Piperine answers all the tests for alkaloids.

1. To 1 ml of an alcoholic solution of piperine add 2 ml of Meyer’s reagent. A dull white precipitate is formed.
2. To 1 ml of the above piperine solution add 1 ml of Dragendorff’s reagent. An orange red precipitate is formed.
3. To 1 ml of piperine solution add 3 ml of Hager’s reagent. A yellow precipitate is formed.
4. To 1 ml of piperine solution add 2 ml Wagner’s solution. A reddish brown precipitate is formed.
5. TLC identification: Spot the initial dichloromethane extract and the isolated piperine and piperine reference sample (both dissolved in acetone) on a silica gel plate and develop in a solvent system of acetone and hexane (3:2). Air dry the plates and spray with vanillin sulphuric acid and activate the plates at 105°C for 15 min. Piperine is visualized as yellow spots.

Estimation

Several methods of estimation of piperine, such as gravimetry, titrimetry, spectrophotometry, HPTLC, HPLC are reported in the literature. While spectrophotometric methods are largely the official methods many newer methods of estimation involving extraction with super fluid carbon dioxide are also being reported.

A. Method I (Spectrophotometric assay)

1. Take 1 g finely ground pepper of #40 mesh in a 100 ml conical flask. Add 15 ml methanol and heat to boiling on a steam-heated water bath.
2. The solution is filtered hot and the residue is treated with further 4 × 15 ml quantities of methanol successively for the complete extraction of alkaloids.
3. The filtrates are collected in a 100 ml standard flask and the filter washed with 2 × 5ml portion of methanol. The rinsings are collected in the same flask and the volume is made up to mark with methanol.
4. After mixing well, 5 ml of the methanolic solution is transferred to another 100 ml standard flask and its volume made up with methanol. This is the test solution.
5. Standard sample: A 200 µg/ml solution of reference sample of piperine is prepared in methanol and it is further diluted to yield a final concentration of 4, 5, 8 and 16 µg/ml.
6. Absorbances of the four standard solutions and that of the test sample are measured at 343 nm using methanol as blank.
7. The percentage of piperine in pepper is calculated from the standard curve of reference piperine.

B. Method II (HPTLC method)

1. HPTLC was performed on 10 cm × 20 cm aluminum-backed silica gel F₂₅₄ HPTLC plates. They are prewashed with methanol, dried and activated for 30 min at 110°C with the plates being placed between two sheets of glass to prevent deformation of the aluminum during heating.
2. 10 µl each of the prepared four standard solutions of piperine and the test solution (from method I) are applied on silica gel plates as 6 mm bands 6 mm apart and 1 cm from the edge of the plate by means of the automatic sample applicator fitted with Hamilton syringe. A methanol blank is applied to the parallel track.
3. Mobile phase of hexane: ethyl acetate: glacial acetic acid (3:1:0.1) is taken in the TLC developing chamber and left to equilibrate for 20 min. The spotted plates are then developed in it to a distance of 90 mm.
4. After developing, the plates are removed and dried in a current of hot air and scanned at 343 nm by means of densitometric scanner under optimized scanning parameters. Peak height and peak area are integrated for the entire track.
5. A calibration curve is plotted using peak area versus concentration of piperine. Piperine content of the sample is determined from the calibration curve from the peak area of the test sample.