CRUDE DRUGS CONTAINING QUINOLINE,

ISOQUINOLINE AND MORPHINAN ALKALOIDS

Content
1.

MACROMORPHOLOGICAL EVAULATION
Cinchonae cortex
Ipecacuanhae radix
Chelidonii herba
Papaveris maturi fructus
Opium crudum
Boldi folium
Fumariae herba

2.

MICROSCOPICAL TESTS
Cross section:

Cinchonae cortex
Ipecacuanhae radix

Powder preparation: Cinchonae cortex

3.

PHYSICO-CHEMICAL AND CHEMICAL TESTS

3.1.

General alkaloid reactions

(Cinchonae cortex)

3.2
3.2.l

Specific alkaloid reactions

Grahe test
(Cinchonae cortex)
3.2.2 Emetine test
(Ipecacuanhae radix)
3.2.3. Marquis test
(Papaveris maturi fructus)
3.2.4. Meconic acid test
(Papaveris maturi fructus)
4.
4.1.

QUANTITATIVE DETERMNATIONS
Determination of alkaloid content in Cinchonae cortex (Ph.Eur.)

4.2

Determination of morphine in ripped capsules of poppy by TLCdensitometry

Quantitative estimation of alkaloids in Chelidonii herba (Ph.Eur.)

4.3.

1.

MACROMORPHOLOGICAL EVALUATION
Cinchonae cortex
Cinchona pubescens Vahl.
(Cinchona succirubra Pavon)
C. calisaya (Weddell)
C. ledgeriana (Moens ex Trimen)

Cinchona bark
Rubiaceae

Ph. Eur., Ph.Hg.VIII.

The bark generally coils to a tube. The
drug consists of pieces 2 to 8 mm thick
and 30 to 40 cm long. The suber is
grayish or grayish brown, roughly
wrinkled lengthwise and often cracked
transversally. Sporadically covered
with silvery grey lichen. The bark is
reddish brown inside and finely striated
lengthwise. The fracture of its internal
part is short fibrous, the external part is
even.

Ipecacuanhae radix
Cephaelis ipecacuanha (Brot.) A.Rich.
Cephaelis acuminata Karsten

Ipecacuanha root
Rubiaceae

Ph. Eur., Ph.Hg.VIII.

Karlten
About 20 cm in length, the root is
marketed in pieces not longer than 3 to 6
cm and not thicker than 5 mm. The roots
are tortuous, seldom ramified. Grey to
grayish brown bark, hard as horn, easily
exfoliating from the cylindrical greenish
white xylem. Unevenly developed and
sometimes thickened, the bark surrounds
the
wood
concentrically
or
semicircularly. Therefore the drug seems
to be knotty or segmented as a worm;
besides it is wrinkled lengthwise.
Transversally cut at the thickened part of
the bark, the root is covered by bark 2 to
3 times as wide as the xylem. Short
cylindrical rhizome, about 2 mm in
diameter,
somewhat
wrinkled
lengthwise, the xylem represents 1/6 of
the diameter. Smooth fracture; dusting
when broken.

Chelidonii herba
Chelidonium majus L.

Greater Celandine
Papaveraceae

Ph.Eur., Ph.Hg.VIII.
A perennial plant with a branched woody
rootstock. The stems are rather fragile, branched,
20-60 cm tall, with scattered hairs, and freshly
contain an orange latex.The leaves are deeply
pinnatisect, with up to 7 oblong or ovate leaflets,
bluish-green beneath, the margins crenate-dentate.
The flowers are up to 2.5 cm in diameter terminal.
The sepals number 2, usually hairy. The petals
number 4, broad-obovate, bright yellow. The
staments are numerous, yellow, with thickened
filaments. The fruit (a capsule) is linear in outline,
1-celled, up to 5 cm long. The seeds are black with
a white appendage.

Papaveris mature fructus

Papaver somniferum L.

Poppy capsules, Poppy heads

Papaveraceae

Ph.Eur., Ph.Hg.VIII.
The capsules very much in size and shape, being
ovoid or globular or depressed at the apex and
base. They are about 3 to 4 cm in diameter, but the
depressed ones are often as much as 8 cm across.
Thay are glabrous, pale yellowish brown and often
marked with darker spots. At the base is a short
piece of peduncle and the thalamus which appears
as a slight swelling on the stalk and is marked with
scars left by the fall of the perianth parts. At the
apex is a persistent sessile radiate stigma with
about eight to sixteen rays.
The capsule is unilocular with eight to fifteen parietal plancentae which extend
towards the centre of the loculus in the form of thin plates, to the surface of
which the numerous small seeds are attached. The capsule dehisces by pores just
beneath the stigma; there is one pore to each carpel. The pericarp is odourless,
but has a bitter taste. The seeds are white to slate-grey the darker coloured
seeds are known as maw seed sub-reniform and about 1 to 1.25 mm long. The
surface is covered with polygonal reticulations about nine in the length and five
in the width of the seed; the hilum and micropyle are situated in the slight
depression near the smaller end. The embryo is curved and is embedded in an
abundant oil endosperm. They are inodorous and the taste is oily.

Opium crudum
Papaver somniferum L.

Opium, Raw
Papaveraceae

Ph.Eur., Ph.Hg.VIII.
More or less rounded or conical, somewhat
flattened, masses weighing usually between 250
and 1000 g; soft when fresh becoming hard and
brittle, especially near the surface, on keeping;
internally brown or dark brown and nearly
smooth or somewhat granular.

Boldi folium
Peumus boldus Molina

Boldo leaf
Monimiaceae

Ph. Eur., Ph.Hg.VIII.

Leaves ovate or elliptical, 4 to 8 cm long,
shortly
petiolate,
grayish-green,
coriaceous, brittle; margin entire, slightly
revolute; both surfaces with numerous
emergences each crowned with a group of
one-celled, thick-walled hairs (easily
broken off); in the mesophyll numerous
oil-cells; aromatic odour; pungent,
camphoraceous, bitter taste.

Fumariae herba
Fumaria officinalis L.

Fumitory
Fumariaceae

Ph. Eur., Ph.Hg.VIII.

2.

MICROSCOPICAL TESTS

Chinae succirubrae cortex (cross section)

On the periphery of the cross section a suber layer of thin walls, filled here and there
with brownish red colouring substance. They primary cortex consists of wide reddish or
reddish yellow thin-walled parenchyma. These cells contain round small starch
granules, 8 to 10 m in diameter, and tannin turning brownish green from a drop of Riron (III) chloride solution. Some parenchymatous cells contain calcium oxalate crystal
sand. At the internal border of the primary cortex there is a disconnected row of round
or elliptic latex tubes, whose major diameter is tangential. The phloem contains 1 to 3
cell rows of alternating medullary rays and bast rays. In the latter conspicuous bast
fibres by ones, twos or threes, scattered, or lying in radial rows interrupted by the
parenchyma. The bast fibres are thick-walled, golden yellow in colour. In longitudinal
section the fibres are spindle shaped, 0.5 to 1.3 mm long, 0.5 to 0.7 mm thick, with slitlike pits.
Chinae cortex pulvis
They powder is reddish brown. The microscope shows short, spindle shaped, yellow
shining bast fibres or their fragments. The fibre walls are penetrated by thin ducts,
widening in ward in a funnel-like way. In the powder, reddish brown parenchyma
fractions also occur containing some starch, as sporadically calcium oxalate sand.
Ipecacuanhae radix (cross section)
The root is covered by a cork layer consisting of tangentially elongated thin-walled
radially arranged cells. Paracambium consists of a single cell layer; no phelloderm or
consisting of a few cell rows of parenchyma. Extensive cortical tissue with large thinwalled and irregularly arranged parenchymatous cells in the external layer. Further
inside they are smaller and radially arranged. Most of the cells contain a starch grain of
24 um size, composed often of 2 to 7 granules. Some of the cells contain a raphide
bundle of 45 to 80 um length, consisting of calcium oxalate. The cells of the phloem
parenchyma are like those of the internal parenchyma of the bark, arranged somewhat
radially together with the sieve tubes and their companion cells. No medullary rays can
be discerned.
The cambium is as wide as a row of cells. Most of the xylem consists of radially
arranged tracheid-like trachea thickened by bordered pits; wood fibres; simple pithily
thickened libriform fibres, 10 um in size, filled with starch; transitory xylem elements;
thick-walled wood parenchyma cells with starch content, and septate fibres. Every
xylem element is lignified. The medullary rays of the xylem are 2 cell rows wide,
contain starch, but are not characteristic.

CINCHONA

IPECACUANHA

3.

IDENTIFICATION TEST

3.1

Grahe test
(Cinchonae cortex)
Heat carefully 0.5 g of bark powder in a test tube on a small flame. Within a
short time a carmine red tart ring will appear on the colder parts of the test tube.
Dissolve the sublimate in 10 ml of 70% ethanol. The filtered liquid shows a
bluish fluorescence. After adding a few drops of 2% sulphuric acid the
fluorescence increases.

3.2.

Emetine test
(Ipecacuanhae radix)
Boil 0.2 g powdered root with 5 ml of 2 n hydrochloric acid. Filter the mixture.
After adding some drops of ammonium molybdate solution filtrate acquires an
orange red color.

3.3.

Marquis test
(Papaveris maturi fructus)
Boil 0.2 g of powdered and defatted crude drug with 20 ml of 2% sulphuric acid
for a few minutes. After filtration, the extract is alkalized with 10% ammonia
solution (pH 9) and extracted with 3x10 ml of chloroform-isopropanol (3:1). The
united organic phase is dried over anhydrous sodium sulphate and evaporated at
40 oC to 2 ml. Half of the residue is dried in a white, small dish, than 2 drops of
formaldehyde-sulphuric acid reagent (1 drop of formaldehyde in 1 ml of
concentrated sulphuric acid) is added. The mixture acquires a violet color.

3.4.

Meconic acid test

(Papaveris maturi fructus)
0.5 g of powdered crude drug is boiled with 5 ml of water. After filtration 2-3
drops of R iron (III) chloride solution is added to the extract, when a red color
appears.

4.

QUANTITATIVE DETERMINATIONS

4.1.

Determination of alkaloid content in Cinchone cortex (Ph.Eur.)

(Cinchonae cortex)
In a 250 ml conical flask mix 1.000 g of the powdered drug (180) with 10 ml of
water and 7 ml of dilute hydrochloric acid R (7.3% m/V). Heat in a water-bath
for 30 min, allow to cool and add 75 ml of chloroform R, 5 ml of a 200 g/l
solution of sodium hydroxide R. Shake the mixture repeatedly for 30 min, add
3g of powdered tragacanth R and shake until the mixture becomes clear. Filter
through a plug of absorbent cotton and rinse the flask and the cotton five times
with 20 ml of chloroform R. Combine the filtrate and washings, evaporate to
dryness and dissolve the residue in 10.0 ml of ethanol R.
10

Evaporate 2.5 ml of the solution to dryness, dissolve the residue in 0.1 M

hydrochloric acid and dilute to 500.0 ml with the same acid. Prepare two
reference solutions by dissolving separately 15.0 mg of quinine R and 15.0 mg
of cinchonine R in 0.1 M hydrochloric acid and diluting each solution to 500.0
ml with the same acid. Measure the absorbance of the 3 solutions at 316 nm and
348 nm.
Calculate the percentage content of alkaloids from the following equations:

( A316 x A348c ) - ( A316c x A348 ) 100

2
x = __________________________________________ x _____ x ______
( A316q x A348c ) - ( A316c x A348q )
m 1000

( A316 x A348q ) - ( A316q x A348 ) 100

2
y = ___________________________________________ x ____ x ______
( A316c x A348q ) - ( A316q x A348c ) m
1000

m
x
y
A316
A348
A316c

=
=
=
=
=
=

A 316q =
A348c =
A 348q =

mass of the drug used in grams,

percentage content of quinine-type alkaloids,
percentage content of cinchonine-type alkaloids,
absorbance of the test solution at 316 nm,
absorbance of the test solution at 348 nm,
absorbance of the reference solution containing cinchonine at
316 nm, corrected to a concentration of 1 mg per 1000 ml,
absorbance of the reference solution containing quinine at 316 nm,
corrected to a concentration of 1 mg per 1000 ml.
absorbance of the reference solution containing cinchonine at
348 nm, corrected to a concentration of 1 mg per 1000 ml.
absorbance of the reference solution containing quinine at 348 nm,
corrected to a concentration of 1 mg per 1000 ml.

Calculate the content of total alkaloids, (x + y), and the relative content of quinine-type
alkaloids, from the following expression:
100x
x+y

11

UV spectrum of (1) quinine and (2) cichonine strandards in 0.1 N hydrochloric acid
and, (3)extract of Cinchonae cortex

4.2.

Determination of morphine in ripped capsules of poppy by TLC-densitometry

Extraction and purification

1000.0 g dried pulverized plant material is extracted with 15 ml of 2% sulphuric acid in
ultrasound bath for 15 min. After filtration the plant material is reextrated with 15 ml of
2% sulphuric acid for 15 min. The united extract is alkalized with 10% ammonia
solution (pH9) and extracted with 2x10 ml of chloroform isopropanol (3:1). The
united organic phase is dried over anhydrous sodium sulphate and evaporated to dryness
at 40 oC. The residue is dissolved in 2.00 ml of chloroform methanol (1:1).
TLC
Sorbent: Kieselgel-60 F254 (Merck) 0.2 mm (100x200 mm)
Developing system: acetone 25% ammonia (9+1).
Length of development: 85 mm
Sample application: 6 and 9 l of sample solution and 6, 9, 12 l of 0.1% morphine
test solution is injected by using microsyringe
Quantitative determination of morphine by SHIMADZU CS-930 DENSITOMETER
at 254 nm

12

Quantitative thin-layer chromatography using densitometry

Since thin-layer chromatography has been generally accepted as an analytical
separation method there is a steadily increasing demand for quantitative techniques.
Methods including the removal of the separated compounds from the plate are timeconsuming and painstaking.
Therefore, the trend in quantitative TLC is towards in situ methods.
Apart form the so-called spot area measurement, which is based entirely on the
apparent size of the spot disregarding its concentration, there are four optical methods for
quantitative in situ scanning.
1.
2.
3.
4.

The transmission or densitometric methods

The reflection or remission technique
Measurements of the fluorescence
The fluorescence quenching method

Densitometry is a method whereby the intensity of colour of a substance is measured

directly on the chromatogram.
In densitometry two sets of parameters need to be considered in relation to precision and
reproducibility. The first set are those relating to the instrument used and these can all be
made constant; the second set are those relating to the chromatogram which will always
remain variable.
The Shimadzu CS-930 is a refined, microcomputerized dual-wavelength scanner,
furnished with a data processor having recording function, as its integral part.

13

Dual-wavelength method eliminates baseline fluctuations caused by local irregularities

of layer thickness and allows extremely trace components to be detected reliably.
Zigzag scanning method with a very small light beam eliminates errors caused by
irregular shape of developed spots.
It works with curve linearization and background correction (eliminates the influence of
the background contamination of the TLC plates).
The wavelength scanning function provides absorption spectrum of a TLC spot.
Quantitative calculation function allows direct concentration printout
- Concentrations of components or concentration ratio of any two components
can be directly printed out only by calibrating with a standard sample.
- Concentrations are calculated either by the external or by the internal standard
method.
Calibration of morphine content in poppy capsules is based on calculation of peak areas
for standard morphine solution and plant samples on the same plate.

4.3.

Quantitative estimation of alkaloids in Chelidonii herba (Ph.Eur.)

0.7500 g of the powdered drug is extracted with 200 ml of dilute acetic acid (12 % g/v)
by refluxing on a water bath (100 C) for 30 min, shaking frequently. The cool extract is
filtered to a volumetric flask (250.00 ml). The first 20 ml of the filtrate is discarded.
Further 30 ml of the acidic filtrate is alkalized with cc NH4OH (pH 8-9) and extracted
with 3x30 ml of chloroform. The chloroformic phases are unified, dried (Na2SO4
siccum) and filtered, than evaporated to dryness under reduced pressure. The residue is
dissolved in 2.5 ml of ethanol (96 %, v/v) and transferred into a volumetric flask (25.00
ml). The flask is washed with small portions of diluted sulphuric acid (9.8%, g/v).
(25.00 ml of alkaloid solution, representing 0.09 g of Chelidonii herba).
5.00 ml of this solution (0.018 g drug) is transferred to a volumetric flask (25.00 ml)
and mixed with 5.00 ml chromotropic acid reagent and diluted to 25.00 ml with cc.
sulphuric acid.
Blank solution is prepared by mixing 5.00 ml diluted sulphuric acid (9.8 % g/v), 5.00
ml chromotropic acid reagent and diluted to 25.00 ml with cc. sulphuric acid.
Both reaction mixtures are kept on boiling water bath (100 C) for 10 min.
Adsorption of alkaloid containing solution is measured at 570 nm against blank solution
after cooling to about 20 C.
A1%1cm (chelidonin) = 933 (Chelidonin % = 2.98 x A)
(A violet xanthen derivate is composed).

14

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