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 Specific alkaloid reactions 3.2.l 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. QUANTITATIVE DETERMNATIONS 4.1. Determination of alkaloid content in Cinchonae cortex (Ph.Eur.)

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

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

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1. MACROMORPHOLOGICAL EVALUATION

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

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 Ipecacuanha root Cephaelis ipecacuanha (Brot.) A.Rich. Rubiaceae Cephaelis acuminata Karsten

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.

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Chelidonii herba Greater Celandine Chelidonium majus L. 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 Poppy capsules, Poppy heads Papaver somniferum L. 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.

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Opium crudum Opium, Raw Papaver somniferum L. 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 Boldo leaf Peumus boldus Molina 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 Fumitory Fumaria officinalis L. Fumariaceae Ph. Eur., Ph.Hg.VIII.

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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 µm in diameter, and tannin turning brownish green from a drop of R-iron (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 slit-like 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 thin-walled 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.

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CINCHONA

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IPECACUANHA

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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.

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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 = mass of the drug used in grams, x = percentage content of quinine-type alkaloids, y = percentage content of cinchonine-type alkaloids, A316 = absorbance of the test solution at 316 nm, A348 = absorbance of the test solution at 348 nm, A316c = absorbance of the reference solution containing cinchonine at 316 nm, corrected to a concentration of 1 mg per 1000 ml, A 316q = absorbance of the reference solution containing quinine at 316 nm, corrected to a concentration of 1 mg per 1000 ml. A348c = absorbance of the reference solution containing cinchonine at 348 nm, corrected to a concentration of 1 mg per 1000 ml. A 348q = 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

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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 µl of sample solution and 6, 9, 12 µl of 0.1% morphine

test solution is injected by using microsyringe Quantitative determination of morphine by SHIMADZU CS-930 DENSITOMETER

at 254 nm

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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 time-consuming 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. The transmission or densitometric methods 2. The reflection or remission technique 3. Measurements of the fluorescence 4. 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.

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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).

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