Melissa leaf
01/2011:1447
MELISSA LEAF
Melissae folium
DEFINITION
Dried leaf of Melissa officinalis L.
Content : minimum 1.0 per cent of rosmarinic acid (C18H16O8 ;
Mr 360.3) (dried drug).
CHARACTERS
Odour reminiscent of lemon.
IDENTIFICATION
A. The leaves have a petiole of varying length ; the lamina is
broadly ovate, up to about 8 cm long and 5 cm wide, acute
at the apex and rounded to cordate at the base ; the margins
are crenate to dentate. The upper surface is intense green,
the lower surface is paler green and shows a conspicuous
midrib and a raised, reticulate venation ; scattered hairs
occur on the upper surface and along the veins on the lower
surface, which is also finely punctuate.
Figure 1447.-1.– Illustration for identification test B of
powdered herbal drug of melissa leaf
B. Microscopic examination (2.8.23). The powder is greenish. (50 per cent V/V) R. Boil in a water-bath under a reflux
Examine under a microscope using chloral hydrate
solution R. The powder shows the following diagnostic
characters (Figure 1447.-1) : fragments of the upper
epidermis, in surface view, with sinuous walls [A, B, G],
sometimes accompanied by palisade parenchyma [Aa] ;
fragments of the lower epidermis [D] with diacytic stomata acid CRS in ethanol (50 per cent V/V) R and dilute to 100.0 mL
(2.8.3) [Db] ; short, straight, unicellular, conical covering with the same solvent. Dilute 20.0 mL of this solution to
trichomes with a finely striated cuticle, free [E] or attached 100.0 mL with ethanol (50 per cent V/V) R.
to an epidermis [Da] ; multicellular, uniseriate covering
trichomes with pointed ends and thick, warty cuticles
[C] ; eight-celled secretory trichomes of lamiaceous type
1536
EUROPEAN PHARMACOPOEIA 10.
(surface view [Ga]) ; secretory trichomes with unicellular to
tricellular stalks and unicellular or, more rarely, bicellular
heads (surface view [Ba], transverse section [F]).
C. Thin-layer chromatography (2.2.27).
Test solution. Place 2.0 g of the powdered herbal drug
(355) (2.9.12) in a 250 mL round-bottomed flask and add
100 mL of water R. Distil for 1 h using the apparatus for the
determination of essential oils in herbal drugs (2.8.12) and
0.5 mL of xylene R in the graduated tube. After distillation
transfer the organic phase to a 1 mL volumetric flask,
rinsing the graduated tube of the apparatus with the aid of
a small portion of xylene R, and dilute to 1.0 mL with the
same solvent.
Reference solution. Dissolve 1.0 μL of citronellal R and
10.0 μL of citral R (composed of neral and geranial) in
25 mL of xylene R.
Plate : TLC silica gel plate R (5-40 μm) [or TLC silica gel
plate R (2-10 μm)].
Mobile phase : ethyl acetate R, hexane R (10:90 V/V).
Application : 20 μL [or 4 μL] as bands.
Development : in an unsaturated tank over a path of 15 cm
[or 6 cm].
Drying : in air.
Detection : spray with anisaldehyde solution R and heat at
100-105 °C for 10-15 min ; examine in daylight.
Results : see below the sequence of zones present in the
chromatograms obtained with the reference solution and
the test solution. Furthermore, other zones may be present
in the chromatogram obtained with the test solution.
Top of the plate
_______ _______
Citronellal : a grey or A grey or greyish-violet zone
greyish-violet zone at the border (citronellal) at the border between
between the upper and middle the upper and middle thirds
thirds
A reddish-violet zone
_______ _______
Citral : 2 greyish-violet or 2 greyish-violet or bluish-violet
bluish-violet zones at the border zones (citral) at the border
between the middle and lower between the middle and lower
thirds thirds
Reference solution Test solution
TESTS
Foreign matter (2.8.2) : maximum 10 per cent of stems with a
diameter greater than 1 mm and maximum 2 per cent of other
foreign matter, determined on 20 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.
ASSAY
Liquid chromatography (2.2.29).
Test solution. Use brown-glass flasks. Disperse 0.100 g of
the powdered herbal drug (355) (2.9.12) in 90 mL of ethanol
condenser for 30 min, cool, and filter into a 100 mL volumetric
flask. Rinse the flask and the filter with 10 mL of ethanol
(50 per cent V/V) R and dilute to 100.0 mL with the same
solvent. Filter through a 0.45 μm filter.
Reference solution (a). Dissolve 20.0 mg of rosmarinic
Reference solution (b). Dissolve 5.0 mg of ferulic acid R in
reference solution (a) and dilute to 50.0 mL with the same
solution.
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 10.0 Melissa leaf dry extract

Column : Test solution. To 0.2 g of the extract to be examined add 5 mL

– size : l = 0.25 m, Ø = 4.6 mm ; of methanol R. Sonicate for 5 min and filter.
– stationary phase : octadecylsilyl silica gel for Reference solution. Dissolve 1.0 mg of hyperoside R, 1.0 mg
chromatography R (5 μm). of rutoside trihydrate R and 5.0 mg of rosmarinic acid R in
Mobile phase : 10 mL of methanol R.
– mobile phase A : phosphoric acid R, acetonitrile R, water R Plate : TLC silica gel plate R (5-40 μm) [or TLC silica gel plate R
(1:19:80 V/V/V) ; (2-10 μm)].
– mobile phase B : phosphoric acid R, methanol R, acetonitrile R Mobile phase : anhydrous formic acid R, water R, ethyl acetate R
(1:40:59 V/V/V) ; (6:6:90 V/V/V).
Application : 10 μL [or 2 μL] as bands of 15 mm [or 8 mm].
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V) Development : over a path of 8 cm [or 6 cm].
0 - 20 100 → 55 0 → 45 Drying : in air.
20 - 25 55 → 0 45 → 100 Detection : heat at 100 °C for 5 min, spray the plate whilst still
hot with a 5 g/L solution of diphenylboric acid aminoethyl
25 - 30 0 → 100 100 → 0 ester R in ethyl acetate R, and examine in ultraviolet light at
365 nm.
Flow rate : 1.2 mL/min.
Results : see below the sequence of fluorescent zones present
Detection : spectrophotometer at 330 nm. in the chromatograms obtained with the reference solution
Injection : 20 μL. and the test solution. Furthermore, other weaker fluorescent
Relative retention with reference to rosmarinic acid (retention zones may be present in the chromatogram obtained with the
time = about 11 min) : ferulic acid = about 0.8. test solution.
System suitability : reference solution (b) : Top of the plate
– resolution : minimum 4.0 between the peaks due to ferulic
acid and rosmarinic acid.
Calculate the percentage content of rosmarinic acid using the Rosmarinic acid : a light blue An intense light blue fluorescent
fluorescent zone zone (rosmarinic acid)
following expression :
A blue fluorescent zone
A1 ´ m 2 ´ p ´ 0.2 _______ _______
A 2 ´ m1
A blue fluorescent zone
A1 = area of the peak due to rosmarinic acid in the _______ _______
chromatogram obtained with the test solution ;
Hyperoside : an orange or
A2 = area of the peak due to rosmarinic acid in greenish-yellow fluorescent
the chromatogram obtained with reference zone
solution (a); A light blue fluorescent zone
m1 = mass of the herbal drug to be examined used to Rutoside : an orange or
prepare the test solution, in grams ; greenish-yellow fluorescent
m2 = mass of rosmarinic acid CRS used to prepare zone
reference solution (a), in grams ; Reference solution Test solution
p = percentage content of rosmarinic acid in rosmarinic
acid CRS. TESTS
Loss on drying (2.8.17) : maximum 6.0 per cent.

01/2010:2524 ASSAY
Liquid chromatography (2.2.29).
Test solution. Use brown glass flasks. To 0.200 g of the extract
to be examined add 50 mL of ethanol (50 per cent V/V) R.
Sonicate for 10 min and dilute to 100.0 mL with ethanol
MELISSA LEAF DRY EXTRACT (50 per cent V/V) R. Filter through a membrane filter (nominal
pore size 0.45 μm).
Melissae folii extractum siccum Reference solution (a). Dissolve 20.0 mg of rosmarinic
acid CRS in ethanol (50 per cent V/V) R and dilute to 100.0 mL
DEFINITION with the same solvent. Dilute 20.0 mL of this solution to
Dry extract produced from Melissa leaf (1447). 100.0 mL with ethanol (50 per cent V/V) R.
Content : minimum 2.0 per cent of rosmarinic acid (C18H16O8 ; Reference solution (b). Dissolve 5 mg of ferulic acid R in
Mr 360.3) (dried extract). reference solution (a) and dilute to 50 mL with reference
solution (a).
PRODUCTION
Column :
The extract is produced from the herbal drug by a suitable
procedure using either hot water (not less than 70 °C) or a – size : l = 0.25 m, Ø = 4.6 mm ;
hydroalcoholic solvent that is at most equivalent in strength – stationary phase : octadecylsilyl silica gel for
to ethanol (70 per cent V/V). chromatography R (5 μm).
CHARACTERS Mobile phase :
Appearance : brown or greenish-brown, amorphous powder. – mobile phase A : phosphoric acid R, acetonitrile R, water R
(1:19:80 V/V/V) ;
IDENTIFICATION – mobile phase B : phosphoric acid R, methanol R, acetonitrile R
Thin-layer chromatography (2.2.27). (1:40:59 V/V/V) ;

General Notices (1) apply to all monographs and other texts 1537