Pharmacognosy Lab 3-4-5 PDF

Pharmacognosy Lab 3 Cardioactive Glycosides Part II Lab assignment: Identification of Cardio-active Glycoside by preliminary qualitative tests Thin Layer Chromatography (TLC) Identification using preliminary qualitative tests:...

Pharmacognosy Lab 3 Cardioactive Glycosides Part II Lab assignment: Identification of Cardio-active Glycoside by preliminary qualitative tests Thin Layer Chromatography (TLC) Identification using preliminary qualitative tests: 3 Kedde’s Reaction Test 1 Baljet’s Test (unsaturated lactone ring): (unsaturated lactone ring): Cardenolide Take 1 ml of the extract (fraction A) Take 3 ml of the (fraction B) in a test add 2 drops of picric acid tube and make it alkaline with 1 m1 NaOH add 2 ml of 1% 3,5-dinitrobenzoic acid, (sodium hydroxide solution) using and Add 1 ml of 0.5 N aqueous litmus paper. methanolic KOH (50%). The reaction mixture immediately turns This results in a turbid purple-violet, which color disappears after yellow to orange color. a few minutes. 2 Raymond’s Reaction Test (unsaturated lactone ring): 4 Legal's Reaction Test (unsaturated lactone ring): Take 1 ml of the extract(fraction B)in a Dissolve a few ml of the fraction B in test tube, 2 ml of pyridine, add l to 2 drops of 10% sodium add 1 ml of Sodium nitroprusside, hydroxide solution (NaOH), followed by adding 1 ml of sodium and Add a few drops of alcoholic hydroxide NaOH, solution of 1 %3,5-dinitrobenzene. A transient blood red color develops. This results is pink color. PAGE 1  Pharmacognosy 5 Lieberman-Burchard Sterol Test (aglycone/sterol part): 6 Keller Killian’s Test (glycone part): Take I ml of the extract (fraction B) in Take 1 ml of the extract (fraction A)in test tube a test tube add 5 ml of anhydrous acetic acid, add 2 ml of Glacial acetic acid and shake well add 1 drop of 0.1% of ferric chloride take 4 drops of the above mixture solution (FeCls), and place in a porcelain dish take 1 ml of concentrated H2SO4 and Add one drop of Concentrated add it to the above mixture in drops HzSO4. to make two layers. A change of color from rose, through red, violet, and blue to green. Two layers are formed: The color is slightly different from 1. The upper one is a light bright green compound to compound. color. 2. The lower layer has a transparent clear color (H2SO4 Layer) 3. the junction appears as a reddish-brown ring. PAGE 2  Pharmacognosy The common aglycones related to these structures 1. aloe-emodin Lab 4 2. emodin 3. rhein 4. chrysophanol Anthraquinone Part I 5. physcion. Anthraquinones are glycosides that possess The sugars present are usually anthracene nucleus or their derivatives 1. arabinose aglycone in which 2 keto groups are attached 2. rhamnose to the benzene ring (anthraquinone) 3. glucose The Glycosides upon hydrolysis yield aglycones that are di-, tri-, or tetrahydroxy anthraquinone or modifications of these compounds. Glycosides of anthranols, dianthrones and oxanthrones (reduced derivatives of anthraquinones) also occur in plant materials and contribute to the therapeutic activity (Figure 9). PAGE 3  Pharmacognosy Medicinal use and Drugs containing anthraquinone Mechanism of action: glycosides: Anthraquinone and related glycosides, are Cascara bark stimulant cathartics and Frangula Senna leaves exert their action are the most widely used. by increasing the tone of the smooth muscle in the wall of the colon Rhubarb and Aloe are not recommended as cathartics due to their irritating actions which and stimulating the secretion of water increase the chance for a gripping effect. and electrolytes into the large intestine. Senna Leaves After the oral administration, the anthraquinone (Cassia acutifolia F. Leguminosae) glycosides are hydrolyzed in the colon are used as an example of the anthraquinone - by the action of enzymes of the microflor Glycosides. The main active constituents of Senna are to the pharmacologically active free aglycones dimeric glycosides whose which usually produce their effect in 8-12 hours aglycones are composed of after administration. aloe-emodin and/or rhein. These agents are indicated for constipation in These are presented patients who do not respond to milder drugs as Sennosides A, B, C and D, and for bowel evacuation before investigational the major concentration being Sennoside A and procedures or surgery. B (Figure 11). Stimulant laxatives are habit-forming so long-term use may result in laxative dependence and loss of normal bowel function. The glycosides of anthranols and anthrones elicit a more drastic reaction than do corresponding anthraquinone glycosides and cause discomfort gripping action. PAGE 4  Pharmacognosy Lab assignment: Extraction and isolation of anthraquinone Glycoside from dry leaves of Cassia acutifolia applying Decoction as a method of extraction. ‫د‬ ‫ددددددد‬ PAGE 5  Pharmacognosy ❖Fraction A contains the glycoside ❖Fraction B contains the genin part aglycone (dianthrone) ❖Fraction C contain the genin part aglycone (monoanthrone) The O-glycoside is hydrolyzed to free anthraquinone by heating with HCl acid. The C- glycoside releases the free anthraquinone only after oxidative cleavage in the presence of FeCl3 in an acidic medium, releasing free anthraquinone (or rhein in the case of anthraquinone dimmers) and the sugar moiety PAGE 6  Pharmacognosy Lab 5 Anthraquinone Part II Lab assignment A. Identification of Anthraquinone Glycoside by preliminary qualitative tests B. Thin Layer Chromatography (TLC). A Identification using preliminary qualitative tests: Borntrager test for anthraquinones Put 5 ml of each of the three fractions obtained in the previous lab; fractions A, B and C in a test tube, add 2 ml of 10% Ammonia solution and observe the color of each test tube Take 5 ml of chloroform in a test tube add 2 ml of 10% ammonia and use it as a control to compare the different colors. The pink color will be produced, Fraction C (monoanthrones) will appear more transparent (clear) than Fraction B (dianthrones). Fraction A has the unhydrolyzed aglycone thus will give a negative result. PAGE 7