To ensure quality of herbal drugs, it is necessary not only to establish the identity, but also to ensure batch-to-batch reproducibility. Apart from macroscopic and microscopic evaluation, tests to identify principal chemical groups are the next priority. These cover identification and characterization of the crude drug with respect to phytochemical constituents. Several analytical techniques are to be employed for this.

1. Physicochemical standards: applicable to groups of specific constituents
   1. Fixed oils, volatile oils and related substancesPhysical constants such as specific gravity, optical rotation, viscosity and refractive index are especially valuable for oils and fats, oleoresins, balsams, gums and similar substances. A number of quantitative chemical tests such as acid value, iodine value, saponification value, ester value, unsaponifiable matter, acetyl value, volatile acidity (Reichert Meissl value) are mainly applicable to fixed oils. Hence some of these tests are also useful in the evaluation of resins (acid value, sulphated ash) volatile oils (acid value, acetyl value, ester value) and gums (methoxyl determination, volatile acidity). The pharmacopoeias prescribe range of values of these physical and chemical parameters for different fixed oils, oleoresins, volatile oils, balsams etc. as appropriate for each of them. Experimental determination of these indices for the crude drug/derived product to evaluate if the values are within the pharmacopoeial limits will thus indicate their standard.
   2. Swelling index/swelling factorCrude drugs especially gums and those containing an appreciable amount of mucilage, pectin or hemicellulose are of specific therapeutic or pharmaceutical utility because of their swelling properties. The swelling index is the volume (in ml) taken up by the swelling of 1g of herbal material under specified conditions. Its determination is based on the addition of water or a swelling agent as specified in the test procedure for each individual herbal drug (either whole, cut or powdered). Using a glass-stoppered measuring cylinder, the material is shaken repeatedly for one hour and then allowed to stand for a required period of time. The volume of the mixture (in ml) is then read. The mixing of whole herbal material with the swelling agent is easy to achieve, but cut or pulverized material requires vigorous shaking at specified intervals to ensure even distribution of the material in the swelling agent. BP prescribes stand ard for agar as ≤10, linseed ≥4 for whole drug, ≥ 4.5 for powdered drug, Ispaghula husk ≥10, powder ≥90.
   3. Foaming indexSaponin-rich plant drugs can cause persistent foam when an aqueous decoction is shaken. The foaming ability of an aqueous decoction of herbal materials and their extracts is measured in terms of foaming index; 1 g of the herbal material (powdered and passed through #1250 sieve) is transferred to a 500 ml conical flask containing 100 ml of boiling water. After 30 minutes of moderate boiling, cool, filter into a 100 ml standard flask and make up the volume with sufficient water. Transfer successive portions of 1 ml, 2 ml, 3 ml up to 10 ml of the decoction respectively into 10 stoppered test tubes and make up to 10 ml volumes with water. Stopper and shake them for 15 seconds and set aside for 15 minutes. Measure the height of the foam. If it is less than 1 cm in all tubes, the foaming index is taken as less than 100. If it is more than 1 cm in all tubes, the foam index is over 1,000 and the test is to be repeated by dilution, such that foam height of about 1 cm is got in at least one of the tubes. The volume (in ml) (a) of the decoction used for preparing the dilution in the tube where foaming of 1 cm height is observed is used to determine the foaming index using the formula 1000/a.
   4. Determination of haemolytic activityAbility to cause haemolysis is a characteristic property of saponins. Many herbal drugs, especially those derived from families Caryophyllaceae, Araliaceae, Sapindaceae, Primulaceae and Dioscoreaceae, contain saponins. Saponins when added to a suspension of blood produce changes in erythrocyte membranes, causing haemoglobin to diffuse into the surrounding medium.The haemolytic activity of herbal materials, or a preparation containing saponins, is determined by comparison with that of reference saponin drug R, which has a haemolytic activity of 1,000 units/g. A suspension of erythrocytes is mixed with equal volumes of a serial dilution of the herbal material extract. The lowest concentration to effect complete haemolysis is determined after allowing the mixtures to stand for a given period of time. A similar test is carried out simultaneously with the reference drug R. Procedures proposed for the determination of the haemolytic activity of saponin-containing drugs are all based on the same principle, although the details with respect to the source of erythrocyte suspension, preparation or experimental method may change. In order to obtain reliable results, it is essential to standardize the experimental conditions and especially determine the haemolytic activity by comparison with that of the reference saponin drug R.
   5. Determination of bitterness valueHerbal drugs such as gentian that have a strong bitter taste called ‘bitters’ are employed therapeutically mostly as appetizing agents. Their bitterness is said to stimulate gastric secretions. Bitters can be determined chemically. However since they are composed of two or more constituents with various degrees of bitterness, it is to be measured by taste.The bitter properties of herbal material are determined by comparing the threshold bitter concentration of an extract of the materials with that of a dilute solution of quinine hydrochloride R. The bitterness value is expressed in units equivalent to the bitterness of a solution containing 1g of quinine hydrochloride R in 2,000 ml. Safe drinking water should be used as a vehicle for the extraction of herbal materials and for mouth wash after each testing. Taste buds dull quickly if distilled water is used. The hardness of water rarely has any significant influence on bitterness. Sensitivity of bitterness varies from person to person, and even for the same person it may be different at different times. Therefore the same person should taste both the material to be tested and the quinine hydrochloride solution within a short space of time. The bitter sensation is not felt by the whole surface of the tongue, but is limited to the middle section of the upper surface of the tongue. A certain amount of training is required to perform this test. A person who does not appreciate a bitter sensation when tasting a solution of 0.058 mg of quinine hydrochloride R in 10 ml water is not suitable to undertake this determination.The preparation of the stock solution of each individual herbal material should be specified in the test procedure. In each test series, unless otherwise indicated, the determination should start with the lowest concentration in order to retain sufficient sensitivity of the taste buds. Bitterness value of herbal drugs is to be done only after their identity has been confirmed.
2. Phytochemical screening
   It is essentially a first step in a series of procedures such as extraction, purification and characterization of the active constituents of pharmaceutical importance. These constituents are either of known therapeutic activity or are chemically defined substances or a group of substances generally accepted to contribute substantially to the therapeutic activity of herbal drugs.Phytochemical screening involves a preliminary extraction of the crude drug with a suitable solvent such as alcohol by any of the general methods of extraction such as maceration, percolation etc. The concentration extracted is subjected to a battery of qualitative chemical tests to identify various classes of phytoconstituents such as alkaloids, lipids, anthraquinones, terpenoids, flavonoids, tannins, coumarins, saponins, volatile oils, resins etc. Alternatively the drug is subjected to successive extraction with solvents of increasing polarity to isolate constituents of corresponding polarity in the respective solvents. These upon concentration yield extracts that are qualitatively evaluated for the presence of groups of phytoconstituents. Qualitative chemical tests may identify specific constituent groups like alkaloids, sugars, lipids etc. A combination of techniques, especially chromatography, is now employed for the positive identification of phytoconstituents.
3. Quantitative chemical evaluation
   Assaying herbal drugs with known active principals is another method of ensuring product identity and purity. An assay is established to set criterion for the minimum accepted percentage of active substances. A crude drug may be assayed for a particular group of constituents, for example, total alkaloids in cinchona or total sennosides in senna. Alternatively it may be necessary to evaluate specific components like reserpine content in Rauwolfia species. A plethora of analytical methods based on chemical and physical assays are available for routine standardization.
   • Gravimetric assays are used for drugs such as colchicines in colchicum corm, podophyllum resin, total balsamic esters, caffeine in tea etc.
   • Simple physical separation assay based on complex formation with cresol is used for determining cineole content in eucalyptus oil.
   • Acid-base titration based assays are routinely employed to determine the total alkaloid content of tobacco, opium or cinchona as also are strychnine in nux vomica, cinnamaldehyde in cinnamon, free alcohols in peppermint oil, carvone in oil of caraway, citral in lemon oil.
   • Spectrophotometric methods including colorimetric and flourimetric assays are developed for most active ingredients of herbal drugs.
   • Several newer assay techniques based on HPLC, GC, radioimmunoassay, enzymeimmunoassay are being developed for assaying phytoconstituents.