White horehound EUROPEAN PHARMACOPOEIA 11.

1. verbenalin 2. ferulic acid 3. unknown substance (may be absent) 4. acteoside

Figure 1854.-2. – Chromatogram for the assay of verbena herb : test solution

Calculate the percentage content of verbenalin using the
following expression :
A1 ´ A4 ´ m 2 ´ 1000
A 2 ´ A3 ´ m1
A1 = area of the peak due to verbenalin in the
chromatogram obtained with the test solution ;
A2 = area of the peak due to verbenalin in the
chromatogram obtained with the reference
solution ;
A3 = area of the peak due to ferulic acid in the
chromatogram obtained with the test solution ;
A4 = area of the peak due to ferulic acid in the
chromatogram obtained with the reference
solution ;
m1 = mass of the herbal drug used to prepare the test
solution, in grams ;
m2 = mass of verbenalin in the reference solution, in
grams.
01/2019:1835
WHITE HOREHOUND
Marrubii herba
DEFINITION
Whole or fragmented dried flowering aerial parts of
Marrubium vulgare L.
Content : minimum 0.7 per cent of marrubiin (C20H28O4 ;
Mr 332.4) (dried drug).
IDENTIFICATION
A. The stems are up to 50 cm long, quadrangular, up to 7 mm
wide, young stems are densely covered with whitish downy
hairs, older stems are greenish-grey and less hairy. The
lower leaves are broadly ovate to almost orbicular, upper
leaves less broadly ovate, both petiolate ; lamina 1.5-4 cm
long, 1-3.5 cm wide, apex sub-acute, base tapering or
somewhat cordate, margin dentate to crenate, petiole up
to 3 cm long ; venation pinnate, prominent on the lower
surface, distinctly depressed on the upper surface. Both leaf
surfaces are densely covered with fine, white, woolly hairs,
older leaves having fewer hairs on the dark greyish-green
upper surface. The flowers are small, sessile in dense
axillary clusters. The calyx is 5 mm long, persistent, with
5 long and 5 short, alternating, hooked, recurved fringing
spines ; throat of calyx with an internal ring of long silky
hairs ; corolla 7 mm long, dull white, 4-lobed, upper lobe
2-lipped, lower-lobe 3-lipped ; 4 short stamens ; style with
bifid stigma.
B. Microscopic examination (2.8.23). The powder is
greyish-green. Examine under a microscope using chloral
hydrate solution R. The powder shows the following
diagnostic characters (Figure 1835.-1) : numerous covering
trichomes of different types, isolated or associated with
fragments of the epidermis of the leaves : a) covering
trichomes in tufts [B, H] with 3-20 rigid branches of various
sizes with smooth, regularly thickened walls, sometimes
sessile [B], sometimes arising from a multicellular
stalk [H], some branches are unicellular [Ba, Ha], others are
multicellular (2-6 cells), 100-200 μm long, thick-walled and
swollen at the junctions [Bb, Hb] ; b) unicellular covering
trichomes ; c) multicellular covering trichomes similar to
the branches of the trichomes in tufts ; glandular trichomes
of different types : a) glandular trichomes with a unicellular
stalk and a unicellular [D], bicellular [Hc] or quadricellular
head (surface view [Gb]) ; b) glandular trichomes with a
multicellular stalk and a unicellular head [A] ; c) glandular
trichomes of lamiaceous type with a unicellular stalk and
an 8-celled head covered with a cuticle (surface view [Gd]) ;
fragments of the epidermis of the leaves [G] consisting of
polygonal cells with slightly sinuous walls [Ga], stomata
of the diacytic type (2.8.3) [Gc], covering trichomes and
glandular trichomes ; covering trichomes from the inner
surface of the calyx, twisted or coiled, up to 1000 μm long,
bi- or tricellular, thickened at the cell junctions, with
a flexuous, very elongated distal cell [C] ; fragments of
palisade parenchyma (surface view [Bc]) containing small
needle-shaped crystals of calcium oxalate ; fragments of
vascular tissue from the stems and veins [F] ; fragments of
petals with papillose cells [J] ; spherical pollen grains, about
25 μm in diameter with a smooth exine [E].

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EUROPEAN PHARMACOPOEIA 11.0 White horehound

solution (b). During extraction with hydrochloric acid and

methanol, conversion of pre-marrubiin to marrubiin takes
place which leads to an increase in intensity of the zone.

Top of the plate

Guaiazulene : a A bluish-violet zone A bluish-violet zone
reddish-violet zone
_______ _______

A bluish-violet zone A bluish-violet zone

_______ _______

Cholesterol : a An intense bluish-violet A bluish-violet zone

bluish-violet zone zone (marrubiin) (marrubiin)
A bluish-violet zone A bluish-violet zone

A bluish-violet zone A bluish-violet zone

Reference solution Test solution (a) Test solution (b)

TESTS
Loss on drying (2.2.32) : maximum 10.0 per cent, determined
on 1.000 g of the powdered herbal drug (710) (2.9.12) by
drying in an oven at 105 °C for 2 h.
Total ash (2.4.16) : maximum 15.0 per cent.
Ash insoluble in hydrochloric acid (2.8.1) : maximum 3.0 per
cent.

ASSAY

Figure 1835.-1. – Illustration for identification test B of Liquid chromatography (2.2.29).

powdered herbal drug of white horehound
C. Thin-layer chromatography (2.2.27).
Test solution (a). To 1.0 g of the powdered herbal drug
(710) (2.9.12) add 2 mL of dilute hydrochloric acid R and
8 mL of methanol R. Heat under a reflux condenser for
30 min, cool and filter.
Test solution (b). To 1.0 g of the powdered herbal drug
(710) (2.9.12) add 10 mL of methanol R. Heat under a
reflux condenser for 30 min, cool and filter.
Reference solution. Dissolve 10 mg of cholesterol R and
10 mg of guaiazulene R in 10 mL of methanol R.
Plate : TLC silica gel plate R (5-40 μm) [or TLC silica gel
plate R (2-10 μm)].
Mobile phase : methanol R, toluene R (5:95 V/V).
Test solution. Reduce 50 g of the herbal drug to a powder (250)
(2.9.12) and homogenise. To 1.00 g of the powdered herbal
drug in a 50 mL round-bottomed flask add 15 mL of a mixture
of 2 volumes of dilute hydrochloric acid R and 8 volumes
of methanol R. Heat in a water bath at 80 °C under a reflux
condenser for 30 min. Allow to cool to room temperature
and filter through a plug of adsorbent cotton into a 25 mL
volumetric flask. Dilute to 25.0 mL with methanol R by rinsing
the round-bottomed flask and the filter.
Reference solution. Dissolve 2.0 mg of marrubiin CRS in
methanol R and dilute to 10.0 mL with the same solvent.
Column :
– size : l = 0.25 m, Ø = 4 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for
chromatography R (5 μm).
Mobile phase :

Application : 20 μL [or 5 μL] of test solutions (a) and (b) – mobile phase A : acetonitrile R1 ;
and 10 μL [or 2 μL] of the reference solution, as bands. – mobile phase B : dilute 0.5 mL of phosphoric acid R to
1000 mL with water for chromatography R ;
Development : over a path of 10 cm [or 6 cm].
Time Mobile phase A Mobile phase B
Drying : in air. (min) (per cent V/V) (per cent V/V)
0 - 15 40 → 90 60 → 10
Detection : treat with a 5 g/L solution of vanillin R in a
mixture of 20 volumes of ethanol (96 per cent) R and 15 - 20 90 → 40 10 → 60
80 volumes of sulfuric acid R and examine in daylight
20 - 25 40 60
immediately after heating at 130 °C for 5-10 min.

Results : see below the sequence of zones present in the
chromatograms obtained with the reference solution
and test solutions (a) and (b). Further zones in the
chromatograms obtained with test solutions (a) and
(b) may be present. The zone due to marrubiin in the
chromatogram obtained with test solution (a) is more
intense than that in the chromatogram obtained with test
Flow rate : 1.5 mL/min.
Detection : spectrophotometer at 217 nm.
Injection : 20 μL.
Locate the peak due to marrubiin by comparison with the
chromatogram obtained with the reference solution.

General Notices (1) apply to all monographs and other texts 1775
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Wild pansy (flowering aerial parts) EUROPEAN PHARMACOPOEIA 11.0

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

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