EUROPEAN PHARMACOPOEIA 10.

0 White horehound

Calculate the percentage content of verbenalin using the the branches of the trichomes in tufts ; glandular trichomes
following expression : of different types : a) glandular trichomes with a unicellular
stalk and a unicellular [D], bicellular [Hc] or quadricellular
A1 ´ A4 ´ m 2 ´ 1000 head (surface view [Gb]) ; b) glandular trichomes with a
A 2 ´ A3 ´ m1 multicellular stalk and a unicellular head [A] ; c) glandular
trichomes of lamiaceous type with a unicellular stalk and
A1 = area of the peak due to verbenalin in the an 8-celled head covered with a cuticle (surface view [Gd]) ;
chromatogram obtained with the test solution ; fragments of the epidermis of the leaves [G] consisting of
A2 = area of the peak due to verbenalin in the polygonal cells with slightly sinuous walls [Ga], stomata
chromatogram obtained with the reference of the diacytic type (2.8.3) [Gc], covering trichomes and
solution ; glandular trichomes ; covering trichomes from the inner
A3 surface of the calyx, twisted or coiled, up to 1000 μm
= area of the peak due to ferulic acid in the
long, bi- or tricellular, thickened at the cell junctions, with
chromatogram obtained with the test solution ;
a flexuous, very elongated distal cell [C] ; fragments of
A4 = area of the peak due to ferulic acid in the palisade parenchyma (surface view [Bc]) containing small
chromatogram obtained with the reference needle-shaped crystals of calcium oxalate ; fragments of
solution ; vascular tissue from the stems and veins [F] ; fragments of
m1 = mass of the herbal drug used to prepare the test petals with papillose cells [J] ; spherical pollen grains, about
solution, in grams ; 25 μm in diameter with a smooth exine [E].
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 Figure 1835.-1. – Illustration for identification test B of
upper surface. The flowers are small, sessile in dense powdered herbal drug of white horehound
axillary clusters. The calyx is 5 mm long, persistent, with C. Thin-layer chromatography (2.2.27).
5 long and 5 short, alternating, hooked, recurved fringing Test solution (a). To 1.0 g of the powdered herbal drug
spines ; throat of calyx with an internal ring of long silky (710) (2.9.12) add 2 mL of dilute hydrochloric acid R and
hairs ; corolla 7 mm long, dull white, 4-lobed, upper lobe 8 mL of methanol R. Heat under a reflux condenser for
2-lipped, lower-lobe 3-lipped ; 4 short stamens ; style with 30 min, cool and filter.
bifid stigma.
Test solution (b). To 1.0 g of the powdered herbal drug
B. Microscopic examination (2.8.23). The powder is (710) (2.9.12) add 10 mL of methanol R. Heat under a reflux
greyish-green. Examine under a microscope using chloral condenser for 30 min, cool and filter.
hydrate solution R. The powder shows the following
diagnostic characters (Figure 1835.-1) : numerous covering Reference solution. Dissolve 10 mg of cholesterol R and
trichomes of different types, isolated or associated with 10 mg of guaiazulene R in 10 mL of methanol R.
fragments of the epidermis of the leaves : a) covering Plate : TLC silica gel plate R (5-40 μm) [or TLC silica gel
trichomes in tufts [B, H] with 3-20 rigid branches of various plate R (2-10 μm)].
sizes with smooth, regularly thickened walls, sometimes Mobile phase : methanol R, toluene R (5:95 V/V).
sessile [B], sometimes arising from a multicellular Application : 20 μL [or 5 μL] of test solutions (a) and (b)
stalk [H], some branches are unicellular [Ba, Ha], others are and 10 μL [or 2 μL] of the reference solution, as bands.
multicellular (2-6 cells), 100-200 μm long, thick-walled and
swollen at the junctions [Bb, Hb] ; b) unicellular covering Development : over a path of 10 cm [or 6 cm].
trichomes ; c) multicellular covering trichomes similar to Drying : in air.

General Notices (1) apply to all monographs and other texts 1667
Wild pansy (flowering aerial parts) EUROPEAN PHARMACOPOEIA 10.0

Detection : treat with a 5 g/L solution of vanillin R in a Locate the peak due to marrubiin by comparison with the
mixture of 20 volumes of ethanol (96 per cent) R and chromatogram obtained with the reference solution.
80 volumes of sulfuric acid R and examine in daylight Calculate the percentage content of marrubiin from the
immediately after heating at 130 °C for 5-10 min. following expression :
Results : see below the sequence of zones present in the
A1 ´ m 2 ´ p ´ 2.5
chromatograms obtained with the reference solution
and test solutions (a) and (b). Further zones in the A 2 ´ m1
chromatograms obtained with test solutions (a) and
(b) may be present. The zone due to marrubiin in the A1 = area of the peak due to marrubiin in the
chromatogram obtained with test solution (a) is more chromatogram obtained with the test solution ;
intense than that in the chromatogram obtained with test A2 = area of the peak due to marrubiin in the
solution (b). During extraction with hydrochloric acid and chromatogram obtained with the reference
methanol, conversion of pre-marrubiin to marrubiin takes solution ;
place which leads to an increase in intensity of the zone. m1 = mass of the herbal drug to be examined used to
Top of the plate prepare the test solution, in milligrams ;
m2 = mass of marrubiin CRS used to prepare the
Guaiazulene : a A bluish-violet zone A bluish-violet zone
reddish-violet zone reference solution, in milligrams ;
_______ _______ p = percentage content of marrubiin in marrubiin CRS.
A bluish-violet zone A bluish-violet zone
_______ _______ 01/2008:1855
corrected 6.0
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) WILD PANSY
(FLOWERING AERIAL PARTS)
TESTS
Loss on drying (2.2.32) : maximum 10.0 per cent, determined Violae herba cum flore
on 1.000 g of the powdered herbal drug (710) (2.9.12) by DEFINITION
drying in an oven at 105 °C for 2 h.
Dried flowering aerial parts of Viola arvensis Murray and/or
Total ash (2.4.16) : maximum 15.0 per cent. Viola tricolor L.
Ash insoluble in hydrochloric acid (2.8.1): maximum 3.0 per Content : minimum 1.5 per cent of flavonoids, expressed as
cent. violanthin (C27H30O14 ; Mr 578.5) (dried drug).
ASSAY IDENTIFICATION
Liquid chromatography (2.2.29). A. The stem is angular and hollow. The leaves are oval,
Test solution. Reduce 50 g of the herbal drug to a powder (250) petiolate, with a cordate base or elongated and obtuse, with
(2.9.12) and homogenise. To 1.00 g of the powdered herbal lyrate stipules, divided in the middle. The flowers, with a
drug in a 50 mL round-bottomed flask add 15 mL of a mixture long peduncle, are zygomorphic, with 5 oval, lanceolate
of 2 volumes of dilute hydrochloric acid R and 8 volumes of sepals, an appendage pointed outwards and 5 petals of
methanol R. Heat in a water bath at 80 °C under a reflux which the lower one bears a spur ; in Viola arvensis, the
condenser for 30 min. Allow to cool to room temperature petals are shorter than the calyx, the lower petal is cream
and filter through a plug of adsorbent cotton into a 25 mL coloured, with black lines, the 4 upper petals may be
volumetric flask. Dilute to 25.0 mL with methanol R by rinsing cream coloured or violet blue ; in Viola tricolor, the petals
the round-bottomed flask and the filter. are longer than the calyx and violet coloured, more or
less tinged with yellow. The androecium consisting of
Reference solution. Dissolve 2.0 mg of marrubiin CRS in 5 stamens bears at the apex a membranous connective
methanol R and dilute to 10.0 mL with the same solvent. appendage with 2 spurs. The trilocular ovary shows a
Column : short style and globular stigmata. The fruit are navicular
– size : l = 0.25 m, Ø = 4 mm ; capsules, three-lobed, yellowish brown, 5 mm to 10 mm
– stationary phase : end-capped octadecylsilyl silica gel for long. The pale yellow, pyriform seeds are about 1 mm long,
chromatography R (5 μm). bearing a caruncle.
Mobile phase : B. Microscopic examination (2.8.23). The powder is greenish.
Examine under a microscope using chloral hydrate
– mobile phase A : acetonitrile R1 ; solution R. The powder shows the following diagnostic
– mobile phase B : dilute 0.5 mL of phosphoric acid R to characters : fragments of the epidermis of the leaves in
1000 mL with water for chromatography R ; surface view with wavy-walled cells and anomocytic
Time
stomata (2.8.3) ; conical unicellular covering trichomes,
Mobile phase A Mobile phase B
widened at the base and sharply pointed at the apex, with
(min) (per cent V/V) (per cent V/V)
a striated cuticle ; glandular trichomes with a multicellular
0 - 15 40 → 90 60 → 10
head, and a short, multicellular stalk in the indentations
15 - 20 90 → 40 10 → 60 of the leaf margins ; cluster crystals of calcium oxalate,
sometimes included in parenchyma ; fragments of the
20 - 25 40 60
corolla with wavy-walled epidermal cells, those from the
mid-region papillose and with some extended to form flask
Flow rate : 1.5 mL/min.
or bottle-shaped projections, those from the base of the
Detection : spectrophotometer at 217 nm. petals with covering trichomes up to about 300 μm long
Injection : 20 μL. with characteristic hump-like swellings along their length ;

1668 See the information section on general monographs (cover pages)