The study was conducted in New Zealand white rabbits weighing 1.65 ± 0.22 kg, divided into two groups with eight rabbits in each group. The rabbits were acclimatized for three weeks to laboratory conditions before initiating the experiment. They were housed in individual cages and fed with antibiotic free diet. Feed and water were provided ad libitum. Feed was withheld for at least 6-8 h before and until 4 h after drug administration. Necessary approval from the Institutional Animal Ethics Committee was obtained to carry out the investigation.
Norfloxacin (Aravind Pharma, India) was dissolved in 0.1 N HCl to obtain a 3.33% solution (50 mg of norfloxacin in 1.5 mL 0.1 N HCl). The required amount of curcumin (Sigma-Aldrich, USA) was dissolved in a mixture of distilled water and Tween-20 at a 2 : 1 ratio restricting the total volume to 4-5 mL. Group-I rabbits (control) received norfloxacin at the rate of 100 mg/kg body weight as a single oral dose. The rabbits in group-II were administered a similar dose of norfloxacin after pre-treatment with curcumin (60 mg/kg body weight; p.o) for three days at an interval of 24 h. Blood samples (1.0-1.5 mL) were aseptically drawn from the marginal ear vein into heparin-coated tubes (Hi-Media, India) immediately before (0) at 5, 10, 15 and 30 min, and 1, 2, 4, 6, 8, 12 and 24 h after the administration of norfloxacin. Plasma samples were obtained by centrifugation of each blood sample (1,250 ×g, 10 min) and were stored at -20
(for not more than 24 h) until being assayed.
Plasma norfloxacin concentrations were determined using high performance liquid chromatography (HPLC; Shimadzu, Japan). Dilutions of norfloxacin (E. Merck, India) ranging from 0.01-4 mg/mL were carried out with the mobile phase to obtain a standard curve. The HPLC system consisted of double pump (LC-20AT), rheodyne manual injector with 20 µL loop, dual wavelength ultraviolet detector (SPD-20A) and LC Solution software for data analysis. Chromatography was carried out using a reverse phase C18
column (250 × 4.5 mm, particle size 5 ± 0.3 µm, pore diameter 100 ± 10 A°; Phenomenax, USA) as a stationary phase. The mobile phase consisted of 0.1% v/v orthophosphoric acid (pH adjusted to 2.0) and acetonitrile mixed at a v/v ratio of 850 : 150. Chromatography was carried out at a flow rate of 1 mL/min at room temperature and the absorbance of norfloxacin at 275 nm was measured. The cleaned-up plasma samples [16
] were analyzed for 8 min; there were no interfering peaks in the chromatogram at the retention time (Rt
= 4.90 ± 0.14 min) of norfloxacin. The quantification limit was 0.015 µg/mL and the standard curve was linear in the range 0.015-4 µg/mL with a R2
value of 0.999. Extraction recovery was determined to be 94.17% by comparing peak areas obtained for plasma-based standards and those obtained for mobile phase-based standards. The intra- and inter-day assay coefficients of variations were < 8.0%.
The plasma concentration-time profile of norfloxacin of each experimental animal was used to determine its pharmacokinetics. The pharmacokinetic data of norfloxacin was analyzed using the 'method of least square' and 'method of residual yields' [8
]. The compartmental analysis of the data was undertaken using the mono-exponential equation:
Ctp = Be-βt - Ae-Kat
where, Ctp = plasma drug concentration, B is the zero-time intercept of regression line of elimination phase, A is the zero-time plasma drug concentration intercept of regression line of absorption phase, Ka is the absorption rate constant, β is the overall elimination rate constant, t is the time and e is the natural logarithm base.
The total AUC and area under the first moment of plasma drug concentration-time curve (AUMC) were calculated as described previously [18
]. The volume of distribution (Vd(area)
) and clearance from the body (ClB
) were calculated as previously described [8
] for a non-vascular route of administration.
The loading and maintenance dosage schedules were selected to maintain a MIC of 0.1, 0.5 and 1.0 µg/mL in plasma [12
The difference between the means of the two treatments was determined by student's t
] and the data were analyzed using GraphPad Instant software (GraphPad Software, USA).