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       Belladonna Leaf consists of the dried leaf or the dried leaf and flowering, and occasionally fruit-bearing, tops of Atropa belladonna L. (Family Solanaceae). It contains not less than 0.35 per cent of the alkaloids of Belladonna Leaf.

Origin of plant Belladonna is native to Eurasia, Mediterranean, Central and Southern Europe, including Britain, Southern and Eastern North Africa and Iran.

Constituents Belladonna Leaf contains the main alkaloid, (S)-hyoscyamine, which undergoes rapid racemization to atropine or R,S-hyoscyamine. It also contains small quantities of scopolamine or hyoscine, apoatropine and flavonoids.

Description Odour, slightly nauseous; taste, slightly bitter.
       Macroscopical Entire or broken leaves green to greenish brown, often crumpled, rolled or partly matted together; when whole, lamina, elliptic to ovate, 5 to 25 cm long, 3 to 12 cm wide, apex acute to acuminate, base often decurrent, margin entire, young leaves pubescent, older leaves slightly pubescent along the vein; petiole 0.5 to 4 cm long; under a lens microsphenoidal crystal cells visible between veins as dark points by transmitted light or bright points by reflected light. Flowering tops: stems, flattened and hollow, bearing leaves in pair of unequal sizes at each node, flowers or occasionally fruits occur in the leaf axils; flowers: campanulate corolla about 2 cm long, about 1.5 cm wide, purple or yellow-brown lobes 5, short, reflexed; stamens 5, epipetalous; ovary superior, 2-lobed, containing numerous ovules; fruits, if found, occur as berries, globular, up to 12 mm in diameter, green, dark yellow, yellowish brown, to dusky red or brownish black, sometimes subtended by persistent calyx and containing numerous flattened, reniform seeds.
       Microscopical
       Leaf Epidermal cells with sinuous anticlinal walls and distinctly striated cuticle. Stomata anisocytic, more frequent on the lower epidermis. Non-glandular trichomes, uniseriate, up to 6-celled. Short club-shaped glandular trichomes with a one-celled stalk and multicellular head and long glandular trichomes with a uniseriate stalk and unicellular head are found on both upper and lower epidermises. Mesophyll, a layer of palisade parenchyma underlying with spongy parenchyma containing microcrystals. Midrib, an arc of bicollateral bundles, collenchyma and scattered parenchyma cells with microcrystals.
       Stem Epidermis with striated cuticle and few trichomes. Endodermis distinct. Pericycle fibres, long, thin-walled, slightly lignified. A circle of bicollateral bundles presents. Parenchyma of cortex and pith contains scattered crystal cells.
       Flower Calyx with numerous glandular trichomes with uniseriate stalks and 1- to 3-celled glandular heads. Corolla possesses papillose inner epidermis and outer epidermis with glandular trichomes similar to those of the calyx. Pollen grains mounted in chloral hydrate solution occurs in spherical shape, 40 μm in diameter, tricolpate, bearing 3 germinal furrows and rows of pits between the ridges on the exine.
       Fruit If found, epicarp consists of polygonal epidermal cells with striated cuticle and stomata. Mesocarp composed of large pulp cells some of which contain rosette aggregate crystals.
       Seed If found, large wavy-walled epidermal cells with prominent ridges over the anticlinal walls.

Packaging and storage Belladonna Leaf shall be kept in well-closed containers, protected from light, and stored at a temperature not exceeding 25°.

Identification
       A. Shake 1 g of the powdered drug with 10 mL of 0.05 M sulfuric acid for 2 minutes, filter, and add 1 mL of strong ammonia solution and 5 mL of water to the filtrate. Extract cautiously with 15 mL of ether to avoid the formation of an emulsion, dry the ether layer over anhydrous sodium sulfate, filter, and evaporate the ether in a porcelain dish. Add 0.5 mL of fuming nitric acid, evaporate to dryness over a small flame, add 10 mL of acetone and, dropwise, a 3 per cent w/v solution of potassium hydroxide in ethanol: a deep violet colour develops.
       B. Carry out the test as described in the “Thin-Layer Chromatography” (Appendix 3.1).
       Standard solution Dissolve 50 mg of hyoscyamine sulfate in 9 mL of methanol. Dissolve 15 mg of hyoscine hydrobromide in 10 mL of methanol. Mix 1.8 mL of the hyoscine hydrobromide solution and 8 mL of the hyoscyamine sulfate solution.
        Test solution To 600 mg, in fine powder, add 15 mL of dilute sulfuric acid, shake for 15 minutes and filter. Wash the filter with dilute sulfuric acid until 20 mL of the filtrate is obtained. To the filtrate add 1 mL of strong ammonia solution and shake with two 10-mL portions of peroxide-free ether. If necessary, separate by centrifugation. Dry the combined ether layers over anhydrous sodium sulfate, filter, and evaporate to dryness on a water-bath. Dissolve the residue in 0.5 mL of methanol.
       Adsorbent Silica gel G
       Mobile phase Strong ammonia solution, water and acetone (3:7:90)
       Application Apply 10 μL and 20 μL of each solution, as 20-mm bands.
       Development and drying Allow the solvent fronts to ascend 10 cm above the line of application. Dry the developed plate at 105° for 15 minutes and allow to cool.
       Detection A Spray the plate with potassium iodobismuthate TS, until orange or brown bands become visible against a yellow background, and observe the result.
       Results A When examine under visible light, the test solution shows a band of hyoscyamine in the lower third and a band of hyoscine at the upper third of the chromatogram, corresponding in colour, size and Rf to the band shown by the standard solution of the same volume. Faint secondary bands may appear, particularly in the middle of chromatogram obtained with 20 μL of the test solution or near the point of application in the chromatogram obtained with 10 μL of the test solution.
       Detection B Spray with a 10 per cent w/v solution of sodium nitrite until the coating is transparent and examine after 15 minutes. Observe the result.
       Results B When examine under visible light, the bands of hyoscyamine in the chromatograms obtained with the test solution and the standard solution change from brown to reddish brown but not to greyish blue (atropine), and any secondary bands disappear.

       Foreign matter Not more than 3 per cent of stems with a diameter exceeding 5 mm (Appendix 7.2).

       Acid-insoluble ash Not more than 4.0 per cent w/w (Method II, Appendix 7.6).

       Total ash Not more than 16.0 per cent w/w (Appendix 7.7).

       Assay Carry out the determination as described in the “Gas Chromatography” (Appendix 3.4).
       pH 9.5 Phosphate buffer Dissolve 34.8 g of dipotassium hydrogenphosphate in 900 mL of water, and adjust to a pH of 9.5, by the addition of 3 M hydrochloric acid or sodium hydroxide, with mixing.
       Diluent Diluted sulfuric acid (1 in 350)
       Internal standard solution Dissolve about 40 mg of Homatropine Hydrobromide RS, accurately weighed, in about 25 mL of Diluent in a 50-mL volumetric flask, add Diluent to volume, and mix. (Note Prepare freshly on the day of use.)
       Standard preparation Dissolve about 10 mg of Scopolamine Hydrobromide RS, accurately weighed, in about 5 mL of Diluent in a 10-mL volumetric flask, add Diluent to volume, and mix (Solution A). Dissolve about 20 mg of Atropine Sulfate RS, accurately weighed, in about 25 mL of Diluent in a 50-mL volumetric flask, add 2.0 mL of Solution A, and mix. Add Diluent to volume, and mix. (Note Prepare freshly on the day of use.)
       Standard curve Prepare three standard solutions as follows. Pipette into three separate 60-mL separators 1.0-, 2.0-, and 3.0-mL portions, respectively, of Standard preparation, and add 9.0, 8.0, and 7.0 mL, respectively, of Diluent. Proceed as directed under Assay preparation, beginning with “add 1.0 mL of Internal standard solution”.
       Assay preparation Moisten 10 g, in moderately coarse powder, with a mixture of 8 mL of strong ammonia solution, 10 mL of ethanol, and 20 mL of ether, and extract the alkaloids by either Method I or Method II as follows. If necessary, reduce the volume of the extract to 100 mL by evaporation on a water-bath.           METHOD I Place the moistened drug in a continuous-extraction thimble, and allow maceration to proceed overnight, then extract with ether for 3 hours, or longer if necessary, to effect complete extraction.
       METHOD II Place the moistened drug in a small percolator, and allow maceration to proceed overnight. Percolate slowly with a mixture of three volumes of ether and one volume of chloroform. Continue the percolation until the residue from 3 to 4 mL of percolate last passed, when dissolved in diluted sulfuric acid (1 in 70) and treated with mercury(II) iodide TS, shows not more than a faint turbidity.
       Transfer the extract from Method I or Method II to a separator with the aid of ether. Extract with five 15-mL portions of diluted sulfuric acid (1 in 70), filtering each portion drawn off into a 100-mL volumetric flask. Wash the filter with diluted sulfuric acid (1 in 70), and collect the washings in the flask. Add diluted sulfuric acid (1 in 70) to volume, and mix. Dilute 20.0 mL of the resulting solution with the same dilute acid to 100.0 mL. Pipette 10 mL of this solution into a 60-mL separator. Add 1.0 mL of Internal standard solution, then add 15 mL of chloroform, shake vigorously, allow the layers to separate, and discard the chloroform layer. (Note If emulsions are formed, a mixture of 10 volumes of chloroform and 3 volumes of 2-propanol may be substituted for chloroform throughout the extraction procedure.)
       Add another 15 mL of chloroform, and extract again, discarding the chloroform phase. Add 15 mL of Phosphate buffer and sufficient 1 M sodium hydroxide to yield a final pH between 9.0 and 9.5. Add 15 mL of chloroform, shake vigorously, and allow the layers to separate. Filter the organic phase through 10 g of anhydrous sodium sulfate, previously washed with chloroform and supported in a funnel with a small pledget of glass wool, into a suitable container. Extract again with two 15-mL portions of chloroform, again collecting the clarified organic phase. Wash the sodium sulfate and the tip of the funnel with 5 mL of chloroform. Evaporate the combined organic phases under reduced pressure at a temperature below 45°, add 1 mL of chloroform, and mix to dissolve the alkaloids, taking care to wet the sides of the container.
       Extraction blank Place 10 mL of Diluent in a 60-mL separator. Prepare as directed under Assay preparation, beginning with “then add 15 mL of chloroform”. The blank chromatogram contains no significant interferences at the locus of atropine, scopolamine, or homatropine.
       Chromatographic system
       DETECTOR Flame ionization
       COLUMN A glass column (1.2m × 4 mm) packed with 3 per cent of 50 per cent phenyl-50 per cent methylpolysioxane on acid-washed and alkali-washed diatomaceous support (100- to 120-mesh)
        TEMPERATURES
        Column 215°
        Injection port 215°
        Detector 240°
       CARRIER GAS Helium
       FLOW RATE 65 mL per minute
       System suitability
       SAMPLE Standard preparation
       Suitability requirements
       RESOLUTION Not less than 3.0 between atropine and homatropine peaks
       SYMMETRY FACTOR Not more than 2.0 for the atropine peak
       RELATIVE STANDARD DEVIATION Chromatograph six to ten injections of Assay preparation, and record peak areas. The analytical system is suitable for conducting this Assay if the relative standard deviation for the ratio, RA, is not more than 2.0 per cent for the atropine peak.
       Procedure Inject a portion (about 5 L) of each standard solution described under Standard preparations into the chromatograph. Measure the areas, aA, aH, and aS, of the atropine, homatropine and scopolamine peaks, respectively, in each chromatogram, and calculate the area ratios RA, and RS by the formulae:

aA/aH and aS/aH, respectively.

       Plot the standard curves of the values of RA and RS against the amounts in mg, of atropine and scopolamine in the solutions. (The ratio of the molecular weight of atropine to that of anhydrous atropine sulfate is 0.8551, and the ratio of the molecular weight of scopolamine to that of anhydrous scopolamine hydrobromide is 0.7894.) Inject a portion of Assay preparation into the chromatograph, measure the peak areas, and calculate the area ratios, as with the standard solutions. Record from the Standard curve the quantities, in mg, of atropine and scopolamine in the weight of sample taken. Add the quantity, in mg, of atropine and scopolamine, and multiply by 50 to obtain the weight, in mg, of alkaloids in the portion of Belladonna Leaf taken.