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          Black Pepper is the dried green mature fruit of Piper nigrum L. (Family Piperaceae), Herbarium Specimen Number:  DMSC 667, Crude Drug Number:  DMSc 0275.

Constituents  Black Pepper contains volatile oil, alkaloids, of which  piperine and  piperettine, are its major components.

Description of the plant  (Figs. 1a, 1b)  Woody perennial climber, aromatic; branches stout,  trailing and rooting at the nodes.  Leaves simple, very variable in length, 12.5 to 17.5 cm long, 5 to 12.5 cm wide, sometimes glaucous beneath, apex acuminate, base acute, rounded or cordate, equal or unequal, basal nerves 5 to 9 with one pair running to apex; petiole 1.2 to  3.7 cm long, stout.  Flower usually dioecious, but often the female bears 2 anthers, and the male a pistillode, anther 2-celled.  Fruiting spike, 10 to 12 cm or more long.  Fruit drupe,  globose or ovoid, sessile, 1-seeded, 4 to 6 mm in diameter, orange-red to reddish when ripe. Seed usually globose; testa thin. 

Description  Odour, aromatic; taste, pungent.

       Macroscopical  (Fig. 1a)  Subspherical, greyish brown to brownish black, superior, 3.5 to  6 mm in diameter; surface deeply and coarsely reticulately-wrinkled; remains of a sessile  stigma visible at the apex; vertical section of the fruit shows a thin, narrow, dark pericarp, within which is the whitish kernel of the single seed, to which the pericarp firmly adheres; kernel consisting of almost entirely of perisperm, hollow at the centre, and, at its apex,  surrounding a scanty endosperm in which the minute embryo is embedded.

      Microscopical  (Figs. 2a, 2b)  Transverse section of the fruit shows epicarp, mesocarp, endocarp, spermoderm, and perisperm.  Epicarp composed of a layer of epidermal cells containing small rectangular crystals about 6 to 10 μm long and 2 to 3 layers of thickened  stone cells with rather large lumina intermingled with thin-walled parenchyma cells.  Mesocarp composed of several layers of polygonal parenchyma cells, some of which are oil cells which occur more in the inner part; vascular bundles located in the middle part of mesocarp.  Endocarp, a layer of light brown, lignified beaker cells.  Spermoderm  composed of 2 to 3 layers of compressed elongated cells, the innermost layer composed of larger cells containing dark brown contents.  Perisperm composed of cells containing numerous angular starch grains up to 5 μm in diameter, some of which containing oil globules, resin and monoclinic crystals.

       Black Pepper in powder possesses the diagnostic microscopical characters of the  unground drug.

Packaging and storage  Black Pepper shall be kept in well-closed containers, preferably of metal or glass, protected from light and stored in a cool and dry place. 

Identification

       A. Reflux 1 g of the sample, in No. 150 powder, with 20 mL of chloroform in a water-bath  for 15 minutes, and filter (solution 1).  Evaporate 2 mL of solution 1 to dryness. Dissolve the residue with 1 mL of ethanol, and add 0.5 mL of sulfuric acid:  a brownish red colour is produced, and after set aside the colour changes to brown.

       B. Evaporate 5 mL of solution 1 to dryness. Dissolve the residue with 0.5 mL of a 10 per cent v/v solution of sulfuric acid, and add a few drops of modified Dragendorff TS2: an orange precipitate is produced

       C. Carry out the test as described in the “Thin-Layer Chromatography” (Appendix 3.1), using silica gel GF254 as the coating substance and a mixture of 60 volumes of hexane and 40 volumes of ethyl acetate as the mobile phase and allowing the solvent front to ascend 12 cm above the line of application.  Apply separately to the plate, 5 μL each of the following two solutions.  Prepare solution (A) by macerating 500 mg of the sample, in No.150 powder, in a 5-mL volumetric flask with chloroform for 10 minutes, and shaking frequently.  Filter and evaporate the filtrate to dryness.  Dissolve the residue in 1 mL of chloroform.  For solution (B) dissolve 1 mg of piperine in 1 mL of chloroform.  After removal of the plate, allow it to dry  in air and examine under ultraviolet light (254 nm), marking the quenching spots.   The chromatogram obtained with solution (A) shows a quenching spot (hR_f value 28 to 38), corresponding to the piperine spot from solution (B), and several spots of higher and lower hR_f values.  Spray the plate with acetic potassium iodobismuthate TS; the spot due to piperine  is dark orange (Table 1); see also Fig. 3.

        Repeat the same procedure on another plate but spray with a 10 per cent v/v solution of sulfuric acid in methanol; the spot due to piperine is yellow.  After spraying, allow the plate to dry in air for 15 minutes and heat at 110° until colours are fully developed.  The  chromatogram obtained with solution (A) shows a brownish yellow spot (hR_f value 28 to 38) corresponding to the piperine spot from solution (B), and several other spots of different colours situated above and below that due to piperine (Table 1); see also Fig. 3. 

Table 1 hRf Values of Components in Chloroform Extract of the Fruits of Piper nigrum L.

       *piperine

Water  Not more than 14.0 per cent v/w (Azeotropic Distillation Method, Appendix 4.12).

Foreign matter  Not more than 2.0 per cent w/w (Appendix 7.2).

Acid-insoluble ash  Not more than 1.5 per cent w/w (Appendix 7.6).

Total ash  Not more than 7.0 per cent w/w (Appendix 7.7).

Volatile oil  Not less than 1.0 per cent v/w (Appendix 7.3H).  Use 50 g, in No. 150 powder, freshly prepared and accurately weighed.  Use 250 mL of water as the distillation  liquid and a 500-mL round-bottomed flask.  Distil at a rate of 2 to 3 mL per minute for 5 hours.  Use 2.0 mL of xylene in the graduated tube.

Alkaloid content  Not less than 5.0 per cent w/w of alkaloids, calculated as piperine, when determined by the following method.

        Standard piperine solution  Transfer about 20 mg of piperine, accurately weighed,  into a 100-mL volumetric flask, add chloroform to dissolve and dilute to volume.

        Standard piperine curve  Transfer into five 25-mL volumetric flasks 0.2 mL, 0.4 mL,  0.6 mL, 0.8 ml, and 1.0 mL, respectively, of Standard piperine solution and add chloroform to  make 1.0 mL.  To each flask add 10.0 mL of chromotropic acid TS, shake vigorously and place in a water-bath for 30 minutes.  Set aside for a few minutes, stopper, and allow to cool.   Then add 10 mL of dilute sulfuric acid, mix well, allow to cool, and dilute with sufficient  dilute sulfuric acid to volume.  Measure the absorbances of the piperine-containing solutions relative to the blank at 570 nm (Appendix 2.2).  Plot the readings and draw the curve of  best fit. 

         Procedure  Place 500 mg of Black Pepper, in No. 150 powder and accurately weighed,  in an extraction thimble and insert the thimble into a soxhlet extractor of appropriate size.Moisten with 0.5 mL of chloroform, mix, allow to stand for about 5 minutes, make alkaline with 0.5 mL of ammonia TS, and mix.  Macerate for 6 to 12 hours or overnight, cover with  a pledget of absorbent cotton, add a sufficient quantity of chloroform, and extract until complete extraction of the alkaloids is effected (Appendix 7.4).  Transfer the total mass to  a 250-mL volumetric flask, dilute with chloroform to volume and filter.  Transfer 1.0 mL of  the filtrate to a 25-mL volumetric flask, and proceed as directed under Standard piperine curve, beginning with “Add 10.0 mL of chromotropic acid TS, ...”.  Read the absorbance of the  resulting solution, and by reference to the Standard piperine curve, calculate the content  of alkaloids as piperine in the sample.

Dose  250 to 500 mg three times a day after meals.