Hop strobile EUROPEAN PHARMACOPOEIA 10.

Results : see below the sequence of zones present in the CHARACTERS

chromatograms obtained with the reference solution and the
test solution. Furthermore, other fluorescent zones may be Characteristic, aromatic odour.
present in the chromatogram obtained with the test solution.
Top of the plate
_______ _______ IDENTIFICATION
A yellowish-green fluorescent zone A. Hop strobiles are generally isolated and 2-5 cm long,
Hyperoside : a yellowish-orange A yellowish-orange fluorescent petiolate, ovoid, made up of many oval, greenish-yellow,
fluorescent zone zone (hyperoside) sessile, membranous, overlapping bracts. The external
Chlorogenic acid : a light blue A light blue fluorescent zone bracts are flattened and symmetrical. The internal bracts
fluorescent zone (chlorogenic acid) are longer and asymmetrical at the base because of a fold
A yellowish-green fluorescent zone generally encircling an induviate fruit (achene). The ovary
_______ _______ or rarely the fruit, the base of the bracts and especially the
induvial fold, are covered with small orange-yellow glands.
Reference solution Test solution

TESTS
Ethanol (2.9.10) : 95 per cent V/V to 105 per cent V/V of the
quantity stated on the label.
ASSAY
Stock solution. Dilute about 0.400 g, accurately weighed, in
ethanol (60 per cent V/V) R and dilute to 100.0 mL with the
same solvent.
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 25 mL volumetric flask. 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 25 mL volumetric flask. Add 10.0 mL of
anhydrous formic acid R and dilute to 25.0 mL with anhydrous
acetic acid R.
After 30 min measure the absorbance (2.2.25) of the test
solution at 410 nm.
Calculate the percentage content of total flavonoids, expressed
as hyperoside, from the following expression :
A ´ 1.235
m
i.e. taking the value of the specific absorbance of hyperoside
to be 405. Figure 1222.-1. – Illustration for identification test B of
powdered herbal drug of hop strobile
A = absorbance at 410 nm,
m = mass of the extract to be examined, in grams. B. Microscopic examination (2.8.23). The powder is
greenish-yellow. Examine under a microscope using
chloral hydrate solution R. The powder shows the following
01/2011:1222 diagnostic characters (Figure 1222.-1) : fragments of
bracts and bracteoles covered by polygonal, irregular or
wavy-walled epidermal cells [D, L, M] ; unicellular, conical,
straight or curved covering trichomes with thin, smooth
walls, fragmented [E, G] or attached to an epidermis [A] ;
HOP STROBILE rare anomocytic stomata (2.8.3) [K] ; glandular trichomes,
usually free, with bicellular biseriate stalks and heads
Lupuli flos consisting of 8 small cells [H, N], rarely attached to an
epidermis [La] ; fragments of mesophyll containing small
DEFINITION calcium oxalate cluster crystals [J] ; many characteristic
Dried, generally whole, female inflorescence of Humulus orange-yellow glandular trichomes with short, bicellular
lupulus L. biseriate stalks, bearing a part widening into a cup,

1474 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 10.0
150-250 μm in diameter, made up of a hemispherical layer
of secretory cells with a cuticle that has been detached and
distended by the accumulation of oleoresinous secretions
(surface view [B], side view [C]) ; fragments of elongated
sclerenchymatous cells of the testa with thick walls showing
striations and numerous pits [F].
C. Thin-layer chromatography (2.2.27).
Test solution. To 1.0 g of the freshly powdered herbal drug
(355) (2.9.12) add 10 mL of a mixture of 3 volumes of
water R and 7 volumes of methanol R ; shake for 15 min
and filter.
Reference solution. Dissolve 1.0 mg of Sudan
orange R, 2.0 mg of curcumin R and 2.0 mg of
dimethylaminobenzaldehyde R in 20 mL of methanol R.
Plate : TLC silica gel F254 plate R.
Mobile phase : anhydrous acetic acid R, ethyl acetate R,
cyclohexane R (2:38:60 V/V/V).
Application : 20 μL as bands.
Development : over a path of 15 cm.
Drying : in air.
Detection A : examine in ultraviolet light at 254 nm.
Results A : the chromatogram obtained with the reference
solution shows 3 quenching zones ; in the lower quarter
is the faint zone due to curcumin, somewhat below the
middle is the zone due to dimethylaminobenzaldehyde, and
above, the zone due to Sudan orange. The chromatogram
obtained with the test solution shows a number of
quenching zones similar in position to the zones in the
chromatogram obtained with the reference solution : at
about the level of the zone due to curcumin is a faint zone
due to xanthohumol, near the level of the zone due to
dimethylaminobenzaldehyde are zones due to humulones,
and near the level of the zone due to Sudan orange are
zones due to lupulones.
Detection B : examine in ultraviolet light at 365 nm.
Results B : in the chromatogram obtained with the test
solution the zones due to lupulones show blue fluorescence,
the zones due to humulones show brown fluorescence
and the zone due to xanthohumol shows dark brown
fluorescence.
Detection C : spray with dilute phosphomolybdotungstic
reagent R ; expose to ammonia vapour and examine in
daylight.
Results C : in the chromatogram obtained with the test
solution the zones due to humulones and to lupulones
are bluish-grey and the zone due to xanthohumol is
greenish-grey ; in the chromatogram obtained with
the reference solution the zones are bluish-grey or
brownish-grey.
TESTS
Matter extractable by ethanol (70 per cent V/V) : minimum
25.0 per cent.
To 10.0 g of the powdered herbal drug (355) (2.9.12) add
300 mL of ethanol (70 per cent V/V) R and heat for 10 min on
General Notices (1) apply to all monographs and other texts
Horse-chestnut
a water-bath under a reflux condenser. Allow to cool, filter,
and discard the first 10 mL of the filtrate. Evaporate 30.0 mL
of the filtrate to dryness on a water-bath and dry in an oven at
100-105 °C for 2 h. The residue weighs a minimum of 0.250 g.
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.
01/2017:1830
HORSE-CHESTNUT
Hippocastani semen
DEFINITION
Whole or fragmented, dried, ripe seeds of Aesculus
hippocastanum L.
Content : minimum 1.5 per cent of triterpene glycosides,
expressed as protoaescigenin (C30H50O6 ; Mr 506.7) (dried
drug).
IDENTIFICATION
A. The whole, spherical or oval, slightly flattened seed is
2-4 cm in diameter. It has a shiny, dark-brown testa with a
broad, round, matt, light-brown spot (hilum) ; particularly
on larger seeds, a short, narrow v-shaped ridge marks the
position of the radicle, with the point terminating close
to the hilum.
The fragmented seed occurs as more or less polyhedral
pieces, about 1-2 cm in diameter, or as slices. The surfaces
corresponding to the cotyledons are matt, light brown, with
a clean fracture. Those corresponding to the testa are shiny,
dark brown, except at the hilum, where they are matt, light
brown. The testa is weakly bound to the cotyledons and is
often detached.
B. Microscopic examination (2.8.23). The powder is
yellowish-brown. Examine under a microscope using
chloral hydrate solution R. The powder shows the following
diagnostic characters (Figure 1830.-1): numerous
different-sized droplets of fatty oil that are either free [D] or
inside the thin-walled colourless cells of the cotyledons [E,
G] ; yellowish-brown fragments of the outer testa consisting
of sclerenchymatous cells with thick walls (surface view
[C], transverse section [A]) ; fragments from the inner
testa consisting of thick-walled, colourless parenchymatous
cells varying in shape [B, H, J] with poorly visible pits and
occasional annular or spiral vessels [F]. Examine under a
microscope using a 50 per cent V/V solution of glycerol R.
Starch [K] is present in 3 forms : pyriform or reniform
simple granules, often with verruciform excrescences,
about 15-25 μm in size, sometimes up to 30 μm ;
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