4.4 MICROBIOLOGICAL METHOD FOR THE ESTIMATION OF. The microbiological assay was performed by using the test

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1 MICROBIOLOGICAL METHOD FOR THE ESTIMATION OF AMOXICILLIN The microbiological assay was performed by using the test organism Staphylococcus aureus. The strain was isolated from soil and allowed to grow on nutrient Agar medium Preparation of Stock Solutions: 100mg of Amoxicillin was dissolved is 100ml of water Procedure:s The solutions of standard concentration of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 μg/ml were obtained by suitably diluting the stock solution with serum. The medium used for the assay was prepared in the laboratory by using the following ingredients. Peptone : 0.5g Sodium Chloride : 0.5g Beef extract : 0.15g Yeast extract : 0.15g Agar :1.5g Distilled water was used to prepare stock solution (100ml) and the ph was maintained at 7.4. The media so prepared were sterilized. After sterilization, they were cooled at 40ºC and inoculated with the culture of S.aureus and were poured in a clean sterile Petri dish and allowed to solidify. After

2 110 solidification, 10 wells of 3mm in diameter were cut. The standard different solutions were poured into the well and incubated. After the incubation, the zone of inhibition was measured and tabulated. 4.5 Microbiological Method for the Estimation of Ciprofloxacin Ciprofloxacin: The microbiological assay was performed by using the test organism, Escherichia coli.the strains were allowed to grow on nutrient agar medium Preparation of stock solution: 100mg of Ciprofloxacin was dissolved in 100ml of water Procedure:- The standard concentrations of 0.5, 1, 1.5 up to 5.0 μg/ml were obtained by suitably diluting the stock solution with serum. The freshly prepared and autoclaved medium was used for this assay. The test organism was inoculated in the medium and it was poured in a sterile Petri dish and allowed to solidify.after solidification, 10 wells of 3mm in diameter were cut. And the solutions of different concentrations of ciprofloxacin solution were poured into the well and incubated and the zone of inhibition was measured

3 MICROBIOLOGICAL METHOD FOR THE ESTIMATION OF AMPICILLIN The microbiological assay was performed by using the test organism, Staphylococcus aureus. The strain was isolated from soil and allowed to grow on nutrient Agar medium Preparation of Stock Solutions: 100mg of Ampicillin was dissolved is 100ml of water Procedure:s The solutions of standard concentration of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 μg/ml were obtained by suitably diluting the stock solution with serum. The medium used for the assay was prepared in the laboratory by using the following ingredients. Peptone : 0.5g Sodium Chloride : 0.5g Beef extract : 0.15g Yeast extract : 0.15g Agar :1.5g

4 112 Distilled water was used to prepare stock solution (100ml) and the ph was maintained at 7.4. The media so prepared were sterilized. After sterilization, they were cooled at 40ºC and inoculated with the culture of S.aureus and were poured in a clean sterile Petri dish and allowed to solidity. After solidification, 10 wells of 3mm in diameter were cut. The standard different solutions were poured into the well and incubated. After the incubation, the zone of inhibition was measured and tabulated. 4.7 Microbiological Method for the Estimation of Ofloxacin Ofloxacin: The microbiological assay was performed by using the test organism, Escherichia coli.the strains were allowed to grow on nutrient agar medium Preparation of stock solution: 100mg of Ofloxacin was dissolved in 100ml of water Procedure: The standard concentrations of 0.5, 1, 1.5 up to 5.0 μg/ml were obtained by suitably diluting the stock solution with serum. The freshly prepared and autoclaved medium was used for this assay. The test organism was inoculated in the medium and it was poured in a sterile Petri dish and allowed to solidify.

5 113 After solidification, 10 wells of 3mm in diameter were cut. And the solutions of different concentrations of ofloxacin solution were poured into the well and incubated and the zone of inhibition was measured Preparation of nanoparticles: The sepia nanoparticles were prepared by controlled gellification process. This method is already in application for the encapsulation of antibiotics. Initially, the sepia powder was taken and it was mixed with the drug Amoxicillin solution. To this, 1 ml of calcium chloride solution was added and mixed thoroughly by stirring with the magnetic stirring for about 15min. Then 1 ml of chitosan solution was added and the stirring was continued for another one hour. Finally, the resultant solution was sonicated in the organ Sonicator for 30min. Then,this mixture was kept for one day at 25-30ºC. The drug-loaded nanoparticles were harvested by centrifugation at rpm for one hour. The flakes formed were collected and stored in a suitable container.the initial concentration of the drug: - polymer ratio was 1:1 w/w. The same method was followed for the preparation of the Ciprofloxacin nanoparticles

6 114 The drug and polymer ratio was as follows Amoxicillin: Sepia Ciprofloxacin:Sepia Ofloxacin:Sepia Ampicillin:Sepia 1:1 1:1 1:1 1:1 1:1.5 1:1.5 1:1.5 1:1.5 1:2 1:2 1:2 1:2 1:2.5 1:2.5 1:2.5 1:2.5 1:3 1:3 1:3 1: :4 1:4 1:4 1:4 4.8 CHARACTERISATION OF NANOPARTICLES: The following In-vitro evaluation tests were performed. (i) (ii) (iii) (iv) In-vitro drug release Drug content analysis Particle size analysis Zeta potential The In-vivo evaluation test was performed with the healthy rabbits IN-VITRO DRUG RELEASE OF AMOXICILLIN NANOPARTICLES 30 mg of drug-loaded nanoparticles were placed in the dissolution rate test apparatus Usp basket type stirring element. The basket was covered with an egg membrane. 900ml of phosphate buffer solution (ph 7.4) at 37ºC was used as dissolution medium. The basket was centrifuged at a speed of 100rpm. 5ml of media was withdrawn at

7 115 various time intervals of 15min, 30min, 1hr, 2hr, 4hr, 8hr, 12hr, and 24 hr with the help of 5ml pipette and replaced by 5ml of phosphate buffer solution (ph 7.4), and the drug content was estimated by UV- Spectrometer at 285nm Ciprofloxacin nanoparticles: 30mg of drug-loaded nanoparticles were placed in the dissolution test equipment Usp. The basket type of apparatus was covered with an egg membrane. 900ml of sodium acetate buffer solution (ph5) at 37ºC was used as a dissolution medium. The basket was centrifuged at a speed of 100 rpm. 5ml of media was withdrawn at various time intervals of 15min, 30min, 1hr, 2hr, 4hr, 8hr, 12hr, and 24 hr with the help of a 5ml pipette and replaced by 5ml of sodium acetate buffer solution (ph5) and the drug content was estimated by UV spectrophotometer at 540nm Drug Content Analysis: Drug content was analysed by adding 100ml of 10% hydrochloric acid with 30 mg of nanoparticles. The latter was kept at a temperature of about 35ºC for 24 hrs. After that, the nanoparticles were separated by centrifugation at rpm. The drug concentration was analysed in the supernatant liquid by UV-Visible spectrophotometer. The amoxicillin nanoparticles were analysed at 285 nm. Whereas, Ciprofloxacin nanoparticles were analysed at 530nm.The ampicillin nanoparticles were analysed at 268 nm.the ofloxacin nanoparticles were analysed at 293 nm

8 116 Drug loading capacity = Amount of drug bound by total amount of nanoparticles X Amount of drug taken Yield of nanoparticles = Amount of particles harvested X 100 Total amount of used drug + polymer PARTICLE SIZE ANALYSIS Particle Size distribution was determined by a Scanning Electron Microscope (SEM). A suspension of the nanoparticles was sprayed on a glass cover slip with the aid of an atomizer. The fine droplets were dried overnight, sputtered with about 10mm thick gold layer and examined IR SPECTRAL STUDIES The IR spectral studies were conducted by using the pressed pellet technique. In this technique, drug samples viz. Amoxicillin,Ciprofloxacin, Ofloxacin, Ampicillin and the polymer sample sepia, physical mixture of drug and the polymer were mixed with potassium bromide and compressed into a thin transparent pellet using a hydraulic press. The IR spectra obtained are shown in graph DIFFERENTIAL SCANNING CALORIMETRY STUDIES Differential Scanning Calorimetry was performed. Amoxicillin, Ciprofloxacin, Ofloxacin, Ampicillin Sepia, the two physical mixtures of drug and polymer and the two formulations prepared by using these two drugs were analysed. The samples were sealed in aluminum pans and the DSC thermograms obtained are shown in graphs

9 ZETA POTENTIAL ANALYSIS: The Zeta potential of nanoparticles was recorded using Zetasizer. The samples which gave good In-vitro release viz the batch of formulations AMN3, CN3, AP5 and OF6. were subjected to zeta potential analysis STABILITY STUDIES ON SELECTED FORMULATION: The batch of formulations viz AMN3, CN3, AP5 and OF6. were subjected to stability testing as follows: The prepared nanoparticle samples were kept in bottle and placed at room temperature, 45ºC and at 4ºC for a period of one month. At weekly intervals, samples were taken and analysed spectrophotometrically and the results are given in Tables BIOPHARMACEUTICAL EVALUATION: The In-vivo evaluation of selected formulations was carriedout with a view to assess the pharmacokinetics of absorption and elimination bioavailability. The following formulations were selected for In-vivo evaluation 1) Formulation batch AMN3 in which, the drug Amoxicillin and polymer ratio was 1:2. 2) Formulation batch CN3 in which, the drug Ciprofloxacin and polymer ratio was 1:2. 3) Formulation batch AP5 in which,the drug Ampicillin and polymer ratio was 1:3

10 118 4) Formulation batch OF6 in which,the drug Ofloxacin and polymer ratio was 1: EXPERIMENTAL DESIGN: Healthy rabbits weighing between 1.5 and 2 kg obtained from the animal house of Sri Padmavathi School of Pharmacy were maintained in air conditioned room. The experimental protocol was approved by the Institution s Ethical Committee. Adult healthy rabbits (n = 4) were used for the experimental study. All animals were kept in fasting for a period of 18hrs (over night fasting) before experimentation. The selected nanoparticle formulation of was poured in a small empty capsule and administrated orally. The dose of the nanoparticle was 10mg/kg with 100ml of water for all the 4 rabbits. Before the administration of the doses, blood samples were collected from the above experimental animals and the same served as blank. During experimentation, 1 ml of blood samples were collected from animals at 15min, 30min, 1hr, 2hr, 4hr, 8hr, 12hr, 24 hr, 36 hr, 48 hr, and 72hrs and centrifuged at 5000 rpm. The serum was collected into drug tubes and all the samples were stored under refrigerated conditions prior to assay. The serum concentration of the drugs namely Amoxicillin, Ciprofloxacin, Ampicillin and Ofloxacin was determined by micro biological method described earlier. The values of concentration of drugs in serum are given in tables. 5.17, 5.19, 5.21 and 5.23

11 119 The various pharmacokinetic parameters such as concentration maxima (max), Tmax, (AUC), elimination rate constant (ke), biological half life (t1/2) and absorption rate constant (ka) were calculated DETERMINATION OF VARIOUS PHARMACOKINETIC PARAMETERS The following important pharmacokinetic parameters were determined. Absorption constant = - ka = logc1 logc2 t1 t2 C1 = Concentration at t1 C2 = Concentration at t2 The elimination constant was determined by The half life t1/2 = ka Y Intercept Slope = ke = logc1 logc t1 t2 Ka = Dissociation constant KaFX0 = 74 Vd (Ka-KE) F = Fraction of drug absorbed into systemic circulation X0 = Dose administered initially Vd = Volume of Distribution Ka = Dissociation Constant

12 120 Ke = Elimination Tmax = In Ka/Ke Ka Ke From the above values, the concentration maximum was determined by = KaFX0 Vd(Ka-KE) STABILITY STUDIES: The nanoparticles containing the batch of formulations viz AMN3, CN3, AP5 and OF6 were subjected to the accelerated stability testing to find their stability and efficacy. Each formulation was divided into three portions of which, one portion was kept at nanotemperature, second at 45ºC and third at 4ºC for a period of one month. At weekly intervals, samples were determined spectrophotometrically.