INTRODUCTION:

Bioavailability refers to the extent and rate at which the active moiety (drug or metabolite) enters systemic circulation, thereby accessing the site of action. Bioavailability of a drug is largely determined by the properties of the dosage form (which depend partly on its design and manufacturing), rather than by the drug's physicochemical properties, which determine absorption potential (Guidance, 2002; EMEA, 2001). Kankasava is an Ayurvedic herbal formulation which contain piperine is one of the active constituent. Recently, it has been patented as bioavailability enhancer, non-nicotinic smoking cessation aid and as an important ingredient of incapacitating composition (Bajad et al., 2001 and Zhixiu et al., 1999). HPLC as well as gas chromatography methods for analysis of piperine in peppers, pepper extract and oleoresin have been reported, but there is no sensitive and reproducible method for quantification of piperine in body fluids of any formulations. A HPTLC method has been reported for its analysis in body fluids with manually prepared silica gel plates. Piperine could be detected in human milk and serum by using complex HPLC method (isocratic plus gradient elution with mixture of four solvents) developed for quantification of curcuminods (Bajad et al., 2003). However, the method does not describe quantification of piperine in these fluids. Therefore method was developed for the analysis of piperine in rat plasma of all the prepared and marketed formulations of kankasava,

MATERIAL AND METHODS:

Chromatography

A Yangling – Yl - 9100 HPLC system with LC-10A pump, SPD-M10A photodiode array detector set at 340 nm was used. The system was fitted with a Rheodyne injector and samples were injected manually. Data were collected and analyzed using computer software Class LC 10A ver 3.1. Separation was achieved using waters symmetry C₁₈column (250 × 4.6 mm, 5µm) preceded by a guard column of the same material, 10 × 3.2 mm. The solvent system, 25 mM KH₂PO₄ (pH 4.5, adjusted with orthophosphoric acid) – acetonitrile (35:65), was pumped isocratically at 1 ml/min.

Collection and analysis of plasma

Piperine 150 mg/kg and Kankasava (10 ml/kg) was administered orally to 12 rats and six rats without treatment served as control group. Blood (0.5 ml) was collected from each animal and immediately extracted with 4 ml of ethyl acetate for 3 minutes in amber colored tubes and centrifuged at 6000 rpm for 5 minutes. Ethyl acetate layer was removed and dried over anhydrous sodium sulphate. The ethyl acetate was then evaporated under a stream of nitrogen at 50˚C. The residue was reconstituted in 0.1 ml of solvent system and 20–50 µl injected into the HPLC system. During all the operations, samples were protected from light due to susceptibility of piperine to photoisomerisation. A standard plot was prepared by injecting 2 – 2000 ng of piperine dissolved in acetonitrile. Recovery, intra-assay and inter-assay studies (3 days) were performed by spiking 0.1 ml of plasma and 1, 50 and 200 ng of piperine (n = 4 for each concentration) and extracting the samples with ethyl acetate as described above.

Plasma concentration – time profile of formulations

Piperine 150 mg/kg and Kankasava 10 ml/kg was administered orally once a day. Blood samples were collected of all the treated and control groups of animals in micro-centrifuged tubes at 0, 0.5, 1, 2, 4, 6, and 8 hours. The blood volume withdrawn was replaced with equal volume of heparinised saline. Plasma was immediately separated by centrifuged and analyzed the same day.

Figure 1 Plasma concentration of standard piperine

Figure2 Plasma concentration of kankasava

RESULT AND DISCUSSION:

Blank plasma gave no interfering peak at the retention time of piperine making the analysis very simple. The calibration plot was linear over the range studied (2–2000ng). The equation for the straight line was y = 0.0001 (x) – 0.7348 (r² = .9984). The solvent system of 25 mM KH₂PO₄ (pH 4.5)–acetonitrile (35:65) was found to be optimal for analysis of piperine in plasma. Good overall recovery (85.5±6%) was obtained with 1x4 ml of ethyl acetate with extraction time for 3 minutes. Lower extraction volume of ethyl acetate as well as decreased extraction time resulted in low and variable recoveries. Limit of detection and limit of quantification were 1 ng/ml and 3 ng/ml respectively. In the plasma concentration – time profile of formulation in rats (n=6) piperine could be detected in plasma from 0.5 to 8 hours after administration with maximum plasma concentration (2.80 – 1.76 µg/ml) at 0.5 – 1 h post dosing (Figure 1 and 2). Piperine is a major alkaloid of peppers, which are widely used as spice. It has received attention due to its potential to increase bioavailability of drugs when co-administered. Considering its effect on drug metabolizing enzymes and gastrointestinal tract, following the method for its determination in body fluid is of obvious significance. Piperine, a lipophilic molecule, could be extracted from plasma using ethyl acetate. For separation of piperine from plasma components to obtain a sharp peak, a balance of organic and aqueous phase was obtained. Use of more than 65% of organic solvent in the mobile phase resulted in the merger of piperine peak with plasma components, whereas increasing the aqueous phase proportion to more than 35% broadened the peak. Monitoring of samples at 340 nm (λ_maxfor piperine) helped in reducing the interference from endogenous plasma components which absorb poorly at this wavelength. The method was used for studying the plasma concentration – time profile of formulation which contains piperine in rats plasma. Piperine is rapidly absorbed through the gastrointestinal tract and could be detected in plasma as early as 50 min after administration.

REFERENCES:

· Bajad S, Bedi K L, Singh A K and Johri R K. Planta Med, 2001, 67; 176.

· Bajad S, Coumer M, Khajuria R, Suri OP and Bedi KL. Characterization of new urinary metabolites of piperine by LC/NMR/MS studies, European J Pharmaceutical Sciences, 2003, (19(5); 413-421.

· EMEA–The European Agency for the Evaluation of Medicinal Products–Evaluation of Medicine for Human Use, London, 2001.

· Guidance for Industry: Bioavailability and bioequivalence studies for orally administered drug products-General considerations, U.S. Department of Health and Human Services Food and Drug Administration, Center for Drug Evaluation and Research (CDER), 2002.

· Zhixiu L, Hoult J R S, Benett DC and Raman A. Planta Med, 1999, 65: 600.

Received on 19.11.2012 Accepted on 20.12.2012

Asian J. Pharm. Res. 3(1): Jan.-Mar. 2013; Page 01-02

*Corresponding Author E-mail: ah_bharti@yahoo.com