Chemical and Reagents
Pure form of Chalcone was bought from Sigma Aldrich and was utilized as in same form considering as a reference standard. The methanol and water used in the experiments were of HPLC analytical grade. The three chalcone derivatives namely,
(Е)-(3,4,5-Trimethoxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one, (2); and
(Е)-1-(3,4,5-Trimethoxyphenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one, (3), Fig. 1, were synthesized as delineated formerly by our group .
The ten healthy adult New Zealand White rabbits weighing between 1.75–2.75 Kg of either sex were procured from the institute Advanced Facility for Small Animal Research, PGIMER Chandigarh, after approval from the Institutional Animal Ethics Committee Ref. No. 69/IAEC/418 as per the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines. The animals were housed in polypropylene cages under standard laboratory conditions at controlled temperature (25◦±°C ) and 12-h light/dark cycles until the end of the experimental period. Animals were given free access to diet and water in a room and were fasten for at least 12 h prior to experiment. To achieve meaningful statistical results, we used a minimally sufficient number of animals in all cases. A fifteen-days washout period was carried out, which allows animals to completely recover and clear off the administered derivatives.
A total of ten healthy adult New Zealand White rabbits were divided into three groups; (i) derivative 1 treated group, (ii) derivative 2 treated group, and (iii) derivative 3 treated group. The whole experiment was performed in two batches with each batches containing n = 3 animals per group. The second batch with same groups were repeated after giving fifteen-days washout period which helps animals to recover and clear off previous administered derivatives. The three chalcone derivatives were administered at effective dose concentration [dose extrapolated from in vitro study,  as per OECD GLP compliance. A single dose of each chalcone derivatives 1, 2 & 3 (suspended in 0.5% carboxymethyl cellulose) were administered to each animal at 0 h and then blood samples of animals were collected at specific time intervals of 0, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, 48 h from marginal ear vein in heparin containing vials. These derivatives were either administered orally through orogastric tube or intraperitoneally. All blood samples were collected from the marginal ear vein after topical anaesthesia with 4% lignocaine solution. No euthanasia procedure was required during the study. Plasma samples for the determination of drug concentration were prepared for RP-HPLC analysis.
1. Preparation of Plasma Sample
Rabbit blood samples were accumulated in heparin contained dry evacuated tubes from marginal ear veins of healthy rabbits as per the standard operating procedure. These samples were centrifuged within 1 h of collection, for segregation of plasma at 1500 rpm for 10 min. The segregated plasma was preserved at 20◦C till assay. Prior experimental analysis the deproteinization of the plasma samples was done in the mixture of methanol and water (90:10 v/v), then vortexed for at least 10–15 min, after while centrifuged for at least 15 min at 6000 rpm, and finally supernatants were pipette-out in a new vial. The supernatants were then spiked with the measured volume of diluted stock solutions of chalcone making up to final concentrations of 10–100 µg/mL. Each sample of 20 µL volume was injected through a Rheodyne injector in the instrument and the effluent was observed at 310 nm.
2. Standard Preparation
A concentrated (100 µg/mL) stock solution was obtained by solubilizing 10 mg of chalcone in 10 mL of methanol and then the final volume was added up to 100 mL with the mobile phase in 100 mL volumetric flask. The working solution of chalcone was prepared by diluting the stock solution with the mobile phase. The same procedure was followed for plotting the calibration curve for all the three chalcone derivatives, (Additional File 1: Figure S1).
3. Sample Preparation
Each sample has been collected from rabbits at a different time interval and plasma was separated and either processed for assay procedure or stored at -20 ◦C in deep freezer till experiment. On the day of the experiment, 100 µL of plasma from each vial was transferred to another and mixed with 100 µL of acetonitrile (HPLC analytical Grade). Then was kept for 5 min at room temperature and then centrifuged at 7000 rpm for 10 min. Supernatant was separated and reconstituted with the mobile phase and finally sterile filter with 0.22 µm membrane filter before the assay procedure.
4. Chromatographic conditions
Chromatographic separation was achieved as mentioned earlier  with slight modification. Briefly, the separation was performed on RP-HPLC at ambient temperature (25 ◦C) by using a mobile phase consisting of methanol and water in the ratio of 90:10 (v/v) for 10 min. The mobile phase was pumped at a rate of 1.0 mL/min. The detector wavelength was set at 310 nm.
Pharmacokinetic variables evaluation:
The experimental data and pharmacokinetic variables are shown as the mean ± standard error mean (SEM). The peak serum concentration (Cmax) and the time to achieve maximum concentration (tmax) were retrieved from the observed concentration versus time data profile. The area under the serum concentration-time curve from time zero to the time of final measurable sample (AUC0 − 48) was calculated using the linear trapezoidal method .