Glycosides are compounds which yield one or more sugars upon hydrolysis i.e., which give sugar and non-sugar moieties. The sugar portion is called glycon and the non-sugar portion is called aglycone. The most commonly occurring sugars are β-D-glucose, ramnose, cymarose and digitoxose. Chemically, these are the acetals in which the hydroxyl of the sugar is condensed with a hydroxyl group of the non-sugar component. The secondary hydroxyl is condensed within the sugar molecule itself to form an oxide ring.
Some of the properties of glycosides are listed below:
1. They are crystalline or amorphous substances.
2. They are soluble in H2O and dilute alcohol with the exception of resin glycosides, but insoluble in organic solvents like benzene, ether, or CHCl3.
3. The aglycone moiety is soluble in organic solvents. Glycosides are easily hydrolysed by water, mineral acids, and enzymes.
Isolation of Glycosides by the Stats-Otto Method:
Isolation of Bitter Glycosides:
Isolation of Picrocrocin:
We take for example the isolation of picrocrocin from Crocus sativus (saffron). Picrocrocin is a colourless bitter glycoside obtained from the dried stigmas and tops of the styles of Crocus sativus, belonging to the family iridaceae. It melts at 156°C.
Picrocrocin undergoes decomposition upon hydrolysis in the presence of alkali to give safranal and glucose.
The dried powder is extracted with petroleum ether to remove lipids, then with dry ether, which removes the picrocrocin. The crude picrocrocin is re-extracted with dry ether and re- crystallised from methanol, ethanol and methanol-chloroform-ether mixtures.
Saffron:
(i) Subjected to thorough drying in a desiccator over soda lime for 10 days.
(ii) Subjected to grinding to a fine powder, by using ball mill.
Dried, powdered saffron:
To Remove Lipids.
Damp Ether Prevents the Crystallisation of Picrocrocin.
(i) Subjected to extraction with light petroleum ether for 15 hrs.
(ii) Petroleum ether layer is rejected; the residue is dried.
(iii) The dried residue is subjected to extraction with dry ether for 10 hrs. During extraction, care must be taken to avoid the entry of water vapour into the extraction flask. For this, the extraction apparatus must be kept closed with a calcium chloride tube. During this stage, crystallisation occurs in the flask (It is crude picrocrocin).
Crude product:
A red-resinous contaminant is left in the thimble.
(i) Purified by another Soxhlet extraction with dry ether for 36—48 hours.
(ii) The solvent is evaporated; crude picrocrocin is dissolved in a minimum quantity of 80% methanol; it is filtered and evaporated under reduced pressure.
(iii) It is then dissolved in methanol and slowly allowed to evaporate in the air. The picrocrocin then crystallises out in thick yellow prisms.
(iv) The non-crystalline material is removed by washing several times with ether: alcohol mixture (3:1) as solvent.
(v) Once again, subjected to purification by dissolving in methanol, chloroform mixture. Ether is added until the solution becomes turbid. It is allowed to stand for several days. The pure picrocrocin crystallises out in large prisms. Crystals of picrocrocin are obtained.
Isolation of Coumarins:
Coumarins like cichoriin, frachinoside, fraxin, etc., are obtained from the dried leaves of Fraxinus chinensis, belonging to the family oleaceae.
The fractionation and isolation scheme for various coumarins is described below:
Isolation of Embelin from Embelia Ribes:
Estimation:
Method I – By Spectrophotometry:
An ultraviolet spectrophotometric method can be used to estimate embelin in the berries of Embelia ribes and in different formulations. The maximum absorption of embelin is found at 291 nm in chloroform and is found to obey the photometric linearity.
Procedure:
1. Ten mg of pure embelin is accurately weighed and taken in a 100 ml volumetic flask. The volume is made up with chloroform. The scanning is performed at 240-400 nm in the ultraviolet region, using a 200-20 UV-Vis. spectrophotometer. The maximum absorbance is found at 291 nm (Fig. 8.41).
2. A standard solution of 0.1 mg/ml of embelin is prepared in chloroform. From the stock solution, different concentrations of embelin solutions—0.005 mg, 0.010 mg, 0.015 mg, 0.020 mg. 0.025 mg, 0.030 mg per ml—are prepared. The absorbances of these solutions are measured at 291 nm.
3. For the estimation of embelin in berries of Embelia ribes, the embelin can be isolated as per the above isolation procedure with each batch containing 10 gms of 40 mesh powdered berries.
For the estimation of embelin in market tinctures, three batches of market tinctures are taken. In each case, 20 ml of the tincture is evaporated on a water bath. The residue is extracted with ether. Embelin is isolated as per the procedure adopted.
Each 500 mg of market tablet contains: Aconitum palmatum (100 mg), Embelia ribes (100 mg), Piper nigrum (20 mg), Mentha arvensis (15 mg), and base (q.s.).
A batch of 10 tablets are powdered and extracted with ether. The extract is processed in the same way as the isolation of embelin.
Method II:
Procedure:
This method involves the complete extraction of the fruit powder in diethyl ether, evaporation of the solvent and washing of the residue with petroleum ether (40-60°). The residue left over is crude embelin. Three sets of 2.0 gm each of the 60 # Embelia ribes powder are processed to isolate crude embelin.
In another experiment for studying percent recovery, 2.0 gm E. ribes fruit powder and 30 mg of pure embelin are taken. The residues are taken in 100 ml methanol. To 2 ml extract, 4 ml aniline (20% in methanol) and 6 ml methanol are mixed. The resultant red colour is measured in a 1 cm cell at 445 nm on a Perkin-Elmer Lambda 12/1.0 nm (1.31) UV/Vis. spectrophotometer.
Method III:
By Development of Colour with 1 N Aqueous Potassium Hydroxide
Procedure:
Embelin forms a violet coloured complex with potassium hydroxide. Potassium hydroxide (1 N) is used for the development of colour. Extraction of embelin is done as before. Three sets of 2.0 gm of E. ribes powder and three sets of 2.0 gm powder with 30 mg embelin are taken and extracted. The residues are re-constituted in 100 ml methanol. 0.5 ml of this methanolic solution is mixed with 10 ml of IN KOH. The violet color complex is measured in a 1 cm cell at 515 nm on a Perkin-Elmer Lambda 12/1.0 nm (1.31) UV/Vis. spectrophotometer.
Isolation of Tannins:
1. Tannin solutions can be precipitated by heavy metals.
2. With FeCl3
(i) A blue-black colour is produced, if gallitannins and ellagitannins are present.
(ii) A brownish green ppt. is obtained, if condensed tannins are present.
3. If very dilute ferric chloride solution is gradually added to an aqueous extract of Hamamelis leaves (containing both types of tannins), a blue colour is initially produced which changes to olive green.
4. Gold beater s skin test – Soak a small piece of Gold beater’s skin in 2% hydrochloric acid, rinse with distilled water and place in test solution for 5 minutes. Wash with distilled water and transfer it to 1% ferrous sulphate solution. Brown or black colour on the skin indicates the presence of tannins.
5. Gelatin test – 0.5 to 1% solution of tannins can precipitate a 1% gelatin solution in 10% sodium chloride.
6. Phenazone test – To 5 ml of an aqueous extracts of crude drug, add 0.5 of a sodium acid phosphate, warm, cool and filter. To the filtrate, add 2% phenazone solution. All tannins are precipitated.
7. Test for catechin – When catechins are heated with acid, phloroglucinol is formed which can be detected by a modification of the test for lignins. Dip a matchstick in the plant extract, dry, moisten with concentrated hydrochloric acid and warm on a flame. The phloroglucinol produced gives a pink or red colour to the matchstick.
8. Test for chlorogenic acid – An extract when treated with aqueous ammonia and gradually exposed to air gives a green colour, if chlorogenic acid is present.
Isolation of Artemisinin, Artemisinic acid and Arteannuin B:
Hala N. Elsohly (1990) isolated artemisinin, artemisinic acid and arteannvin B, terpenoidal drugs, (Scheme 8.13) from Artemisia annua.
Isolation of Aristolochic Acid and Aristolic Acid:
Aristolochic acid and aristolic acid are extracted from dried ground roots of Aritolochia indica (1 kg) in a Soxhlet extractor with 95% ethanol. The ethanolic extracts are concentrated to thick black-brown syrup under reduced pressure to yield about 110 gm of residue. The residue is treated with chloroform (0.5 litre) and 2% sodium bicarbonate solution (3 litres) and the suspension is subjected to vigorous stirring for 24 hours.
The wine-red alkaline solution obtained is decanted and filtered. The combined filtered alkaline solution is then acidified with 5% HCl to yield a yellow-brown precipitate, which is filtered and dried to yield 10-12 gm of total acid fraction.
Extraction with the 2% sodium bicarbonate is repeated several times until no further precipitation results upon acidification of the bicarbonate extract. The crude total acid fraction (1.5 gm) is extracted with chloroform (2 litres) in a Soxhlet apparatus for 10 hours (residue: 0.41 gm). The chloroform solution is added to a column of silicic acid-celite 545 (4:1, 500 gm).
In 4-5 hours, a passage of 2 litres of chloroform through the column results in resolution of 4 major coloured segments:
(A) Highly fluorescent reddish-yellow,
(B) Yellow-green non-fluorescent (the largest),
(C) Reddish-brown mixture of bands, and
(D) Brown-black fluorescent mixture of bands.
Chloroform (5 litres) is used to elute band A (15 mg of a non-crystalline substance) and band B (875 mg) which is aristolochic acid. The aristolochic acid is subjected to purification by crystallisation of the crude acid from a mixture of dimethyl formamide and ethanol; yellow-orange crystals with a melting point of 275-278°C resulted.
Conversion of Aristolochic Acid to Aristolic Acid:
To a solution of aristolochic acid in 1% aqueous ammonia hydroxide solution (pH 12), is added sodium borohydride (for 100 mg of aristolochic acid, 76 mg of sodium borohydride); the mixture is stirred at 25°C for 4 hours. The reaction mixture is poured onto crushed ice, acidified with hydrochloric acid to pH 4 and extracted with ethyl acetate (5 × 25 ml).
The combined ethyl acetate phases are washed with water, dried over sodium sulphate, filtered and concentrated in vaccum to afford a homogeneous residue of artistolic acid (80 mg, 90% yield) which is crystallised from dimethyl formamide and ethanol to afford fine needles with a melting point 255-256°C.
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