Wormwood EUROPEAN PHARMACOPOEIA 11.

PRODUCTION Mobile phase:

The extract is produced from the herbal drug by a suitable – mobile phase A : tetrahydrofuran R, 0.5 per cent V/V
procedure using either water or a hydroalcoholic solvent solution of phosphoric acid R (1.8:98.2 V/V) ;
equivalent in strength to a maximum of 80 per cent V/V – mobile phase B : tetrahydrofuran R ;
ethanol. Time Mobile phase A Mobile phase B
CHARACTERS (min) (per cent V/V) (per cent V/V)
0 - 15 100 0
Appearance : yellowish-brown amorphous powder.
15 - 17 100 → 90 0 → 10
IDENTIFICATION
17 - 23 90 10
Thin-layer chromatography (2.2.27).
23 - 25 90 → 100 10 → 0
Test solution (a). To 0.200 g of the extract to be examined add
5 mL of methanol R. Sonicate for 5 min, filter and dilute to 25 - 40 100 0
10 mL with methanol R.
Test solution (b). To 5.0 mL of test solution (a) add 1.0 mL of Flow rate : 1.0 mL/min.
a 50 g/L solution of anhydrous sodium carbonate R and heat Detection : spectrophotometer at 270 nm.
in a water-bath at about 60 °C for 10 min. Cool and filter if Injection : 10 μL.
necessary. Retention time : salicin = about 6.4 min ; pi-
Reference solution. Dissolve 2.0 mg of salicin R and 2.0 mg of cein = about 7.7 min.
chlorogenic acid R in 1.0 mL of methanol R. System suitability : reference solution :
Plate : TLC silica gel plate R (5-40 μm) [or TLC silica gel plate R – resolution : minimum 1.5 between the peaks due to salicin
(2-10 μm)]. and picein.
Mobile phase : water R, methanol R, ethyl acetate R Calculate the percentage content of total salicylic derivatives,
(8:15:77 V/V/V). expressed as salicin, from the following expression :
Application : 10 μL [or 2 μL] as bands. A1 ´ m 2 ´ p ´ 2
Development : over a path of 15 cm [or 6 cm]. A 2 ´ m1
Drying : in a current of warm air.
A1 = area of the peak due to salicin in the chromatogram
Detection : spray with a mixture of 5 volumes of sulfuric acid R
and 95 volumes of methanol R. Heat at 100-105 °C for 5 min obtained with the test solution ;
and examine in daylight. A2 = area of the peak due to salicin in the chromatogram
Results : see below the sequence of the zones present in the obtained with the reference solution ;
chromatograms obtained with the reference solution and m1 = mass of the extract to be examined used to prepare
test solutions (a) and (b). Furthermore, other zones may be the test solution, in grams ;
present in the chromatogram obtained with test solutions (a) m2 = mass of salicin CRS used to prepare the reference
and (b). solution, in grams ;
Top of the plate p = percentage content of salicin in salicin CRS.
_______ _______
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Several reddish-violet
zones may be present
Salicin : a reddish-violet A weak reddish-violet A reddish-violet zone
zone zone (salicin) (salicin)
_______ _______

Chlorogenic acid : a WORMWOOD

brown zone
Reference solution Test solution (a) Test solution (b)
ASSAY
Liquid chromatography (2.2.29).
Test solution. To 0.300 g of the extract to be examined add
40 mL of methanol R and 40.0 mL of 0.1 M sodium hydroxide.
Heat in a water-bath at about 60 °C under a reflux condenser,
with frequent shaking, for about 1 h. After cooling, add
4.0 mL of 1 M hydrochloric acid. Filter the suspension into a
100 mL volumetric flask, then wash and dilute to 100.0 mL
with a mixture of equal volumes of water R and methanol R.
Filter through a membrane filter (nominal pore size 0.45 μm).
Reference solution. Dissolve 5.0 mg of picein R in 25.0 mL
of a mixture of 20 volumes of water R and 80 volumes of
methanol R (solution A). Dissolve 15.0 mg of salicin CRS in
25 mL of a mixture of 20 volumes of water R and 80 volumes
of methanol R. Add 5.0 mL of solution A and dilute to 50.0 mL
with water R.
Column:
– size : l = 0.10 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl silica gel for
chromatography R (3 μm).
Absinthii herba
DEFINITION
Basal leaves or slightly leafy, flowering tops, or mixture of
these dried, whole or cut organs of Artemisia absinthium L.
Content : minimum 2 mL/kg of essential oil (dried drug).
IDENTIFICATION
A. The leaves are greyish or greenish, densely tomentose on
both surfaces. The basal leaves, with long petioles, have
triangular or oval bipinnatisect or tripinnatisect lamina,
with rounded or lanceolate segments. The cauline leaves
are less segmented and the apical leaves are lanceolate.
The stem of the flower-bearing region is greenish-grey,
tomentose, up to 2.5 mm in diameter and usually with
5 flattened longitudinal grooves. The capitula are arranged
as loose, axillary panicles, inserted at the level of the
lanceolate or slightly pinnatisect leaves ; they are spherical
or flattened hemispherical, 2-4 mm in diameter and consist
of a grey, tomentose involucre, the outer bracts linear, inner
layer ovate, blunt at the apices with scarious margins, a
receptacle with very long paleae up to 1 mm or more long,
numerous yellow, tubular, hermaphroditic florets about
2 mm long and few yellow, ray florets.

1780 See the information section on general monographs (cover pages)

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EUROPEAN PHARMACOPOEIA 11.0
B. Microscopic examination (2.8.23). The powder is
greenish-grey. Examine under a microscope using chloral
hydrate solution R. The powder shows the following
diagnostic characters (Figure 1380.-1.) : many T-shaped
trichomes [A] with a short uniseriate stalk consisting of
1-5 small cells, perpendicularly capped by a very long,
undulating terminal cell tapering at the ends ; fragments of
epidermises (surface view [D]) with sinuous or wavy walls,
anomocytic stomata (2.8.3) [Da], covering trichomes [Db]
and glandular trichomes containing oil [Dc] or not
containing oil [Dd], each with a short, biseriate, 2-celled
stalk and a biseriate head with 2-4 cells ; free glandular
trichomes (side view [C]) ; fragments of the corollas of
the tubular and ray florets, some containing small cluster
crystals of calcium oxalate [H] ; numerous paleae each
composed of a small cell forming a stalk and a very long,
cylindrical and thin-walled terminal cell about 1-1.5 mm
long, either whole [E] or limited to the distal part [B] ;
spheroidal pollen grains, about 30 μm in diameter, with
3 pores and a finely warty exine [G] ; fragments of vascular
tissue from the leaves [F] or the stems [J] consisting of
vessels with spiral or annular thickenings [Fa], or with
bordered pits [Ja], fibres [Fb, Jb] and parenchymatous cells
with pitted, moderately thickened walls [Fc, Jc].
Figure 1380.-1. – Illustration for identification test B of
powdered herbal drug of wormwood
C. Thin-layer chromatography (2.2.27).
Test solution. Place 2 g of the powdered herbal drug (355)
(2.9.12) in 50 mL of boiling water R and allow to stand
for 5 min, shaking the flask several times. After cooling,
add 5 mL of a 100 g/L solution of lead acetate R. Mix and
filter. Rinse the flask and the residue on the filter with
20 mL of water R. Shake the filter with 50 mL of methylene
chloride R. Separate the organic layer, dry over anhydrous
sodium sulfate R, filter and evaporate the filtrate to dryness
on a water-bath. Dissolve the residue in 0.5 mL of ethanol
(96 per cent) R.
Reference solution. Dissolve 2 mg of methyl red R and 2 mg
of resorcinol R in 10.0 mL of methanol R.
Plate : TLC silica gel plate R.
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Yarrow
Mobile phase : acetone R, glacial acetic acid R, toluene R,
methylene chloride R (10:10:30:50 V/V/V/V).
Application : 10 μL as bands.
Development : over a path of 15 cm.
Drying : in air.
Detection A : spray with acetic anhydride - sulfuric acid
solution R and examine in daylight.
Results A : the chromatogram obtained with the test
solution shows a blue zone due to artabsin shortly above a
red zone due to methyl red in the chromatogram obtained
with the reference solution.
Detection B : examine in daylight while heating at
100-105 °C for 5 min.
Results B : the chromatogram obtained with the reference
solution shows in the middle third a red zone due to methyl
red and below it a light pink zone due to resorcinol. The
chromatogram obtained with the test solution shows an
intense red or brownish-red zone due to absinthin with a
similar RF value to that of the zone due to resorcinol in the
chromatogram obtained with the reference solution. Other
zones are visible, but less intense than that due to absinthin.
TESTS
Foreign matter (2.8.2) : maximum 5 per cent of stems with a
diameter greater than 4 mm and maximum 2 per cent of other
foreign matter.
Bitterness value (2.8.15) : minimum 10 000.
Loss on drying (2.2.32) : maximum 10.0 per cent, determined
on 1.000 g of the powdered herbal drug (355) (2.9.12) by
drying in an oven at 105 °C for 2 h.
Total ash (2.4.16) : maximum 12.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1) : maximum 1.0 per
cent.
ASSAY
Essential oil (2.8.12). Use 50.0 g of the cut drug, a 1000 mL
round-bottomed flask and 500 mL of water R as the distillation
liquid. Add 0.5 mL of xylene R in the graduated tube. Distil at
a rate of 2-3 mL/min for not less than 3 h.
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YARROW
Millefolii herba
DEFINITION
Whole or cut, dried flowering tops of Achillea millefolium L.
Content :
– essential oil : minimum 2 mL/kg (dried drug) ;
– proazulenes, expressed as chamazulene (C14H16 ; Mr 184.3) :
minimum 0.02 per cent (dried drug).
IDENTIFICATION
A. The leaves are green or greyish-green, faintly pubescent
on the upper surface and more pubescent on the lower
surface, 2-3 pinnately divided with linear lobes and a
finely pointed whitish tip. The capitula are arranged in a
corymb at the end of the stem. Each capitulum, 3-5 mm
in diameter, consists of the receptacle, usually 4-5 ligulate
ray-florets and 3-20 tubular disk-florets. The involucre
consists of 3 rows of imbricate lanceolate, pubescent green
bracts arranged with a brownish or whitish, membranous
margin. The receptacle is slightly convex and, in the axillae
of paleae, bears ligulate ray-florets with a three-lobed,
whitish or reddish ligule and tubular disk-florets with
1781