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          Java Long Pepper is the dried red mature infructescence of Piper retrofractum Vahl  (P. chaba Hunter) (Family Piperaceae), Herbarium Specimen Number:  DMSC 48, 75, 456, 467. 

Constituents  Java Long Pepper contains piperine, piperlongumine, pyridine alkaloids,  sesamin, volatile oil, etc.

Description of the plant  (Figs. 1a, 1b)  Climber, glabrous, rather fleshy, with the aid of adventitious roots, rarely creeping; stem much branched, stout, cylindrical, thickened  above nodes.  Leaves simple, alternate, 3 to 20 cm long, 2 to 13 cm wide, lower ones ovate or lanceolate with cordate base, upper ones oblong-oval to oblong with obtuse, cordate or cuneate base, unequal, all tapering or acuminate, entire, glabrous, reticulate venation sunk above and raised beneath; petioles of lower leaves 1.5 to 3 cm long, of the upper ones 0.5 to 1.5 cm long; stipule 1 to 1.5 cm long, membranous, lanceolate, obtuse, enclosing the bud but soon falling off.  Inflorescence spike, erect or patent; peduncle 0.7 to 2 cm long; bract  broadly oval-ovate, 1.5 to 2 mm long; flower unisexual, bisexual; male spike 2.5 to 8.5 cm long, stamens 2, rarely 3, very short; female spike 1.7 to 3 cm long, stigmata 2 to 3, short, obtuse, persistent.  Infructescence cylindro-conic; berry connate and adnate to stalk of bract, broadly rounded, bright red.  Seed 2 to 2.5 mm in diameter.

Description  Odour, aromatic; taste, pungent.

          Macroscopical  (Fig. 1a)  Condensed infructescence, reddish brown, subcylindrical, about 2.5 to 7.5 cm long and 5.0 to 8.0 mm in diameter, rather rough surface with persistent stigmata.

          Microscopical  (Figs. 2a, 2b, 2c)  Transverse section of the fruit shows epicarp composed of a layer of epidermal cells of which the outer tangential wall thick, glandular trichomes and cuticle.  Mesocarp composed of 3 to 4 layers of collenchyma of hypodermis in which stone cells scattered followed by zone of thin-walled parenchyma, some of which containing brownish substance, oil droplets and starch grains and a few layers of large oil cells.  Endocarp composed of a single layer of sclerenchyma.  Zone of ground parenchyma scattered with vascular bundles around the cavity at the centre of the fruit.  Spermoderm composed of outer thick-walled epidermis, collapse parenchyma of middle epidermis and inner thin-walled epidermis.  Perisperm, elongated reserve parenchyma cells containing numerous angular starch grains and rod-shaped crystals.  Embryo embedded in the  endosperm.

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

Packaging and storage  Java Long 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 powder, with 25 mL of ethanol for 10 minutes and filter (solution 1).  To 2 mL of solution 1, add a few drops of ninhydrin TS and warm in a water-bath for a few minutes:  a purple colour is produced.

          B. Evaporate 2 mL of solution 1 to dryness.  Dissolve the residue in 2 mL of acetic anhydride, and then slowly add 1 mL of sulfuric acid to form two layers:  a brown ring develops at the zone of contact.

          C. Evaporate 2 mL of solution 1 to almost dryness and add a few drops of  Marquis’reagent, prepared by adding a few drops of formaldehyde solution to 6 mL of sulfuric acid:  a brownish red colour is produced.

          D. To 100 mg of the sample, in powder, add 1 mL of sulfuric acid:  a deep red colour is produced at first, turning to reddish brown and brown.

          E. 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 70 volumes of n-hexane and  30 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 powder, with 25 mL of chloroform for 15 minutes and filtering.  Evaporate the filtrate to dryness and dissolve the residue in 2 mL of chloroform.  For solution (B), dissolve 2 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 from solution (A) shows a quenching spot (hR_f value 20 to 22) corresponding to the piperine spot from  solution (B), and several spots of higher hR_f values.  Spray the plate with modified Dragendorff TS2; the spot due to piperine is orange and one orange spot is observed (Table 1); see also Fig. 3. 

          Repeat the same procedure on another plate but spray with anisaldehyde TS.  The  chromatogram obtained from solution (A) shows a green spot (hR_f value 20 to 22)  corresponding to the piperine spot from solution (B), and several other spots of different colours are also observed (Table 1); see also Fig. 3.

Table 1 hR_f Values of Components in Chloroform Extract of the Fruits of Piper retrofractum Vahl

       *piperine

Water  Not more than 13.0 per cent v/w (Azeotropic Distillation Method, Appendix 4.12).

Acid-insoluble ash  Not more than 0.4 per cent w/w (Appendix 7.6).

Total ash  Not more than 7.5 per cent w/w (Appendix 7.7).

Ethanol-soluble extractive  Not less than 10.0 per cent w/w (Appendix 7.12).

Volatile oil  Not less than 1.0 per cent v/w (Appendix 7.3H).  Use 25 g, in fine 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.

Alkaloids content  Not less than 2.5 per cent w/w of alkaloids, calculated as piperine, when determined by the following method.  (Note  Use light-resistant glassware to reduce photodegradation of piperine in solution.)

          Standard piperine solution  Dissolve about 10 mg of piperine, accurately weighed, in sufficient 1,2-dichloroethane to produce 100.0 mL.

          Standard piperine curve  Transfer into six 100-mL volumetric flasks, 1, 2, 3, 4, 5, and 6 mL, respectively, of Standard piperine solution, dilute to volume with 1,2-dichloroethane, and mix.  Measure the absorbances of the standard solutions relative to the blank at 342 nm (Appendix 2.2).  Plot the readings and draw the curve of best fit.

          Procedure  Place about 500 mg of Java Long Pepper, in fine powder and accurately weighed, in a soxhlet apparatus.  Add a sufficient quantity of 1,2-dichloroethane and extract, until complete extraction of the alkaloids is effected (Appendix 7.4).  Transfer the  dichloroethane extract to a 100-mL volumetric flask and dilute with 1,2-dichloroethane to volume.  Transfer 2.0 mL of this solution to a 100-mL volumetric flask and dilute with 1,2-dichloroethane to volume.  Measure the absorbance of the resulting solution, at the maximun at about 342 nm (Appendix 2.2).  By reference to the standard curve, calculate the content of alkaloids as piperine in the sample.

Dose  200 to 500 mg three times a day.