Depending on various environmental factors, different samples of Kalmegh contain andrographolide varying from 0.5% to 1.5%. While leaves contain the maximum amount of andrographolide, seeds contain the lowest. Aerial parts, preferably stems and leaves, are used for extraction.

1. Freshly collected plant material is air dried under shade for a day and then in a hot-air oven at 60°C. It is powdered to #40 mesh
2. 50 g is exhaustively extracted with a 1:1 mixture of dichloromethane and methanol by cold maceration. It is filtered and the extract concentrated to green crystalline mass in a rotary vacuum evaporator.
3. The green-coloured mass is washed several times with toluene to remove most of the colouring matter.
4. Warm it on a hot plate for complete removal of toluene and dissolve in minimum quantity of hot methanol and refrigerate.
5. Separate the crystals by centrifugation and repeat recrystallization from methanol till colourless plates of constant melting point are obtained.

Identification

1. Baljet test: To a small quantity of the drug add 2 to 3 drops of alkaline sodium picrate reagent (1:1 mix of 1% picric acid in ethanol and 10% sodium hydroxide in water). An orange red colour is obtained indicating the presence of the methylene group of the lactone ring.
2. TLC test: Dissolve 1 mg drug in 1.5 ml methanol and prepare a similar solution with standard sample of andrographolide. Apply 5µl each of the test and standard samples on a precoated silica gel plate and develop it in a solvent system of chloroform: methanol (7:1). Develop the plate to a distance of 15 cm. Spray with 20% sulphuric acid in methanol and heat at 120°C for 10 min. Andrographolide is visible as a brown coloured spot and it is comparable to that of the standard.
3. Prepare a 0.1 mg per ml sample of andrographolide in methanol and determine the UV absorption by taking an absorption spectrum scan. λ_max of andrographolide in methanol is 222 nm.

Estimation

Many methods such as HPLC, HPTLC, gravimetric, spectrophotometric and titrimetric have been reported for quantitative estimation of andrographolides.

A. Method I (Spectrophotometric assay)

This estimation is based on measurement of the absorption of the orange red colour formed due to the condensation of the butenolide ring of andrographolide with picric acid in the alkaline medium of Baljet reagent.

1. 1 g of the dried aerial parts of A. paniculata (1 g) is extracted in hot methanol (4 × 50 ml) and filtered.
2. The filtrate is concentrated on a water bath and the concentrate washed with cold toluene (3 × 25 ml).
3. Excess water is added to toluene insoluble fraction and extracted with ethyl acetate (4 × 50 ml) in a separating funnel.
4. Ethyl acetate is evaporated to dryness on a water bath and residue redissolved in 10 ml methanol. This is the test solution.
5. From this solution, 0.2 ml is taken in a 10 ml volumetric flask, 5 ml of Baljet reagent added, mixed well and made up to volume with methanol.
6. Standard solution: Dissolve 10 mg of standard sample of andrographolide and dissolve it in 100 ml of hot methanol in a volumetric flask. Transfer 0.5, 1, 2, 4, 6, 8 and 10 ml solutions into 10 ml volumetric flasks. Add 5 ml of freshly prepared Baljet reagent to each of the flasks, mix well and make up to volume with methanol.
7. Measure the absorbance of the test and standard solutions against a blank prepared in a similar manner but without the sample or standard. The absorbance values are plotted against their respective concentrations to obtain the calibration curve.
8. The amount of andrographolides in the sample is calculated from the calibration curve.

B. Method II (HPTLC assay)

1. Standard sample: 1mg andrographolide is taken in a 10 ml standard flask, 7 ml methanol is added and mixed well to dissolve. Make up to volume with the same solvent. Transfer 0.2, 0.4, 0.6, 0.8, 1, 2, 3 ml of this solution into individual 10 ml standard flasks and make up to volume with methanol.
2. Take 0.5 g of dried powdered kalmegh in a 10 ml standard flask, add 7 ml methanol, mix well and filter.
3. Collect the filtrate into a 10 ml standard flask and wash the residue with 2 ml methanol. Make up to volume with methanol. Transfer the solution into a separating funnel and extract with 3 × 20 ml ether.
4. Pass the ether layers through a bed of anhydrous sodium sulphate and evaporate in a porcelain dish. Dissolve the residue in 5 ml of methanol. This is the test solution.
5. Apply 10 µl each of the standard and test solutions onto a precoated silicagel 60F₂₅₄ plate and develop it in solvent system, benzene: ethyl acetate (5:5).
6. Air dry the plate and scan it densitometrically using UV reflectance photomode at 220 nm. Determine the andrographolide content of kalmegh from the calibration curve of the peak area versus concentration of the standard sample of andrographolide.