Analytical Pharmacognosy

Introduction Medicinal plants contain active constituents that are used for their pharmacological properties. It is important to maintain the quality and purity of herbal crude drugs to comply with the standards prescribed for authentic drugs. Drug adulteration Adulteration is a practice of substituting the original drug partially or totally with other similar looking substances but the latter is either free from or inferior in chemical and therapeutic properties.

Drug adulteration Adulteration occurs to reduce the price of the drug in the market or by accident. Adulteration involves different conditions such as: Deterioration: Impairment in the quality of the drug Admixture: It is the addition of one articles to another due to ignorance, carelessness or by accident. Sophistication: It is the intentional type of adulteration Substitution: It occurs when a totally different substance is added in place of original drug. Inferiority: It is due to the attack of microorganisms

Types of Adulteration Substitution with substandard commercial varieties. The adulterants here may resemble the original drug but are substandard in nature and hence cheaper in cost. Substitution with superficially similar inferior drugs: These interior drugs may or may not have any of the therapeutic value as the original drug. Due to their morphological resemblance to authentic drug, they are marked as adulterants. Substitution with artificially manufactured drugs: Substances artificially prepared to resemble original drug are used as substituents for high expensive drugs.

Types of Adulteration Substitution of exhausted drugs: The same drug is admixture but it is avoid of any medicinally active constituents as they are already extracted out e.g., volatile oil containing drugs Sometimes, some synthetic chemicals are used to enhance the natural character like the addition of citral to citral oil. Presence of vegetative matter from the same plant: Sometimes, other plants or parts from the same plant growing alone with medicinal plants are allowed to get mixed with drug due to their resembling colour, odour, and in some cases compounds. Harmful adulterants: Sometimes, waste from market are mixed with authentic drugs. Adulteration of powders: Sometimes powdered forms may also be adulterated.

B. Methods of drug Evaluation Drug evaluation mean confirmation of its identity, quality and purity. If adulterated, it also includes detecting the kind of adulterants. Drug evaluation is necessary to check: Biochemical variation in the drug Detrioration from treatment and storage Substitution and adulteration as a result of carelessness or ignorance.

Techniques of standardization of crude drug. Morphological evaluation: Evaluation of drug by colour, odour, taste, size and special features like touch and texture. Microscopic evaluation: Identification of the drug by its known histological characters. Physical evaluation: Determination of physical standards for drugs like moisture content, specific gravity, density, optical rotation and refractive index.

Types of biological evaluation: 4.Chemical evaluation: It includes isolation, purification and identification of active compounds in addition to quantitative and qualitative chemical tests as acid value and saponification value. Preliminary phytochemical screening is a part of chemical evaluation. Qualitative chemical tests are important to detect adulterants. 5. Biological evaluation/assay: Evaluation of drug potency by its effect on living organisms like bacteria, fungal, animal tissue or entire animal. Types of biological evaluation: Toxic Symptomatic Tissue methods

Phytochemical Investigation The technique used to remove active compounds from the plant materials is called extraction which involves the use of different solvents. The plant materials used for extraction should be authenticated and it is chosen according to nature of constituents to be extracted. The dried powdered plant material is commonly used for extraction. The fresh plant parts when used are macerated with a solvent such as alcohol. Qualitative chemical examination The extracts obtained as above are subjected to different chemical tests to identify their various constituents

Detection of alkaloids Dragendorff’ test Mayer’s reagent Hager’s reagent Wagner’s reagent Mayer’s reagent Place 1 g of black pepper powder in 3 mL of ammonia and allow the solution to stand for 10 min. Add 10mL of chloroform. Filter it by using filter paper Take the filtrate and evaporate the chloroform. Add 2mL of Mayer’s reagent. Creamy participate will appear

Detection of carbohydrate Molise’s test to detect carbohydrate Legal’s and Borntrager’s tests to detect glycoside Fehling’s, Barfoed’s and Benedict’s tests to detect sugar Molisch's test:  Prepare 2 ml of carbohydrate solution in a test tube Add few drops of α-naphthol 95% ethanol) Add few drops of concentrated sulphuric acid on the walls of test tube A violet colour is formed at the junction between two layers It gives a positive result with all carbohydrates

Fehling test: Prepare 2 ml of carbohydrate solution in a test tube Add 2 ml of Fehling A solution and 2 ml of Fehling B solution Fehling I consists of 7 g of hydrated copper (II) sulfate dissolved in 100 ml of dist. water. Fehling II is made by dissolving 35 g of potassium sodium tartrate and 10 g of sodium hydroxide in 100 mL of dist. water. Heat in a boiling water bath for five minutes Any change in colour is considered as positive result This test gives positive result with monosaccharides and reducing disaccharides (glucose, fructose, maltose and lactose)

Detection of Fix oils and fats: Press small quantity of pet. ether and benzene extracts between two filter papers. Oil stains on the paper indicates the presence of fix oil Formation of soap or partial neutralization of alkali indicates the presence of fixed oils and fats. Borntrager reaction is used to detect anthraquinone glycoside Powdered drug + alcoholic KOH + boil & filter. Filtrate + Dilute HCl (acidification) + ether + shake. Ethereal layer + FeCl3 + NH3 + shake = aqueous layer rose-red to intense red.

Detection of phytosterols: Liebermann’ test The crude plant extract was dissolved with a few drops of diluted acetic acid then 3 ml of acetic anhydride was added followed by a few drops of conc. H2SO4. The bluish green colour appeared which showed the presence of phytosterol

Detection of saponins: Foam test and haemolysis test The crude extract stock solution was diluted with 20 ml of distilled water and it was agitated in a graduated cylinder for 15 min. The formation of 1 cm foam layer showed the presence of saponins. Detection of phenolic compounds and tannins: Ferric chloride test

f. Detection of protein and free amino acid: Millon’s test To one milliliter of the crude stock extract was added a few drops of Ninhydrin reagent. The purple colour appearance shows the presence of amino acids. g. Detection of gums and mucilage: Swelling properties h. Detection of volatile oils: Hydro-distillation of air dried or fresh plant material.