สารบัญ

          Arcangelisia Flava Stem is the dried stem of Arcangelisia flava (L.) Merr. (Family Menispermaceae), Herbarium Specimen Number: DMSC 857, Crude Drug Number: DMSc 426.

Constituents Arcangelisia Flava Stem contains berberine as the major alkaloidal component. It also contains other isoquinoline alkaloids (e.g., columbamine, palmatine, jatrorrhizine), diterpenoids, etc.

Description of the plant (Figs. 1a, 1b) Large climber, woody, glabrous, dioecious; stem up to 5 cm in diameter, wood yellow, exuding yellow sap when cut, bearing prominent cup-like, petiole-scars. Leaves usually ovate, elliptic-ovate or broadly ovate, 10 to 25 cm long, 5.5 to 19 cm wide, base usually rounded, truncate or slightly cordate, apex abruptly acuminate, palmately 5-nerved at the base and with 1 to 3 pairs of lateral nerves, usually arising from above halfway along the midrib, both surfaces usually with a rather obscure reticulum, coriaceous; petiole 4 to 20 cm long, swollen at both ends, geniculate at base. Inflorescence axillary or cauliflorus paniculate, slender, 10 to 50 cm long, lateral branches spicate to subspicate, 1 to 5 cm long. Male flower sessile or subsessile, subtended by an ovate bracteole, about 2 mm long, strongly thickened at the base; outer sepals 3 to 4, less than 1 mm long; inner sepals 2 whorls of 3, larger, elliptic, ovate or narrowly obovate, 1.5 to 2.5 mm long; synandrium 0.5 to 1 mm long with a globose cluster of 9 to 12 anthers. Female flower main sepals 6, narrowly oblong with the apex becoming reflexed, 2.5 to 4 mm long; staminodes minute, scale-like; carpels 3, 1.5 mm long; stigma broad, sessile, papillose. Infructescence cauliflorous, usually branched, 5 to 45 cm long, with thickened axis and branches, 3 to 6 mm in diameter, the fruits plus carpophores borne on the lateral branches, 1 to 3 borne together on a club-shaped, unbranched carpophore swollen at the apex, up to 4 cm. Fruit drupe, yellow, laterally slightly compressed, transversely subovoid, 2.2 to 3 cm long, 2.5 to 3.3 cm (long axis), 2 to 2.5 cm thick, drying finely rugulose, glabrous; endocarp woody. Seeds broadly ellipsoid, with ruminate endosperm, cotyledons much folded.

Description Odourless; taste, bitter

          Macroscopical (Fig. 1a) Cylindrical, segmented or oblique pieces; externally brownish, rather smooth; sectional view golden yellow to yellowish when fresh and brownish yellow when dried, porous, with several successive concentric and distinctly radiate zones.

          Microscopical (Figs. 2a, 2b) Transverse section of the stem shows several layers of rectangular, brownish cork cells and layers of lignified thick-walled, yellowish rectangular sclerenchymatous cells. Cortex, dark brown band of ovate parenchyma cells. Anomalous vascular tissues, several layers of tissue bands with distinct rays, each layer separated by layers of sclereids; phloem tissues, with groups of thick-walled sclereids and fibres in the outermostregion; xylem tissues, simple pitted and bordered-pitted vessels, xylem fibres and rectangular or oblique or elongated xylem parenchyma containing prisms and starch grains. Pith, parenchyma cells containing prisms and starch grains.

      Arcangelisia Flava Stem in powder possesses the diagnostic microscopical characters of the unground drug.

Packaging and storage Arcangelisia Flava Stem shall be kept in well-closed containers, protected form light, and stored in a dry place.

Identification

          A. Reflux 1 g of the sample, in fine powder, with 25 mL of methanol in a water-bath for 10 minutes, and filter (solution 1). Examine 2 mL of solution 1 under ultraviolet light (366 nm): a yellow fluorescence is observed.

          B. To 2 mL of solution 1, add 0.5 mL of nitric acid and mix well: an orange colour develops.

          C. Evaporate 2 mL of solution 1 to dryness, dissolve the residue in 2 mL of ether, add 0.5 mL of hydrogen peroxide TS (10 volumes), and mix. Carefully add 1 mL of hydrochloric acid to form a layer: a red ring forms at the zone of contact.

          D. Evaporate 2 mL of solution 1 to dryness. Add a few drops of modified Dragendorff TS2 to the residue: an orange precipitate is produced.

          E. Evaporate 5 mL of solution 1 to dryness. Dissolve the residue in 2 mL of 1.5 M sulfuric acid, add a few drops of 0.02 M potassium permanganate, and warm on a water-bath: the colour of potassium permanganate is decolorized.

          F. 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 1-butanol, 20 volumes of water and 10 volumes of glacial acetic acid 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 three solutions. Prepare solution (A) by refluxing 500 mg of the sample, in fine powder, with 20 mL of methanol for 5 minutes. Filter and evaporate the filtrate under reduced pressure to 5 mL. For solution (B), dissolve 1 mg of berberine chloride in 1 mL of water. For solution (C), dissolve 1 mg of palmatine iodide in 1 mL of water. After removal of the plate, allow it to dry in air and examine under daylight, locating the spots. The chromatogram obtained from solution (A) shows two yellow spots (hR_f values 52 to 54 and 44 to 47) corresponding to the berberine chloride spot from solution (B) and the palmatine iodide spot from solution (C), respectively. Subsequently examine the plate under ultraviolet light (254 nm), marking the quenching spots. The chromatogram obtained from solution (A) shows two quenching spots corresponding to the berberine chloride spot from solution (B) and the palmatine iodide spot from solution (C) and other three quenching spots of lower hR_f values. Examine the plate under ultraviolet light (366 nm); the spots due to berberine chloride and palmatine iodide show yellow fluorescences. Other several blue and one yellow spots are also observed. Spray the plate with a 10 per cent w/v solution of phosphomolybdic acid in ethanol and heat at 105° for 5 minutes; the spots corresponding to berberine chloride and palmatine iodide are brown. Other several blue spots are also observed (Table 1); see also Fig. 3.

          Repeat the same procedure on another plate but spray with modified Dragendorff TS; the spots due to berberine chloride and palmatine iodide are orange (Table 1); see also Fig. 3.

Table 1 hRf Values of Components in Methanolic Extract of the Stems of Arcangelisia flava (L.) Merr.

       *palmatine iodide
       **berberine chloride

Loss on drying Not more than 10.0 per cent w/w after drying at 105° to constant weight (Appendix 4.15).

Acid-insoluble ash Not more than 0.5 per cent w/w (Appendix 7.6).

Total ash Not more than 3.0 per cent w/w (Appendix 7.7).

Ethanol-soluble extractive Not less than 4.0 per cent w/w (Appendix 7.12).

Water-soluble extractive Not less than 4.0 per cent w/w (Appendix 7.12).

Chloroform-soluble extractive Not less than 1.0 per cent w/w (Appendix 7.12H).

Berberine content Not less than 2.0 per cent w/w, calculated on the dried basis, when determined by the following method.

          Standard berberine chloride stock solution Transfer about 40 mg of berberine chloride, previously dried at 110° for 4 hours and accurately weighed, to a 50-mL volumetric flask, dissolve in 30 mL of methanol, dilute to volume with a mixture of 1 volume of hydrochloric acid and 100 volumes of methanol, and mix.

          Standard berberine chloride solution Dilute Standard berberine chloride stock solution quantitatively and stepwise with a mixture of 1 volume of hydrochloric acid and 100 volumes of methanol to obtain solutions having known concentrations of 80, 160, 240, 320, and 400 μg per mL, respectively.

          Standard berberine chloride curve Place 2.5 mL of each standard solution in separate aluminium oxide minicolumns (about 15 × 1 cm), pretreated by filling the minicolumns with 2.5 g of neutral aluminium oxide using wet method and washing with about 15 mL of ethanol. Elute each column with 15 mL of ethanol in portions, combine the eluates in a 25-mL volumetric flask and dilute to volume with ethanol. Pipette 10 mL of each solution into separate 25-mL volumetric flasks and dilute to volume with 0.5 M sulfuric acid. Measure the absorbances of the solutions obtained at the maximum at about 345 nm, using 0.5 M sulfuric acid as the blank (Appendix 2.2). Plot the readings and draw the curve of best fit.

         Procedure Place 500 mg of Arcangelisia Flava Stem, in fine powder and accurately weighed, in a soxhlet extractor of appropriate size. Add 25 mL of a mixture of 1 volume of hydrochloric acid and 100 volumes of methanol, extract to colourless and allow to cool.  Quantitatively transfer the extract to a 50-mL volumetric flask and dilute to volume with a mixture of 1 volume of hydrochloric acid and 100 volumes of methanol. Transfer 2.5 mL of this solution to an aluminium oxide minicolumn, and proceed as directed under Standard berberine chloride curve, beginning with “pretreated by...”. Read the absorbance of the resulting solution, and by reference to the Standard berberine chloride curve, calculate the content of berberine in the Arcangelisia Flava Stem taken using 407.85 and 336.37 as the molecular weights of berberine chloride and berberine, respectively.