CRUDE DRUGS CONTAINING QUINOLINE, ISOQUINOLINE, AND PHENANTHRENE ALKALOIDS Content 1. MACROMORPHOLOGICAL EVAULATION Cinchonae cortex Ipecacuanhae radix Chelidonii herba Papaveris maturi fructus Opium crudum Fumariae herba 2. MICROSCOPICAL TESTS Cross section: Cinchonae cortex Ipecacuanhae radix Powder preparation: Cinchonae cortex 3. PHYSICO-CHEMICAL AND CHEMICAL TESTS Specific alkaloid reactions 3.1. Grahe test (Cinchonae cortex) 3.2. Emetine test (Ipecacuanhae radix) 3.3. Marquis test (Papaveris maturi fructus) 3.4. Meconic acid test (Papaveris maturi fructus) 3.5. Chelidonii herba tests (Chelidonii herba) 3.5.1. Chelidonic acid test (Chelidonii herba) 3.5.2. Quaternary amine test (Chelidonii herba) 3.5.3. Chromotropic acid test (Chelidonii herba) 3.5.4. Chelidonii herba alkaloid investigation with TLC 4. QUANTITATIVE DETERMNATIONS 4.1. Determination of alkaloid content in Cinchonae cortex (Ph.Eur.) 4.2 Quantitative determination of morphine in ripped capsules of poppy by TLC 1 1. MACROMORPHOLOGICAL EVALUATION Cinchonae cortex Cinchona bark Cinchona pubescens Vahl. Rubiaceae (Cinchona succirubra Pavon) C. calisaya (Weddell) C. ledgeriana (Moens ex Trimen) Ph. Eur. Whole or cut, dried bark of Cinchona species. Content: minimum 6.5 per cent of total alkaloids, of which 30 %- 60 % consists of quinine type alkaloids (dried drug). Ipecacuanhae radix Ipecacuanha root Cephaelis ipecacuanha (Brot.) A.Rich. Rubiaceae Cephaelis acuminata Karsten Ph. Eur. Karlten Ipecacuanha root consists of the fragmented and dried underground organs of Cephaelis species. It contains not less than 2.0 per cent of total alkaloids, calculated as emetine. The principal alkaloids are emetine and cephaeline. Chelidonii herba Greater Celandine Chelidonium majus L. Papaveraceae Ph.Eur. Dried, whole or cut aerial parts of Chelidonium majus L collected during flowering. Content: minimum 6.0 per cent of total alkaloids, expressed as chelidonine. 2 Papaveris maturi fructus Poppy capsules, Poppy heads Papaver somniferum L. Papaveraceae Opium crudum Opium, Raw Papaver somniferum L. Papaveraceae Ph.Eur. Raw opium is the air-dried latex obtained by incision from the unripe capsule of Papaver somniferum L. It contains not less than 10.0 per cent of morphine and not less than 2.0 per cent of codeine, both calculated with reference to the drug dried at 100-105ºC. Fumariae herba Fumitory Fumaria officinalis L. Fumariaceae 3 2. MICROSCOPICAL TESTS Chinae succirubrae cortex (cross section) Chinae cortex pulvis The powder is reddish brown. calcium oxalate sand short, spindle shaped, yellow shining fibres starch reddish brown parenchyma fractions 4 Ipecacuanhae radix (cross section) 5 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 matter 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. Quinine and quinidine has fluorescence in sulfuric acidic solution. 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 phosphomolybdic acid solution, the filtrate acquires an greenish color. 3.3. Marquis test (Papaveris maturi fructus) Add 10 drops of R NH3 solution to 0.5 g of the crude drug. Shake it in 10 ml dichlormethane, filter over anhydrous sodium sulphate sicc. and evaporate to dryness. Add 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. Condensation of phenol rings occur, diarylmethane dye forms. 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 reddish color appears. 6 3.5. Chelidonii herba tests 3.5.1. Detection of Chelidonic acid (Chelidonii herba) Drop a little amount of Chelidonii herba with a few drops of 20% KOH in a porcelain dish. Yellow colour can be observed. 3.5.2. Detection of quaternary amines (Chelidonii herba) Mix 0,5 g of Chelidonii herba powder with diethyl ether or dichloromethane. After 5 minutes – shaking 2-3 times – the filtrate at 366 nm give pink or yellow fluorescence. Quaternary amines (e.g. berberine, coptisine) have different colour fluorescence in organic solvents. 3.5.3. Chromotropic acid test of chelidonine (Chelidonii herba) Extract 0.5g Chelidonii herba powder in 40 ml of 12% acetic acid on a water bath for 15 minutes in. Add 2 ml cc. NH4OH to 10 ml of the the cooled, filtered extract then shake with 2x10 ml chloroform. After filtering on Na2SO4 sicc., concentrate 10 ml of the chloroform phase in a porcelain dish and add one drop of cc. sulfuric acid and R chromotropic acid. After 15 minutes of purple coloration develops. After adding cc. sulfuric acid, a formaldehyde molecule leaves from the methylendioxy group of chelidonine. Formaldehyde makes a colored product with chromotropic acid. 7 3.5.4. Chelidonii herba alkaloid investigation with TLC 1. Evaporate the other 10 ml chloroformic solution of the previous extract (3.5.3) in a round flask. Dissolve the residue in 1 ml chloroform and use for the TLC investigation of Chelidonii herba. (20 µl) 2. 1 ml 96% ethanol should be added to 0,3 ml of celandine containing solution. 20 µl is used to TLC. 3. Berberis vulgaris twig 0.5/m l extract. Sorbent: Silicagel F254 Developing system 90:9:1 – 1-propanol:water:formic acid Evaluation: UV light 366 nm sample µl alkaloid Rf color Berberis vulgaris 20 berberine ~ 0,31 greenish-yellow Chelidonii herba 20 coptisine ~ 0,2 yellow berberine ~ 0,3 greenish-yellow chlereritrine ~ 0,4 blue sanguinarine ~0,57 yellow chelidonine ~ 0,5 purplish Dr. Milesz celandine 20 solution chelidonine standard 20 ~ 0,5 purplish 1. 2. 3 1. Celandine sol. 2. B. vulg. extract 3. C. majus exctract 8 4. QUANTITATIVE DETERMINATIONS 4.1. Determination of alkaloid content in Cinchone cortex (Ph.Eur.) In a 250 ml conical flask mix 1.000 g of the powdered drug (Cinchonae cortex) with 10 ml of water and 7 ml of diluted hydrochloric acid R (7.3% m/V). Heat in a water-bath for 30 min, allow to cool and add 75 ml of chloroform and 5 ml of a 20% (m/V) solution of sodium hydroxide. Shake the mixture for 30 min on ultrasonic bath, add 3g of powdered tragacanth and shake gently until the mixture becomes clear. Filter through cotton and rinse the flask and the cotton five times with 20 ml of chloroform. Combine the filtrate and washings, evaporate to dryness and dissolve the residue in 10.0 ml of ethanol. Evaporate 0.5 ml of the solution to dryness, dissolve the residue in 0.1 M hydrochloric acid and dilute to 100.0 ml with the same acid. Prepare two reference solutions by dissolving separately 18.5 mg of quinine-HCl and 18.5 mg of cinchonine 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 9 UV spectrum of (1) quinine and (2) cinchonine 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 1. Extraction and purification 1.0000 g dried pulverized plant material is extracted with 15 ml of 2% sulphuric acid in ultrasonic 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 (pH=9) 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). 2. Thin layer chromatography Sorbent: Kieselgel-60 F254 (Merck) Developing system: acetone – 25% ammonia (9+1). Sample application: 3, 6 and 9 μl of sample solution 3, 6, 9 μl of 0.1% morphine test solution is injected using microsyringe Evaluation: UV light, 254 nm (photo) 10 3. Quantitative determination of morphine by CpAtlas program Spot area measurement is based on the apparent size of the spot which is proportional with the amount of substance.