non uniform mixing of the drug solution with the gel base.

TABLE 4: DRUG CONTENT ANALYSIS OF THE PREPARED GEL

Concentration of polymer (%)	Absorbance	Concentration of Metronidazole (µg/ml)
0.5	0.477	32.04
1	0.368	24.63

TABLE 5: PERCENTAGE OF METRONIDAZOLE RELEASED FROM CARBOPOL 934 GEL BASE (0.5%W/V)

Sr. No	Absorbance	Time (min)	µg/ml	µg/5ml	µg/50ml	Cumulative Release	Rate of diffusion	% Release of drug
1	0.005	0	0.6	3.2	32.67	32.67	-	1
2	0.078	5	5.0	25.4	254.9	287.57	50.98	9.5
3	0.088	10	5.7	28.7	287.5	575.07	28.76	19.16
4	0.089	15	5.8	29.0	290.5	865.57	19.36	28.8
5	0.090	30	5.88	29.4	294	1159.57	9.8	38.6
6	0.092	45	6.0	30	300.65	1460.22	6.68	48.6
7	0.097	60	6.3	31.6	316.5	1776.72	5.27	59.2
8	0.099	90	6.4	32.3	323.5	2100.22	3.59	70
9	0.110	120	7.1	35.9	359.45	2459.67	2.99	81.9

TABLE 6: PERCENTAGE OF METRONIDAZOLE RELEASED FROM CARBOPOL 934 GEL BASE (1%W/V)

Sr. No	Absorbance	Time (min)	µg/ml	µg/5ml	µg/50ml	Cumulative Release	Rate of diffusion	% Release of drug
1	0.006	0	0.39	1.93	19.3	19.3	-	0
2	0.011	5	0.71	3.59	35.9	55.5	7.18	0.01
3	0.018	10	1.76	5.88	58.8	144.3	5.88	3.6
4	0.020	15	1.3	6.53	65.35	179.65	4.35	5.6
5	0.025	30	1.63	8.16	81.65	261.3	2.72	8.6
6	0.032	45	2.09	10.4	104.5	365.8	2.32	12
7	0.038	60	2.48	12.4	124	489.8	2.06	16.3
8	0.045	90	3.94	14.7	147	636.8	1.63	23
9	0.054	120	3.52	17.6	176	812.8	1.46	27

3.5. Release of Metronidazole from Carbopol 934 gel base:

The in vitro release of Metronidazole from prepared gel was studied and the obtained results are depicted in Table No 5 and 6.

The results shown that the 0.5 % concentration of the gel base gave maximum release of 81.7 % which is higher than the release from 1%concentration of gel base of 27% at 120 min. Figure No 4 gives the graphical representation of the results. The data also shows that the percentage release of Metronidazole from the Carbopol gel base increases with time up to 120 min.

Fig.4: Percentage of Metronidazole Released from Prepared Gel

Rate of diffusion of Metronidazole from the carbopol 934 gel base using compartment cell model show the results that the rate of diffusion decreases with time. The results are graphically represented in the figure No 5.

Fig.5: Rate of Diffusion of Metronidazole from Prepared Gel

The results also represent that the cumulative release increases with time. Cumulative release of up to 120 min from the prepared gel bases were 32.6, 287.5, 575, 865, 1159, 1460, 1776, 2100, 2459 µg/50ml for 0.5% polymer and for 1% polymer were 19.3, 55.5, 144.3, 179.65, 261.3, 365.8, 489.8, 363. 812.8 µg/50ml which is much lower than the gel of 0.5% polymer as depicted in figure No 6. Hence as the polymer concentration increase the release of the drug from the formulation decreases.

Fig.6: Cumulative Release of Metronidazole from Prepared Gel.

4. CONCLUSSION:

Evaluation of gel formulations for its physicochemical properties like drug release, syringybility, drug content and pH revealed that they can be designed in the treatment of the periodontal disease for the maximum loading of the drug to the periodontal pocket with the help of a syringe or a cannula. The results shown that syringybility and drug release from the Metronidazole gel decreased with the increase in the concentration of Carbopol- 934.

5. ABBREVIATION:

1. pH: Negative log of [H+]

2. μm: Micro meter

3. gm: Gram

4. cm: Centimeter

5. UV: Ultra violet

6. mm: millimeter

7. μg: Micro gram

8. mg: milligram

9. w/v: weight by volume

10. ml: milliliter

6. ACKNOWLEDGEMENT:

My heartily thanks to my beloved friend, Mr. Keyur D. Baruwala, final year student of Smt. B. N. B. Swaminarayan Pharmacy College, Salvav- Vapi for providing his valuable support during my research work.

7. REFERENCES:

1. http://www. Perio.org (viewed on 1^st August 2010).

2. http://www.ada.com (viewed on 20th August 2010).

3. Indian Pharmacopeia (2007).vol2; A-147.

4. Gallardo V., et al. Effect of cellulosic polymer on the release of salicylates in topical formulations. (2001); 50:33-40.

5. Elkababi S. Formulation, stability and rheological studies of certain creams of antifungal and local acting drugs. (Ph.D. Thesis)(2000).

6. Abd E.L., et al. Ahmed S. Preformulation studies on the release of non-steroidal anti-inflammatory drug from different pharmaceutical topical preparations (M. Sc. Thesis). (2007).

7. Tas C., et al. In vitro and ex vivo permeation studies of chlorpheniramine maleate gels prepared by carbomer derivatives. Drug Dev Ind Pharm. (2004); 30; 637-647.

8. Bhahmankar D.M., Biopharamceutics and Pharmacokinetics: A treatise.(2009). Ed: 1;96-106.

9. Dalmora M., et al. Inclusion complex of piroxicam with beta-cyclodextrin and incorporation in cationic microemulsion. In vitro drug release and in vivo topical anti-inflammatory effect. Int J Pharm. (2001); 222; 45-55.

Received on 28.02.2011 Modified on 01.05.2011

Research J. Pharm. and Tech. 4(7): July 2011; Page 1073-1077