EUROPEAN PHARMACOPOEIA 10.

0 Elder flower

section [F]) containing secretory canals with orange-brown Reference solution (b). Dissolve 2 mg of ethyl
contents [Fa] ; bundles of fibres with thickened walls [E] ; parahydroxybenzoate R in reference solution (a) and
pollen grains [G] about 20-25 μm in diameter, with 3 pores dilute to 50 mL with reference solution (a).
and a spiny exine. Column :
C. Thin-layer chromatography (2.2.27). – size : l = 0.15 m, Ø = 4.6 mm ;
Test solution. To 0.5 g of the powdered herbal drug (355) – stationary phase : end-capped octadecylsilyl silica gel for
(2.9.12) add 5 mL of methanol R and sonicate at 60 °C for chromatography R (5 μm).
10 min. Allow to cool, centrifuge and use the supernatant. Mobile phase : acetonitrile R, 0.2 per cent V/V solution of
Reference solution. Dissolve 1 mg of wedelolactone R and phosphoric acid R (24:76 V/V).
1 mg of rosmarinic acid R in 1 mL of methanol R. Flow rate : 1.0 mL/min.
Plate : TLC silica gel F254 plate R (2-10 μm). Detection : spectrophotometer at 249 nm.
Mobile phase : anhydrous formic acid R, acetone R, toluene R Injection : 20 μL.
(1:6:11 V/V/V). Run time : 1.5 times the retention time of wedelolactone.
Application : 10 μL as bands of 8 mm. Relative retention with reference to wedelolactone (retention
Development : over a path of 6 cm. time = about 17 min) : ethyl parahydroxybenzoate = about 1.1.
Drying : in air. System suitability : reference solution (b) :
Detection : heat at 100 °C for 5 min and treat the plate whilst – resolution : minimum 2.0 between the peaks due to
still hot with a 0.5 per cent V/V solution of diphenylboric wedelolactone and ethyl parahydroxybenzoate.
acid aminoethyl ester R in ethyl acetate R ; examine in Calculate the percentage content of wedelolactone using the
ultraviolet light at 365 nm. following expression :
Results : see below the sequence of zones present in the
A1 ´ m 2 ´ p
chromatograms obtained with the reference solution and
the test solution. Furthermore, other faint fluorescent A 2 ´ m1 ´ 10
zones may be present in the chromatogram obtained with
the test solution. A1 = area of the peak due to wedelolactone in the
chromatogram obtained with the test solution ;
Top of the plate
A2 = area of the peak due to wedelolactone in
A red fluorescent zone the chromatogram obtained with reference
A diffuse pale blue fluorescent
solution (a) ;
zone m1 = mass of the herbal drug to be examined used to
A greenish-white fluorescent zone prepare the test solution, in grams ;
_______ _______ m2 = mass of wedelolactone CRS used to prepare
reference solution (a), in grams ;
A pale blue fluorescent zone
p = percentage content of wedelolactone in
wedelolactone CRS.

01/2013:1217
Wedelolactone : a bluish-white A bluish-white fluorescent zone
fluorescent zone (wedelolactone)
A bluish-white fluorescent zone

_______ _______ ELDER FLOWER

Sambuci flos
Rosmarinic acid : a bluish-white
fluorescent zone DEFINITION
A bluish-white fluorescent zone Dried flowers of Sambucus nigra L.
Reference solution Test solution Content : minimum 0.80 per cent of flavonoids, expressed as
isoquercitroside (C21H20O12 ; Mr 464.4) (dried drug).
TESTS IDENTIFICATION
Loss on drying (2.2.32) : maximum 11.0 per cent, determined A. The flower, about 5 mm in diameter, has 3 small bracts,
on 1.000 g of the powdered herbal drug (355) (2.9.12) by visible under a lens, and may have a peduncle. The
drying in an oven at 105 °C for 2 h. 5-toothed calyx is small ; the corolla is light yellow, with 5
Total ash (2.4.16) : maximum 13.0 per cent. broadly oval petals fused at their bases into a tube. The
filaments of the 5 yellow stamens alternate with the petals.
Ash insoluble in hydrochloric acid (2.8.1): maximum 2.0 per The corolla is often isolated or attached to the stamens, to
cent. which it is fused at the base. The ovary is inferior and it
bears a short style with 3 obtuse stigmata.
ASSAY
B. Microscopic examination (2.8.23). The powder is
Liquid chromatography (2.2.29). greenish-yellow. Examine under a microscope using
Test solution. Disperse 0.300 g of the powdered herbal drug chloral hydrate solution R. The powder shows the following
(355) (2.9.12) in 10 mL of ethanol (70 per cent V/V) R in a diagnostic characters (Figure 1217.-1) : numerous spherical,
conical flask and weigh. Heat under a reflux condenser for sometimes ellipsoidal, pollen grains about 30 μm in
1 h, cool and weigh again. Compensate the loss of solvent diameter, with 3 germinal pores and very finely pitted
with ethanol (70 per cent V/V) R, mix well and allow to stand. exine [G] ; cells of the lower epidermis of the sepals often
Filter through a membrane filter (nominal pore size 0.22 μm). containing oil globules and covered by a striated cuticle
Reference solution (a). Dissolve 4.0 mg of wedelolactone CRS (surface view [A]) ; rare fragments of the rim of the sepals
in methanol R and dilute to 100.0 mL with the same solvent. showing unicellular marginal teeth (transverse section [E]) ;

General Notices (1) apply to all monographs and other texts 1421
Elder flower EUROPEAN PHARMACOPOEIA 10.0

petal fragments with numerous small globules of essential Top of the plate
oil [H] ; fragments of upper epidermis of the sepals [B]
or petals [F], in surface view, with slightly and irregularly
thickened walls [Ba, Fa], anomocytic stomata (2.8.3) [Bb, _______ _______
Fb] and a striated cuticle ; mesophyll cells of petals and An orange zone
sepals with idioblasts containing numerous microsphenoid
crystals of calcium oxalate [Bc] ; fragments of anthers Hyperoside : a dark yellow zone
(transverse section [C], surface view [D]) showing the outer
layer [Ca] and the cells of the fibrous layer [Cb, Cc, D].
_______ _______

Rutoside : a dark yellow zone A dark yellow zone

Reference solution Test solution

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

chromatograms obtained with the reference solution and
the test solution. Furthermore, other faint zones may
be present in the chromatogram obtained with the test
solution.
Top of the plate
Caffeic acid : a blue fluorescent
zone
An intense, light blue fluorescent
zone
2 light blue fluorescent zones
_______ _______

An orange fluorescent zone

Hyperoside : an orange fluorescent

zone
Chlorogenic acid : a light blue An intense, light blue fluorescent
fluorescent zone zone

_______ _______

Rutoside : an orange fluorescent An orange fluorescent zone

zone

Figure 1217.-1. – Illustration for identification B of powdered

herbal drug of elder flower Reference solution Test solution

C. Thin-layer chromatography (2.2.27).

TESTS
Test solution. To 0.5 g of the powdered herbal drug (355)
(2.9.12) add 5 mL of methanol R and sonicate for 10 min. Foreign matter (2.8.2): maximum 8 per cent of fragments
Centrifuge for 5 min. of coarse pedicels and other foreign matter and maximum
15 per cent of discoloured, brown flowers. Carry out the
Reference solution. Dissolve 1 mg of caffeic acid R, 1 mg of determination on 10 g.
chlorogenic acid R, 2.5 mg of hyperoside R and 2.5 mg of
rutoside trihydrate R in 10 mL of methanol R. Sambucus ebulus L. Examine the chromatograms obtained in
identification C.
Plate : TLC silica gel plate R (2-10 μm).
Results B : the chromatogram obtained with the test solution
Mobile phase : anhydrous formic acid R, water R, methyl does not show a greenish-white zone above the zone due
ethyl ketone R, ethyl acetate R (10:10:30:50 V/V/V/V). to caffeic acid in the chromatogram obtained with the
Application : 4 μL as bands of 8 mm. reference solution ; in the chromatogram obtained with the
test solution, no green fluorescent zone is seen just below the
Development : over a path of 6 cm. orange fluorescent zone due to rutoside in the chromatogram
Drying : in air. obtained with the reference solution.
Detection : heat the plate for 5 min at 100 °C and treat with Loss on drying (2.2.32) : maximum 10.0 per cent, determined
a 1 g/L solution of diphenylboric acid aminoethyl ester R in on 1.000 g of the powdered herbal drug (355) (2.9.12) by
ethyl acetate R, then treat with a 5 g/L solution of macrogol drying in an oven at 105 °C for 2 h.
400 R in methylene chloride R ; allow to dry in air for Total ash (2.4.16) : maximum 10.0 per cent.
30 min. Examine in daylight (results A) and in ultraviolet
light at 365 nm (results B). ASSAY
Results A : see below the sequence of zones present in the Stock solution. In a 100 mL round-bottomed flask, introduce
chromatograms obtained with the reference solution and 0.600 g of the powdered herbal drug (355) (2.9.12), add 1 mL
the test solution. Furthermore, other faint zones may of a 5 g/L solution of hexamethylenetetramine R, 20 mL of
be present in the chromatogram obtained with the test acetone R and 2 mL of hydrochloric acid R1. Boil the mixture
solution. under a reflux condenser for 30 min. Filter the mixture

1422 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 10.0 Eleutherococcus

through a plug of absorbent cotton into a flask. Add the reticulate and bordered pitted vessels with a wide lumen ;
absorbent cotton to the residue in the round-bottomed flask groups of secretory canals, up to 20 μm in diameter with
and extract with 2 quantities, each of 20 mL, of acetone R, each brown contents ; parenchymatous cells containing cluster
time boiling under a reflux condenser for 10 min. Allow to crystals of calcium oxalate 10 μm to 50 μm in diameter.
cool, filter each extract through the plug of absorbent cotton Examine under a microscope, using a 50 per cent V/V
into the flask. After cooling, filter the combined acetone solution of glycerol R. The powder shows small starch
extracts through a filter paper into a volumetric flask and granules, rounded to slightly angular in outline, single
dilute to 100.0 mL with acetone R by rinsing the flask and compounds or with 2 or 3 components.
the filter paper. Introduce 20.0 mL of this solution into a C. Thin-layer chromatography (2.2.27).
separating funnel, add 20 mL of water R and shake the mixture Test solution. To 1.0 g of the powdered herbal drug (355)
with 1 quantity of 15 mL and then 3 quantities, each of 10 mL, (2.9.12) add 10 mL of alcohol (50 per cent V/V) R and boil
of ethyl acetate R. Combine the ethyl acetate extracts in a under reflux for 1 h. Cool and filter. Evaporate the filtrate
separating funnel, wash with 2 quantities, each of 50 mL, of to dryness on a water-bath. Dissolve the residue in 2.5 mL
water R, and filter the extracts over 10 g of anhydrous sodium of a mixture of 5 volumes of water R and 20 volumes of
sulfate R into a volumetric flask and dilute to 50.0 mL with alcohol (50 per cent V/V) R and filter.
ethyl acetate R.
Reference solution. Dissolve 2.0 mg of esculin R and 2.0 mg
Test solution. To 10.0 mL of the stock solution add 1 mL of of catalpol R in 20 mL of a mixture of 2 volumes of water R
aluminium chloride reagent R and dilute to 25.0 mL with a and 8 volumes of alcohol (50 per cent V/V) R.
5 per cent V/V solution of glacial acetic acid R in methanol R.
Plate : TLC silica gel plate R.
Compensation liquid. Dilute 10.0 mL of the stock solution to
25.0 mL with a 5 per cent V/V solution of glacial acetic acid R Mobile phase : water R, methanol R, methylene chloride R
in methanol R. (4:30:70 V/V/V).
After 30 min, measure the absorbance (2.2.25) of the test Application : 20 μL, as bands.
solution at 425 nm, by comparison with the compensation Development : over a path of 10 cm.
liquid. Drying : in air.
Calculate the percentage content of flavonoids, expressed as Detection A : examine in ultraviolet light at 365 nm.
isoquercitroside, using the following expression : Results A : the chromatogram obtained with the reference
A ´ 1.25 solution shows in the upper half a blue fluorescent zone
(esculin).
m
i.e. taking the specific absorbance of isoquercitroside to Detection B : spray with anisaldehyde solution R and
be 500. examine in daylight while heating at 100-105 °C for
5-10 min.
A = absorbance at 425 nm ; Results B : see below the sequence of the zones present in the
m = mass of the herbal drug to be examined, in grams. chromatograms obtained with the reference solution and
the test solution. Furthermore, other faint zones are present
in the chromatogram obtained with the test solution.
01/2008:1419
corrected 7.0
Top of the plate
A brown zone (eleutheroside B)

Esculin : a blue fluorescent zone

(marked at 365 nm)
ELEUTHEROCOCCUS A reddish-brown zone
(eleutheroside E)
_______ _______
Eleutherococci radix
Catalpol : a violet-brown zone
DEFINITION
Dried, whole or cut underground organs of Eleutherococcus _______ _______
senticosus (Rupr. et Maxim.) Maxim. 2 brown zones
Content : minimum 0.08 per cent for the sum of eleutheroside B
(Mr 372.4) and eleutheroside E (Mr 742.7). Reference solution Test solution

IDENTIFICATION
A. The rhizome is knotty, of irregular cylindrical shape, 1.5 cm TESTS
to 4.0 cm in diameter ; the surface is rugged, longitudinally Foreign matter (2.8.2) : maximum 3 per cent.
wrinkled and greyish-brown to blackish-brown ; the
bark, about 2 mm thick, closely adheres to the xylem ; Loss on drying (2.2.32) : maximum 10.0 per cent, determined
the heartwood is light brown and the sapwood is pale on 1.000 g of the powdered herbal drug (355) (2.9.12) by
yellow ; the fracture shows short thin fibres in the bark and drying in an oven at 105 °C for 2 h.
is coarsely fibrous, especially in the internal part of the Total ash (2.4.16) : maximum 8.0 per cent.
xylem. The lower surface bears numerous cylindrical and
knotty roots, 3.5 cm to 15 cm long and 0.3 cm to 1.5 cm in ASSAY
diameter ; with a smooth, greyish-brown to blackish-brown Liquid chromatography (2.2.29).
surface ; the bark is about 0.5 mm thick, closely adhering Test solution. To 0.500 g of the powdered herbal drug (355)
to the pale yellow xylem ; the fracture is slightly fibrous ; in (2.9.12) in a 100 mL round-bottomed flask, add 30 mL of
places where the outer layer has been removed, the outer a mixture of equal volumes of alcohol R and water R. Heat
surface is yellowish-brown. in a water-bath at 60 °C for 30 min. Allow to cool and filter
B. Microscopic examination (2.8.23). The powder is through a sintered-glass filter (2.1.2). Collect the liquid in a
yellowish-brown. Examine under a microscope, using 250 mL round-bottomed flask. Repeat this operation twice,
chloral hydrate solution R. The powder shows numerous using the residue obtained in the filtration step instead of the
groups of thick-walled, lignified fibres ; fragments of powdered herbal drug. Add both fractions of supernatant

General Notices (1) apply to all monographs and other texts 1423