Standardised Belladonna Leaf Dry Extract

General Notices

(Ph. Eur. monograph 1294)

Ph Eur

DEFINITION
Standardised dry extract obtained from Belladonna leaf (0221).
Content
0.95 per cent to 1.05 per cent of total alkaloids, expressed as hyoscyamine (C17H23NO3; Mr289.4) (dried
extract).

PRODUCTION
The extract is produced from the herbal drug by a suitable procedure using ethanol (70 per cent V/V).

CHARACTERS
Appearance
Brown or greenish, hygroscopic powder.

IDENTIFICATION
A. Thin-layer chromatography (2.2.27).
Test solution To 1 g of the extract to be examined add 5.0 mL of methanol R. Shake for 2 min and filter.
Reference solution Dissolve 1.0 mg of chlorogenic acid Rand 2.5 mg of rutin Rin 10 mL of methanol R.
Plate TLC silica gel plate R.
Mobile phase anhydrous formic acid R, water R, methyl ethyl ketone R, ethyl acetate R(10:10:30:50 V/V/V/V).
Application 20

as bands.

Development Over a path of 15 cm.

Drying At 100-105

Detection Spray the warm plate with a 10 g/L solution of diphenylboric acid aminoethyl ester Rin methanol R,
then spray with a 50 g/L solution of macrogol 400 Rin methanol R; allow to dry in air for 30 min and examine in
ultraviolet light at 365 nm.

Results The chromatograms obtained with the reference solution and the test solution show in the central part a
light blue fluorescent zone (chlorogenic acid) and in the lower part a yellowish-brown fluorescent zone (rutin);
furthermore, the chromatogram obtained with the test solution shows a little above the start a yellowish-brown
fluorescent zone and directly above that a yellow fluorescent zone, and a yellow or yellowish-brown fluorescent
zone between the zone due to rutin and the zone due to chlorogenic acid. Further zones may be present.
B. Examine the chromatograms obtained in the test for atropine.
Results The principal zones in the chromatogram obtained with the test solution are similar in position and
colour to the principal zones in the chromatogram obtained with the reference solution.

TESTS
Atropine
Thin-layer chromatography (2.2.27).
Test solution To 0.20 g of the extract to be examined add 10.0 mL of 0.05 M sulfuric acid, shake for 2 min and
filter. Add 1.0 mL of concentrated ammonia Rand shake with 2 quantities, each of 10 mL, of peroxide-free ether
R. If necessary, separate by centrifugation. Dry the combined ether layers over about 2 g of anhydrous sodium
sulfate R, filter and evaporate to dryness on a water-bath. Dissolve the residue in 0.5 mL of methanol R.
Reference solution Dissolve 50 mg of hyoscyamine sulfate Rin 9 mL of methanol R. Dissolve 15 mg of hyoscine
hydrobromide Rin 10 mL of methanol R. Mix 1.8 mL of the hyoscine hydrobromide solution and 8 mL of the
hyoscyamine sulfate solution.
Plate TLC silica gel plate R.
Mobile phase concentrated ammonia R, water R, acetone R(3:7:90 V/V/V).
Application 20

as bands.

Development Over a path of 10 cm.

Drying At 100-105

for 15 min; allow to cool.

Detection A Spray with potassium iodobismuthate solution R2, until orange or brown zones become visible
against a yellow background.
Results A The zones in the chromatogram obtained with the test solution are similar in position (hyoscyamine in
the lower third, hyoscine in the upper third) and colour to those in the chromatogram obtained with the reference
solution. Other faint zones may be present in the chromatogram obtained with the test solution.
Detection B Spray with sodium nitrite solution Runtil the coating is transparent and examine after 15 min.
Results B The zones due to hyoscyamine in the chromatograms obtained with the test solution and the reference
solution change from orange or brown to reddish-brown but not to greyish-blue (atropine).
Loss on drying(2.8.17)
Maximum 5.0 per cent.

Microbial contamination
TAMC: acceptance criterion 104CFU/g (2.6.12).
TYMC: acceptance criterion 102CFU/g (2.6.12).
Absence of Escherichia coli(2.6.13).
Absence of Salmonella(2.6.13).

ASSAY
At each extraction stage it is necessary to check that the alkaloids have been completely extracted. If the
extraction is into the organic phase this is done by evaporating to dryness a few millilitres of the last organic layer,
dissolving the residue in 0.25 M sulfuric acidand verifying the absence of alkaloids using potassium
tetraiodomercurate solution R. If the extraction is into the acid aqueous phase, this is done by taking a few
millilitres of the last acid aqueous phase and verifying the absence of alkaloids using potassium
tetraiodomercurate solution R.
Disperse 3.00 g in a mixture of 5 mL of ammonia Rand 15 mL of water R. Shake with no fewer than 3 quantities,
each of 40 mL, of a mixture of 1 volume of methylene chloride Rand 3 volumes of peroxide-free ether Runtil the
alkaloids are completely extracted. Concentrate the combined organic layers to about 50 mL by distilling on a
water-bath and transfer the resulting liquid to a separating funnel, rinsing with peroxide-free ether R. Add a
quantity of peroxide-free ether Requal to at least 2.1 times the volume of the liquid to produce a layer having a
density well below that of water. Shake the resulting solution with no fewer than 3 quantities, each of 20 mL, of
0.25 M sulfuric aciduntil the alkaloids are completely extracted. Separate the layers by centrifugation, if
necessary, and transfer the acid layers to a 2ndseparating funnel. Make the combined acid layers alkaline with
ammonia Rand shake with no fewer than 3 quantities, each of 30 mL, of methylene chloride Runtil the alkaloids
are completely extracted. Combine the organic layers, add 4 g of anhydrous sodium sulfate Rand allow to stand
for 30 min with occasional shaking. Decant the methylene chloride and wash the sodium sulfate with 3 quantities,
each of 10 mL, of methylene chloride R. Combine the organic extracts and evaporate to dryness on a water-bath.
Heat the residue in an oven at 100-105
for 15 min. Dissolve the residue in a few millilitres of methylene
chloride R, evaporate to dryness on a water-bath and again heat the residue in an oven at 100-105 for 15 min.
Dissolve the residue in a few millilitres of methylene chloride R, add 20.0 mL of 0.01 M sulfuric acidand remove
the methylene chloride by evaporation on a water-bath. Titrate the excess of acid with 0.02 M sodium
hydroxideusing methyl red mixed solution Ras indicator.
Calculate the percentage content of total alkaloids, expressed as hyoscyamine, using the following expression:
[image: "bp2014_v4_08_1_herbal_drugs" "belladonnaleafdryextractstandardised_1_2012_70_eq.png"]

n
=
volume of 0.02 M sodium hydroxideused, in millilitres;

m
=
mass of drug used, in grams.

Ph Eur