Glycyrrhizin content of liquorice varies from 2% to 9% in different samples. Other constituents are 5% to 15% sugars, 1% to 2% asparigine, 0.04% to 0.06% volatile compounds, β-sitosterol, starch, protein and bitter principals such as glycymarin. Several procedures are reported in the literature on the extraction of glycyrrhizin by various organic solvents, purification by ion-exchange and polymeric resins, adsorption, chromatographic separation, supercritical fluid extraction, foam separation, microwave-assisted extraction and multi-stage counter-current extraction. Due to better water solubility and ease of preparation, glycyrrhizin is isolated in the form of ammonium salt from which glycyrrhizin may be precipitated.

A. Method I

1. Macerate 100 g of liquorice root of # 20 mesh in a mixture of 95 ml of distilled water and 5 ml of solution of ammonia for 24 hours.
2. Filter through Whatman filter paper in Buchner funnel under reduced pressure and wash the residue with sufficient distilled water such that the filtrate measures 100 ml.
3. To the filtrate add sulphuric acid slowly with constant stirring until a precipitate ceases to form.
4. Collect the precipitate on a strainer, wash until free from acid, redissolve in water with the aid of solution of ammonia, filter if necessary.
5. Repeat the precipitation with sulphuric acid; again collect, wash and dissolve the precipitate in a sufficient quantity of solution of ammonia, previously diluted with an equal volume of distilled water;
6. Finally, evaporate the clear solution to a thin syrup which is taken on porcelain tiles or sheets of glass for drying in an oven at temperature not exceeding 60°C.
7. Preserve the dry product in well-closed vessels. The prepared ammonium glycyrrhizin is in the form of dark brown or brownish-red odourless scales, having a very sweet taste. At 100°C the scales become darker in colour, and at a higher temperature melt with decomposition; on complete incineration not more than a trace of ash is left.
8. Dissolve the formed ammonium glycyrrhizinate in distilled water and add sulphuric acid in drops till precipitation is complete.
9. Filter and dissolve precipitated glycyrrhizin in minimum quantity of hot water and set to cool. A jelly-like substance is formed. It is washed with diluted alcohol and dried when an amorphous yellow powder having a strong bitter-sweet taste and an acid reaction is formed.

Identification

1. To a small quantity of glycyrrhizin, add 5 ml water, shake vigorously and set aside. Formation of a persistent foam which does not disappear on shaking indicates the presence of saponins.
2. To a small quantity of glycyrrhizin add 1 ml chloroform, 1ml acetic anhydride and 2 ml concentrated sulphuric acid along the sides of the test tube. Formation of a violet colour at the junction of two layers indicates the presence of a triterpenoid.
3. TLC test: Reflux 5 mg of glycyrrhizin with 20 ml 0.5 M sulphuric acid. Cool and extract with 2 × 10 ml chloroform. Evaporate the combined chloroform extract and dissolve the residue in 1 ml of 1:1 mixture of chloroform and methanol. Repeat the same for standard sample of glycyrrhizin. Apply 5µl each of the test and standard samples in two different tracks on a precoated silica gel GF₂₅₄ plate and develop in a solvent system of toluene: ethyl acetate: glacial acetic acid (12.5:7.5:0.5). Air dry the plate and spray with anisaldehyde-sulphuric acid reagent and heat at 100°C for 5–10 min. Glycyrrhizin is visible as a dark violet spot in both the test and standard tracks.

Estimation

Several analytical methods based on HPTLC, HPLC and capillary electrophoresis are reported in the literature for the estimation of glycyrrhizin in extracts and formulations.

A. Method I (HPTLC assay)

1. 500 mg of dried powdered liquorice is treated with 2 × 10 ml of 70% ethanol, each time sonicated for 10 min and filtered. The filtrate is taken as test sample.
2. Dissolve 5 mg of standard sample of glycyrrhizin in 50 ml 70% ethanol in a standard flask. This is the standard sample.
3. Apply 5 and 10µl of the test and 2, 5, 7 and 10 µl of standard sample in different tracks on a precoated silica gel GF₂₅₄ plate and develop in a solvent system of butanol: acetic acid: water (5:1: 4-upper layer)
4. Air dry and scan at 260 nm in absorbance mode using a HPTLC scanner.
5. Determine the content of glycyrrhizin in liquorice from the calibration curve drawn for standard sample of glycyrrhizin using concentration versus peak area.

B. Method II (Spectrophotometric assay)

1. 500 mg of dried powdered liquorice is macerated in 70% ethanol overnight, filtered and evaporated to a pasty mass.
2. Transfer 20 mg of this extract into a 10 ml standard flask, dissolve in a solution of phosphate buffer (pH 6.8) : ethanol (7:3) and make up to volume with the same solvent. Filter it through Whatman filter paper (#44) and dilute 1 ml of the filtrate to 10 ml with the same solvent. This is the test sample.
3. Dissolve 10 mg glycyrrhetenic acid in phosphate buffer: ethanol (7:3) solvent in a 10 ml standard flask and make up to volume. Transfer 50, 100, 150, 200, 250 and 350 µl into individual 10 ml standard flasks and make up to volume with the same solvent and label them appropriately as S1 to S6.
4. Determine the absorbance of the standard and test solutions at 254 nm and calculate the content of glycyrrhetenic acid in liquorice from the calibration curve of concentration versus absorbance prepared for the standard.