Lecture 3

physical , microscopic and macroscopic evaluation of herbal drugs

1. November 2015 Prepared by D Manal Sabry Diploma of Quality control of Natural Products (1213) Lecture 3

2. STANDARDIZATION OF HERBAL DRUGS

3. Standardization of natural Products Different techniques of standardization Macroscopical methods Microscopic methods ( Microscopical meajurements ) Physical methods

4. STANDARDIZATION Standardization of drug means confirmation of its identity and determination of its quality and purity and detection of nature of adulterant by various parameters like: Morphological Microscopical Physical Chemical Biological observations

5. Different techniques involved in standardization of crude drugs Macroscopic methods Microscopic methods Physical methods Chemical methods Biological methods

6. Visual inspection provides the simplest and quickest means establish Identity purity quality. Macroscopic identity of medicinal plant include materials is based on Shape Size Color surface characteristics Texture Fracture Macroscopic study

7. Detail of cell structure and arrangement of the cells useful for differentiating similar species. Select a representative sample of the material & If it is dried parts of a plant than it may require softening before preparation for microscopy, preferably by being placed in a moist atmosphere, or by soaking in water. Any water-soluble contents can be removed from the cells by soaking in water. Microscopic study

8. Measurement of specimen Stomatal number Stomatal index Palisade ratio Vein-islet number Vein termination number Lycopodium spore method

9. Types of Stomata: There are several types of stomata, distinguished by the form and arrangement of the surrounding cells. - Anomocytic (irregular-celled) - Anisocytic (unequal-celled) - Diacytic (cross-celled) - Paracytic (parallel-celled) Microscopic method of Analysis: Determination of Stomatal number

10. Palisade ratio is the average number of palisade cells under one epidermal cell. For each sample of leaf make not fewer than ten determinations and calculate the average number 18.4 Palisade ratio —— = 4.5 4 Microscopic method of Analysis: Determination of Palisade Ratio

11. The stomatal index is the percentage of the number of stomata formed by the total number of epidermal cells, including the stomata, each stoma being counted as one cell. S x 100 Stomatal index = ———— E + S Where S = the number of stomata in a given area of leaf ; and E = the number of epidermal cells (including trichomes ) in the same area of leaf. - For each sample of leaf make not fewer than ten determinations and calculate the average index. Microscopic method of Analysis: Determination of Stomatal Index

12. Microscopic method of Analysis: Determination of Vein-Islet Number The mesophyll of a leaf is divided into small portions of photosynthetic tissue by anastomosis of the veins and veinlets; such small portions or areas are termed “Vein-Islets”. The number of vein-islets per square millimeter is termed the “Vein-Islet number”. This value has been shown to be constant for any given species and, for full-grown leaves, to be unaffected by the age of the plant or the size of the leaves. The vein-islet number has proved useful for the critical distinction of certain nearly related species.

13. Histochemical detection Starch grains Aleurone grains Fats, fatty oils, volatile oils and resins Calcium oxalate/carbonate crystals Lignified cell wall Cellulose cell wall Mucilage Tannin

14. PHYSICAL STANDARDIZATION OF HERBAL DRUGS: Bitterness value Hemolytic activity Swelling index Foaming index Ash value Astringency Organoleptic testing Determination of Ash value pH estimation Estimation of foreign materials Viscosity Melting point Solubility Moisture content and volatile matter Specific gravity Density Optical rotation Refractive index Particle size determination Bulk density

15. VISCOSITY Viscosity of a liquid is constant at a given temperature and is an index of its composition. Hence, it can be used as a means of standardizing liquid drugs.

16. MELTING POINT In case of pure photochemical, melting points are very sharp and constant. The crude drugs from plant or animal origin, containing the mixed chemicals, are described with certain range of melting point. Their purity can be ascertained by determining their melting points in that range for E.g. Colophony- 75-80˚c Cocoa butter- 30-33˚c

17. SOLUBILTY The presence of adulterant could be indicated by solubility studies e.g. pure Asafoetida is soluble in carbon disulphide

18. MOISTURE CONTENT AND VOLATILE MATTER The moisture content of the drug should be minimized in order to prevent decomposition of crude drug either due to chemical change or microbial contamination. The moisture content is determined by heating a drug at 105˚c in an oven to a constant weight. For the drugs containing volatile constituents, toulene distillation method is used E.g. – Aloe should have moisture content not more than 10% w/w

19. OPTICAL ROTATION Optically active compounds have the property of rotating the plane of polarized light. This property is known as optical rotation. Normally, the optical rotation is determined at 25˚c using sodium lamp as the source of light. E.g. castor oil has optical rotation from +3.5˚to +6 ˚ Caraway oil +74 to 80 Lemon Oil +57 to +70 Terpene less lemon oil -5 to +2 Cinnamon oil 0 to -2 Peppermint oil -10 to -30 Spearmint oil -45 to -60 Citronella oil, ceylon -9 to -18 Citronella oil, Java -5 to +2

20. REFRACTIVE INDEX When a ray of light passes from one medium to another of different density It is constant for a pure drug and varied with wavelength of incident light, temperature and pressure E.g. Castor oil has refractive index 1.4758-1.527

21. ASH VALUES AND EXTRACTIVES The residue remaining after incineration is the ash content of drug Total ash method is used to measure the total amount of material remaining after incineration Is useful for detecting of: Low grade products Exhausted products Excess of sand Earthy matter Total ash usually consists of carbonates, phosphates, silicates and silica.

22. Acid insoluble ash is the residue obtained after boiling the total ash with dil. HCl and igniting the remaining insoluble matter . Water soluble ash is the difference in weight between total ash and residue after treatment of total ash with water.

23. DETERMINATION OF EXTRACTABLE MATTER Amount of the active constituents present in crude drug material when extracted with specific solvent. There are different methods for determination of Extractive value: Cold method Hot method Soxhlet method

24. COLD MARCERATION Place the powdered material in a conical flask. Macerate with 100ml of solvent specified for 6hrs, shake then allowed to stand for 18hrs. Filter and transfer the filtrate to flat bottomed disk and evaporate to dryness on a water bath. Dry at 105 o C for 6hrs, cool and weigh immediately. Calculated the content of extractable matter in mg per g of air dried material.

25. HOT EXTRACTION: place 4 g powdered material in a conical flask. Add water and weigh to obtain total weight. Shake and allowed to stand for 1hr. attach the reflux condenser and boil for 1hr. Readjust to the original weight with solvent. Shake and filter. Transfer the filter to a flat bottomed disk and evaporate to dryness on a water bath. Dry at 105˚ c for 6hrs, cool and weigh immediately. Calculate the content of extractable matter in mg per g of air dried material.

26. Hot Continuous Extraction ( Soxhlet ) In this method, the finely ground crude drug is placed in a porous bag made of strong filter paper, which is placed in chamber E of the Soxhlet apparatus. The extracting solvent in flask A is heated and its vapors condense in condenser D. The condensed extractant drips into the thimble containing the crude drug, and extracts it by contact. When the level of liquid in chamber E rises to the top of siphon tube C, the liquid contents of chamber E siphon into flask A. This process is continuous and is carried out until a drop of solvent from the siphon tube does not leave residue when evaporated. The advantage of this method, compared to previously described methods, is that large amounts of drug can be extracted with a much smaller quantity of solvent.

27. Infusion Fresh infusions are prepared by macerating the crude drug for a short period of time with cold or boiling water . These are dilute solutions of the readily soluble constituents of crude drugs. Decoction In this process, the crude drug is boiled in a specified volume of water for a defined time; it is then cooled and filtered. This procedure is suitable for extracting water-soluble, heat-stable constituents.

28. BITTERNESS VALUE Medicinal plants having strong bitter taste are therapeutically used as appetizing agents The bitterness is determined by comparing the threshold bitter concentration of an extract material with that of quinine hydrochloride The bitterness value is expressed as units equivalent to the bitterness of a solution containing 1gm of quinine hydrochloride in 2000ml. 0.1gm of quinine hydrochloride is dissolved in 100ml drinking water and the stock solution is prepared. Then it is diluted and tested and compared with drug. Bitterness value in unit per gm = 2000*c A*B Where , A = concentration of stock solution B = volume of test solution in tube with . threshold bitter concentration C = quantity of quinine hydrochloride in the tube with threshold bitter concentration

29. HAEMOLYTIC ACTIVITY Haemolytic activity of plant material is determined by comparison with that of reference material, Saponin R , having haemolytic activity of 1000units/g.   Method of preparation of standard: Fill a glass stopper flask to 1/10 of its volume with sodium citrate. Ass sufficient volume of blood freshly collected from healthy ox and shake, this can be stored for about 8 days at 2-4̊ c. place 1ml of citrated blood in a volumetric flask with phosphate buffer pH 7.4 . Haemolytic activity = 1000* a/b Where , 1000 = defined haemolytic activity of Saponin standard a = quantityof saponin standard that produce total haemolysis (g ) b = quantity of plant material that produce total haemolysis (g)

30. SWELLING INDEX The swelling index is the volume in ml taken up by the swelling of 1gm of plant material under specified conditions. Its determination is based on addition of water or a swelling agent as described in test procedure.

31. FOAMING INDEX : Many medicinal plant materials contain saponins that can cause a persistent foam when an aqueous decoction is shaken. The foaming ability of an aqueous decoction of plant materials and their extracts is measured in terms of a foaming index. Calculate the foaming index using the following formula: where a = the volume in ml of the decoction used for preparing the dilution in the tube where foaming to a height of 1 cm is observed. foaming index =

32. WATER AND VOLATILE MATTER: Azeotropic method is used to directly measure the water present in a material . (Loss on drying) In order to measure volatile matter, plant is diluted with water and distillate is collected in a graduated tube. The aqueous portion separates and returns to distillation flask. A solvent of low mass density with a suitable boiling point may be added to measuring tube to easily separate the volatile oil.

33. Foreign organic matter Parts of the medicinal plant material or materials other than those named with the limits specified for the plant material concerned; Any organism, part or product of an organism, other than that named in the specification and description of the plant material concerned; Mineral admixtures that is adhering to the medicinal plant materials, such as soil, stones, sand, and dust .