Wild pansy (flowering aerial parts) EUROPEAN PHARMACOPOEIA 11.

Calculate the percentage content of marrubiin from the arvensis) or 4 pores (Viola tricolor) ; occasional fragments
following expression : of spiral and reticulate vessels and groups of fibres from
the stem.
A1 ´ m 2 ´ p ´ 2.5
C. Thin-layer chromatography (2.2.27).
A 2 ´ m1
Test solution. Heat in a water-bath at 65 °C for 5 min, with
A1 = area of the peak due to marrubiin in the frequent stirring, 2.0 g of the powdered herbal drug (355)
chromatogram obtained with the test solution ; (2.9.12) in 10 mL of alcohol (70 per cent V/V) R. Cool and
filter.
A2 = area of the peak due to marrubiin in the
chromatogram obtained with the reference Reference solution. Dissolve 2.5 mg of rutoside trihydrate R,
solution ; 2.5 mg of hyperoside R and 1.0 mg of caffeic acid R in
methanol R and dilute to 10 mL with the same solvent.
m1 = mass of the herbal drug to be examined used to
Plate : TLC silica gel plate R.
prepare the test solution, in milligrams ;
m2 Mobile phase : anhydrous formic acid R, acetic acid R,
= mass of marrubiin CRS used to prepare the water R, ethyl acetate R (11:11:27:100 V/V/V/V).
reference solution, in milligrams ;
Application : 10 μL, as bands.
p = percentage content of marrubiin in marrubiin CRS. Development : over a path of 12 cm.
Drying : at 100-105 °C.
01/2008:1855 Detection : spray with a solution containing 10 g/L of
corrected 6.0 diphenylboric acid aminoethyl ester R and 50 g/L of
macrogol 400 R in methanol R. Allow the plate to dry in air
for 30 min. Examine in ultraviolet light at 365 nm.
Results : see below the sequence of the zones present in the
chromatograms obtained with the reference solution and
WILD PANSY the test solution. Furthermore, other zones may be present
in the chromatogram obtained with the test solution.
(FLOWERING AERIAL PARTS)
Top of the plate

Violae herba cum flore Caffeic acid : a greenish-blue to

light blue fluorescent zone
DEFINITION A blue fluorescent zone
Dried flowering aerial parts of Viola arvensis Murray and/or
Viola tricolor L.
_______ _______
Content : minimum 1.5 per cent of flavonoids, expressed as
violanthin (C27H30O14 ; Mr 578.5) (dried drug).
IDENTIFICATION Hyperoside : a yellowish-brown
fluorescent zone
A. The stem is angular and hollow. The leaves are oval, A yellowish-green fluorescent
petiolate, with a cordate base or elongated and obtuse, with zone
lyrate stipules, divided in the middle. The flowers, with a
long peduncle, are zygomorphic, with 5 oval, lanceolate
sepals, an appendage pointed outwards and 5 petals of _______ _______
which the lower one bears a spur ; in Viola arvensis, the Rutoside : a yellowish-brown An intense yellowish-brown
petals are shorter than the calyx, the lower petal is cream fluorescent zone fluorescent zone (rutoside)
coloured, with black lines, the 4 upper petals may be
cream coloured or violet blue ; in Viola tricolor, the petals A yellowish-green fluorescent
are longer than the calyx and violet coloured, more or zone
less tinged with yellow. The androecium consisting of A yellowish-green fluorescent
zone
5 stamens bears at the apex a membranous connective A yellowish-green fluorescent
appendage with 2 spurs. The trilocular ovary shows a zone
short style and globular stigmata. The fruit are navicular
capsules, three-lobed, yellowish brown, 5 mm to 10 mm
Reference solution Test solution
long. The pale yellow, pyriform seeds are about 1 mm long,
bearing a caruncle.
B. Microscopic examination (2.8.23). The powder is greenish. TESTS
Examine under a microscope using chloral hydrate Foreign matter (2.8.2) : maximum 3 per cent.
solution R. The powder shows the following diagnostic Swelling index (2.8.4) : minimum 9, determined on the
characters : fragments of the epidermis of the leaves in powdered herbal drug (355) (2.9.12).
surface view with wavy-walled cells and anomocytic
stomata (2.8.3) ; conical unicellular covering trichomes, Loss on drying (2.2.32) : maximum 12.0 per cent, determined
widened at the base and sharply pointed at the apex, with on 1.000 g of the powdered herbal drug (355) (2.9.12) by
a striated cuticle ; glandular trichomes with a multicellular drying in an oven at 105 °C for 2 h.
head, and a short, multicellular stalk in the indentations Total ash (2.4.16) : maximum 15.0 per cent.
of the leaf margins ; cluster crystals of calcium oxalate,
sometimes included in parenchyma ; fragments of the ASSAY
corolla with wavy-walled epidermal cells, those from the Stock solution. In a 200 mL flask, introduce 0.300 g of the
mid-region papillose and with some extended to form flask powdered herbal drug (250) (2.9.12) and 40 mL of alcohol
or bottle-shaped projections, those from the base of the (60 per cent V/V) R. Heat in a water-bath at 60 °C for 10 min,
petals with covering trichomes up to about 300 μm long shaking frequently. Allow to cool and filter through a plug of
with characteristic hump-like swellings along their length ; absorbent cotton into a 100 mL volumetric flask. Transfer the
spherical or polyhedral pollen grains, 60 μm to 80 μm absorbent cotton with the drug residue back into the 200 mL
in diameter, with finely pitted exines and 5 pores (Viola flask, add 40 mL of alcohol (60 per cent V/V) R and heat again

1776 See the information section on general monographs (cover pages)

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EUROPEAN PHARMACOPOEIA 11.0
in a water-bath at 60 °C for 10 min, shaking frequently. Allow B. Microscopic examination (2.8.23). The powder is
to cool and filter into the same 100 mL volumetric flask as
used previously. Rinse the 200 mL flask with a further quantity
of alcohol (60 per cent V/V) R, filter and transfer to the same
100 mL volumetric flask. Dilute to volume with alcohol (60 per
cent V/V) R and filter.
Test solution. Introduce 5.0 mL of the stock solution into
a round-bottomed flask and evaporate to dryness under
reduced pressure. Take up the residue with 8 mL of a mixture
of 10 volumes of methanol R and 100 volumes of glacial acetic
acid R and transfer into a 25 mL volumetric flask. Rinse the
round-bottomed flask with 3 mL of a mixture of 10 volumes
of methanol R and 100 volumes of glacial acetic acid R
and transfer into the same 25 mL volumetric flask as used
previously. Add 10.0 mL of a solution containing 25.0 g/L of
boric acid R and 20.0 g/L of oxalic acid R in anhydrous formic
acid R and dilute to 25.0 mL with anhydrous acetic acid R.
Compensation liquid. Introduce 5.0 mL of the stock solution
into a round-bottomed flask and evaporate to dryness under
reduced pressure. Take up the residue with 8 mL of a mixture
of 10 volumes of methanol R and 100 volumes of glacial
acetic acid R and transfer into a 25 mL volumetric flask.
Rinse the round-bottomed flask with 3 mL of a mixture of
10 volumes of methanol R and 100 volumes of glacial acetic
acid R and transfer into the same 25 mL volumetric flask as
used previously. Add 10.0 mL of anhydrous formic acid R and
dilute to 25.0 mL with anhydrous acetic acid R.
Measure the absorbance (2.2.25) of the test solution at 405 nm
after 30 min.
Calculate the percentage content of total flavonoids, expressed
as violanthin from the expression :
A ´ 1.25
m
taking the specific absorbance of violanthin to be 400.
A = measured absorbance at 405 nm,
m = mass of the herbal drug to be examined, in grams.
07/2014:1891
WILD THYME
Serpylli herba
DEFINITION
Whole or cut, dried, flowering aerial parts of Thymus
serpyllum L.
Content : minimum 3.0 mL/kg of essential oil (dried drug).
IDENTIFICATION
A. The stem is much branched, up to about 1.5 mm in
diameter, cylindrical or indistinctly quadrangular, green,
reddish or purplish, the older stems brown and woody, the
younger stems pubescent. The leaves are opposite, 3-12 mm
long and up to 4 mm wide, elliptical to ovate-lanceolate
with an obtuse apex, cuneate and shortly petiolate at the
base ; the margin is entire and markedly ciliate, especially
near the base ; both surfaces are more or less glabrous but
distinctly punctate. The inflorescence is composed of about
6-12 flowers in rounded to ovoid, terminal heads. The calyx
is tubular, 2-lipped with the upper lip dividing to form
3 teeth, the lower lip with 2 teeth, edged with long hairs ; C. Thin-layer chromatography (2.2.27).
the inner surfaces are strongly pubescent, the hairs forming
a closed tube after flowering. The corolla is purplish-violet
or red, 2-lipped, the lower lip with 3 lobes and the upper
lip notched, the inner surface is strongly pubescent ; 4
epipetalous stamens project from the corolla tube.
General Notices (1) apply to all monographs and other texts
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Wild thyme
greyish-green or greenish-brown. Examine under a
microscope using chloral hydrate solution R. The powder
shows the following diagnostic characters (Figure 1891.-1) :
fragments of the leaf epidermises [A, B, F] covered by a
finely striated cuticle and consisting of cells with sinuous
anticlinal walls [Aa, Ba, Fa] and diacytic stomata (2.8.3)
[Ab, Bb, Fb] ; cells of the adaxial leaf epidermis [B] with
wavy, irregularly thickened anticlinal walls [Ba] ; numerous
covering trichomes on both epidermises and the leaf
margins, with some of the cells containing very small
crystals of calcium oxalate [Af, Ca, Fd], the majority are
short, conical, unicellular, with thickened and warty walls
(surface view [Bc], side view [Fc]) ; fewer multicellular
covering trichomes, long, tapering to a point, composed
of up to 8 cells, slightly swollen at the joints, with finely
pitted walls, on an epidermis [Ae] or fragmented [C] ;
abundant glandular trichomes, mostly multicellular of
the lamiaceous type [Ac] with a unicellular stalk and a
glandular head consisting of 12 inconspicuous cells, others
with a unicellular stalk and a unicellular globular or ovoid
head [Ad] ; purplish-violet fragments of the corolla whose
inner epidermis consists of cells with rounded papillae [D]
and whose outer epidermis [E], with a striated cuticle,
consists of cells with lobed walls [Ea], unicellular [Eb] or
multicellular [Ec] uniseriate covering trichomes, glandular
trichomes with a unicellular head and a unicellular stalk
[Ed] and glandular trichomes of the lamiaceous type ;
relatively rare pollen grains, spherical, about 30 μm in
diameter, with a finely pitted exine and 6 germinal pores
[G].
Figure 1891.-1.– Illustration for identification test B of
powdered herbal drug of wild thyme
Test solution. To 0.5 g of the powdered herbal drug (355)
(2.9.12) add 5 mL of methanol R. Sonicate for 10 min.
Centrifuge or filter ; use the supernatant or the filtrate.
Reference solution. Dissolve 1 mg of rutoside trihydrate R
and 1 mg of rosmarinic acid R in 5 mL of methanol R.
1777