EUROPEAN PHARMACOPOEIA 10.

0 Agrimony

Top of the plate
Caffeic acid : a light blue fluorescent An orange fluorescent zone and a
zone white or pale yellow fluorescent
zone, partly superimposed
_______ _______
Several light blue fluorescent zones
1 or 2 yellow fluorescent zones
_______ _______
A light blue fluorescent zone
A1 = area of the peak due to casticin in the chromatogram
obtained with the test solution ;
A2 = area of the peak due to casticin in the chromatogram
obtained with the reference solution ;
m1 = mass of the extract to be examined used to prepare
the test solution, in grams ;
m2 = mass of agnus castus fruit dry extract HRS used to
prepare the reference solution, in grams ;
p = percentage content of casticin in agnus castus fruit
dry extract HRS.

Homoorientin : a yellow fluorescent A yellow fluorescent zone

zone (homoorientin)

01/2011:1587
A yellow fluorescent zone

Reference solution Test solution

ASSAY AGRIMONY
Liquid chromatography (2.2.29).
Agrimoniae herba
Test solution. Suspend 0.200 g of the extract to be examined
in 15 mL of methanol R, sonicate for 5 min and dilute to
DEFINITION
20.0 mL with the same solvent. Filter through a membrane
filter (nominal pore size 0.45 μm). Dried flowering tops of Agrimonia eupatoria L.

Reference solution. Suspend a quantity of agnus castus fruit dry Content : minimum 2.0 per cent of tannins, expressed as
extract HRS corresponding to 0.10 mg of casticin in 7.5 mL pyrogallol (C6H6O3 ; Mr 126.1) (dried drug).
of methanol R, sonicate for 5 min and dilute to 10.0 mL with
the same solvent. Filter through a membrane filter (nominal IDENTIFICATION
pore size 0.45 μm). A. The stem is green or, more usually, reddish, cylindrical
Column : and infrequently branched. It is covered with long, erect
or tangled hairs. The leaves are compound imparipennate
– size : l = 0.125 m, Ø = 4.0 mm ; with 3 or 6 opposite pairs of leaflets, with 2 or 3 smaller
leaflets between. The leaflets are deeply dentate to serrate,
– stationary phase : end-capped octadecylsilyl silica gel for dark green on the upper surface, greyish and densely
chromatography R (5 μm). tomentose on the lower face. The flowers are small and
form a terminal spike. They are pentamerous and borne
Mobile phase : in the axils of hairy bracts, the calyces closely surrounded
by numerous terminal hooked spires, which occur on the
– mobile phase A : 5.88 g/L solution of phosphoric acid R ; rim of the hairy receptacle. The petals are free, yellow and
– mobile phase B : acetonitrile R ; deciduous. Fruit-bearing obconical receptacles, with deep
furrows and hooked bristles, are usually present at the base
Time
of the inflorescence.
Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V) B. Microscopic examination (2.8.23). The powder is
0 - 30 70 → 45 30 → 55 yellowish-green or grey. Examine under a microscope using
chloral hydrate solution R. The powder shows the following
diagnostic characters (Figure 1587.-1): numerous straight
Flow rate : 1.0 mL/min.
or bent, unicellular, long, thick-walled (about 500 μm)
Detection : spectrophotometer at 348 nm. covering trichomes [Ab, Ca, F], finely warty, and sometimes
spirally marked, often fragmented [F] ; fragments of the
Injection : 10 μL. epidermis of the stems [A] with stomata [Aa], covering
trichomes [Ab] and glandular trichomes [Ac] ; fragments
Identification of peaks : use the chromatogram supplied with of upper leaf epidermis (surface view [C]) with straight
agnus castus fruit dry extract HRS and the chromatogram walls bearing covering trichomes [Ca], accompanied
obtained with the reference solution to identify the peaks due by palisade parenchyma [Cb], with some of the cells
to penduletin and casticin. containing calcium oxalate prisms [Cc] ; fragments of
lower leaf epidermis in surface view [J] with sinuous walls
System suitability : reference solution : and abundant stomata [Ja], mostly anomocytic (2.8.3) but
occasionally anisocytic, and glandular trichomes [Jb] ;
– resolution : minimum 1.5 between the peaks due to
ovoid to subspherical pollen grains, with 3 pores and a
penduletin and casticin.
smooth exine [D] ; glandular trichomes with a multicellular,
Calculate the percentage content of casticin using the uniseriate stalk and a unicellular to quadricellular head [B,
following expression : Jb] ; fragments of the stems [H] with groups of fibres [Ha]
and parenchymatous cells, some of which contain cluster
crystals of calcium oxalate [Hb] ; small spiral vessels from
A1 ´ m 2 ´ p ´ 2 the leaflets [G] ; fragments of large, spiral or bordered-pitted
A 2 ´ m1 vessels from the stem [E].

General Notices (1) apply to all monographs and other texts 1297
Akebia stem EUROPEAN PHARMACOPOEIA 10.0

Total ash (2.4.16) : maximum 10.0 per cent.

ASSAY
Tannins (2.8.14). Use 1.000 g of the powdered herbal drug
(180) (2.9.12).

01/2019:2472
corrected 10.0

AKEBIA STEM
Akebiae caulis
DEFINITION
Whole or fragmented, dried stem of Akebia quinata (Houtt.)
Decne. or Akebia trifoliata (Thunb.) Koidz., or a mixture of
the 2 species.
Content : minimum 0.15 per cent of oleanolic acid (C30H48O3 ;
Mr 456.7) (dried drug).
IDENTIFICATION
A. The whole drug is cylindrical, often slightly twisted,
30-70 cm long and 0.5-2 cm in diameter. Externally light
grey or greyish-brown, rough, with irregular longitudinal
and horizontal cracks and irregular longitudinal furrows.
The outer bark is sometimes easily removed ; the exposed
inner surface is yellowish-brown or reddish-brown.
Lenticels are distinct. Nodes are sometimes swollen,
Figure 1587.-1. – Illustration for identification test B of with scars of lateral branches. The texture is light and
powdered herbal drug of agrimony compact. The fracture is uneven ; the bark is relatively
thick and easily distinguished from the wood, sometimes
C. Thin-layer chromatography (2.2.27). with arched hollows around the outside of the wood ; it
Test solution. To 2.0 g of the powdered herbal drug (355) is yellowish-brown or reddish-brown, with pale yellow,
(2.9.12) add 20 mL of methanol R. Heat with shaking at granular dots visible ; the wood is yellowish-white, with
40 °C for 10 min. Filter. distinct radial striations, the pith is small, occasionally
Reference solution. Dissolve 1.0 mg of isoquercitroside R hollowed, yellowish-white or yellowish-brown.
and 1.0 mg of rutoside trihydrate R in 2 mL of methanol R. The fragmented drug occurs as rounded, elliptical or
Plate : TLC silica gel plate R. irregular slices, 0.5-2 cm in diameter. Externally light
grey or greyish-brown, rough, with irregular longitudinal
Mobile phase : anhydrous formic acid R, water R, ethyl and horizontal cracks and occasional lenticels ; the outer
acetate R (10:10:80 V/V/V). bark is easily removed, the inner exposed surface is
Application : 10 μL as bands. yellowish-brown or reddish-brown. The texture is light,
Development : over a path of 12 cm. compact and easily broken. The cut surface has a relatively
Drying : at 100-105 °C. thick, yellowish-brown, greyish-brown or reddish-brown
bark that is easily distinguished from the wood and appears
Detection : spray the still-warm plate with a 10 g/L solution granular and sometimes lamellar in transverse section.
of diphenylboric acid aminoethyl ester R in methanol R Hollows appearing as arches may be present around the
and then with a 50 g/L solution of macrogol 400 R in outside of the wood. The wood is yellowish-white, with
methanol R ; allow the plate to dry in air for 30 min and distinct radial striations and distinct xylem vessels. The
examine in ultraviolet light at 365 nm. pith is small, occasionally hollowed, yellowish-white or
Results : see below the sequence of zones present in the yellowish-brown.
chromatograms obtained with the reference solution and B. Microscopic examination (2.8.23). The powder is pale
the test solution. yellow to light brown. Examine under a microscope
Top of the plate using chloral hydrate solution R. The powder shows the
following diagnostic characters (Figure 2472.-1) : groups
An orange fluorescent zone may of fibres, frequently broken [C], whose narrow lumens
be present (quercitrin)
contain small prisms of calcium oxalate [Ca] ; these groups
Isoquercitroside : an orange An orange fluorescent zone
fluorescent zone (isoquercitroside)
are usually surrounded by crystal sheaths of calcium
oxalate [Cb] ; groups of parenchymatous cells with thick,
An orange fluorescent zone
(hyperoside) pitted walls [F], some containing compact masses of large
Rutoside : an orange fluorescent An orange fluorescent zone
polyhedral or prismatic crystals of calcium oxalate [Fa] ;
zone (rutoside) some of these cells are isolated [G] ; cork fragments
Reference solution Test solution consisting of reddish-brown polygonal cells with brown
contents (surface view [A]); fragments of parenchyma
consisting of cells with thin colourless walls (transverse
TESTS section [K], longitudinal section [L]) ; isolated cubic or
Loss on drying (2.2.32) : maximum 10.0 per cent, determined prismatic crystals of calcium oxalate [D] ; rare tetragonal,
on 1.000 g of the powdered herbal drug (355) (2.9.12) by polygonal, elongated or rounded sclereids with very thick
drying in an oven at 105 °C for 2 h. walls and granular contents [B, M] ; abundant single or

1298 See the information section on general monographs (cover pages)