Microscopical Methods in Pharmacognosy PDF

# **Pharmacognosy** ## **Microscopical Methods** These methods allow more detailed examination of a drug and can be used to identify organized drugs by their known histological characters. Schleiden (1847) used the microscope for the examination of drugs. Prior to examination through a microscope,...

# **Pharmacognosy** ## **Microscopical Methods** These methods allow more detailed examination of a drug and can be used to identify organized drugs by their known histological characters. Schleiden (1847) used the microscope for the examination of drugs. Prior to examination through a microscope, the material must be suitably prepared. This can be done by powdering, cutting thin sections of the drug, or preparing a macerate. After this, mounting and staining techniques can be followed to differentiate particular tissues from adulterants. In the identification of whole crude drugs, it is often necessary to examine the anatomical structure for the particular morphological group. In some drugs, transverse and longitudinal sections are taken and the arrangement of tissues is observed. The arrangement of tissues is best ascertained by observation of transverse sections. The basic layout of tissues in stems, roots, leaves, etc. is fairly constant, but there is a considerable variation in the amount or extent of different regions, in the extent of lignifications, in the detailed appearance of the cells, etc., between different plants while in some cases, definite anomalous arrangement of the tissues occurs. The cells, which are most useful for the purpose of identification are those least affected by drying, e.g., fibres, sclereids, tracheids, vessels, and cork cells. For example, lignified trichomes in nux-vomica, warty trichomes of senna, wavy medullary rays of cascara bark, glandular trichomes of mint, etc. For effective results, various reagents or stains can be used to distinguish cellular structure. Microscopic evaluation also covers the study of the constituents by application of chemical methods to small quantities of the drug in powdered form or to histological sections of the drug (micrometry or chemomicroscopy). A drop of phloroglucinol and concentrated hydrochloric acid give red stain with lignin. Mucilage is stained pink with ruthenium red and also when treated with corallin soda and a few drops of sodium carbonate solution. Cellulose swells and dissolves in cuoxam, while N 50 iodine solution stains starch and hemicelluloses blue. Microscopic linear measurements and quantitative microscopy are also covered under this technique of evaluation. In the identification of powdered crude drugs, a microscopical examination is essential, and a detailed study of the fragments present must be made. In powdered drugs, the fragments may consist of parts of cells or groups of cells; so it is essential to recognize the cells and tissues under these conditions. Cell contents will be either in free state or present within the cells. The cells of significant diagnostic value are those which are least affected by communication, for example, thick-walled cells, and cells with lignified walls. During microscopic examination, careful drawings should be made of the important features present. ## **Analytical Pharmacognosy** If it is the establishment of authenticity that is required (to determine whether the material is correctly labeled or not) the illustrated report would be compared with a description of the drug in question from any reference book. Powdered cloves do not contain sclereids or calcium oxalate crystals, but both of them are present in powdered clove stalks. Powdered clove fruits show presence of starch, while it is absent in cloves. Presence of non-lignified vessels in powders of rhubarb and ginger indicate adulteration. The presence or absence of crystals of aloin indicates different varieties of aloes. ## **Quantitative Microscopy** Walles (1916) published the use of quantitative microscopy in pharmacognosy, which helps to determine the percentage of extraneous organic matter in powdered drugs, in addition to the measurements of the size of cells, tissues and cell contents. The other important histological aspect is quantitative microscopy and linear measurements. The various parameters studied here are stomatal number and index, palisade ratio, vein-islet number, size of starch grains, length of fibres, etc. Senna varieties are distinguished by differing stomatal number and palisade ratio. The diameter of starch grains in Cinnamomum and Cassia is 10 microns and hence useful for detecting adulterants. The number of sclerenchymatous cells per square mm of cardamom is useful for detecting different varieties of cardamom seeds. ## **Leaf constants** The average number of palisade cells beneath each epidermal cell is called palisade ratio. It can be determined with powdered drugs with the help of camera lucida. Vein-islet number is defined as the number of vein-islets per sq. mm of the leaf surface midway between the midrib and the margin. Levin in 1929 determined the vein-islet numbers of several dicot leaves. Vein-termination number is defined as the number of veinlet terminations per sq. mm of the leaf surface midway between midrib and margin. Stomatal number is the average number of stomata per sq mm of the epidermis of the leaf. Stomatal index (SI) is the percentage proportion of the number of stomata forms to the total number of ordinary epidermal cells of a leaf. It is calculated by using the following formula: $SI = \frac{S}{E+S}$ where, * SI = Stomatal Index * S = Number of stomata per unit area * E = Number of ordinary epidermal cells in the same unit area The technique of determination of leaf constants can be used for microscopic evaluation of several leaf drugs such as senna, datura, digitalis, buchu, coca, belladonna, etc. ## **Water pores** Water pores and stomata resemble each other to some extent in structure and functions. Water pores are immovable unlike stomata and they are present on the teeth of the margin. ## **Trichomes** There are other important diagnostic characters helpful in the identification of drugs and detection of adulterants. Trichomes are the tubular elongated or outgrowth of the epidermal cells. Trichomes are also called plant hairs. Trichomes consist of two parts viz., root (in the epidermis) and body (outside the epidermis). Trichomes are present in most of the parts of the plant such as leaves (senna and digitalis), seeds (nux-vomica and strophanthus), fruits (Helicteris isora and Lady's finger, etc.) and are absent in roots. Trichomes are as such functionless, but sometimes, perform secretory function, and in xerophytes, hairs act as a screen against light to cut short the light intensity. The trichomes excrete, at times, volatile oil as in case of peppermint. They are absent in coca and hemlock, and are rarely present in buchu, and henna. Depending upon the structure and the number of cells present in trichomes, they are classified into the following types: 1. Covering trichomes or non-glandular trichomes or clothing trichomes 2. Glandular trichomes 3. Hydathodes and emergence **Trichomes** | | | | | |:-------------|:-----------|:----------------|:---------------| | **Covering trichomes** | **Glandular trichomes** | **Hydathodes and emergence** | | | | | | | | **Unicellular** | **Multicellular** | | | | * Unbranched* | * Unbranched* | | | | * Uniseriate* | * Biseriate* | | | | * Biseriate* | * Multiseriate * | | | | | * Branched * | | | **Unicellular hair types** 1. Nux-vomica 2. Cannabis 3. Lobelia 4. Senna 5. Gossypium **Multicellular unbranched hair types** 1. Datura 2. Stramonium 3. Digitalis 4. Belladonna 5. Calendula **Multicellular branched hair types** 1. Hamamelis 2. Cascarilla 3. Verbascum thapsus 4. Artemisia **Figure 15.15** Types of covering hairs **Covering trichomes** 1. **Unicellular trichomes** * Lignified trichomes, e.g., nux-vomica, Strophanthus * Short, sharply pointed, curved, e.g., Cannabis * Large, conical, strongly shrunken, e.g., Lobelia * Short, conical, unicellular, e.g., tea, buchu * Strongly waved, thick-walled, e.g., Yerba santa 2. **Multicellular, unbranched trichomes** * **Uniseriate** * Bi-cellular, conical, e.g., Datura * Three-celled, long, e.g., Stramonium * Three- to four-celled long, e.g., Digitalis * Four- to five-celled long, e.g., Belladonna * **Biseriate** * Calendula officinalis * **Multiseriate branched trichomes** * Stellate, e.g., Hamamelis, Helicteris isora * Peltate (platelike arrangement of surrounded cells), e.g., Humulus * Candelabra (Uniseriate, branched axis), e.g., Verbascum thapus * T-shaped trichomes, e.g., Artemisia **Glandular trichomes** These are characterized by the presence of glandular (spherical) cells at the top of the trichome. These are sub-classified as follows: 1. **Unicellular glandular trichomes** The stalk is absent, e.g. Piper betel, Vasaka 2. **Multicellular glandular trichomes** * Unicellular head and unicellular stalk, e.g., Digitalis purpurea. * Unicellular head and uniseriate multicellular stalk, e.g., Digitalis thapsi, Atropa, etc. * Multicellular head, multicellular, biseriate stalk, e.g., sunflower * Unicellular stalk and biseriate head, e.g., Digitalis purpurea * Short stalk with secreting head formed of rosette or club-shaped cells, e.g., Mentha species. * Trichomes with multicellular, multiseriate, cylindrical stalk and rosette of secretory cells, e.g., Cannabis sativa. * Multicellular multiseriate head and multicellular uniseriate stalk, e.g., Indian hemp and tobacco. **Figure 15.16** Types of glandular trichomes 1. Lantana 2. Digitalis lutea 3. Digitalis purpurea 4. Cannabis sativa 5. Atropa belladonna 6. Datura stramonium 7. Primula vulgaris 8. Artemisia maritima 9. Hyoscyamus niger ## **Hydathode and Emergences** These are the organs of absorption or secretion of water developed in certain plants, e.g., Piper betel, London pride, etc. Emergences are otherwise called prickles and are small outgrowths on the epidermal walls of the aerial parts of the plant. They are epidermal and sub-epidermal in origin. They may be present on the stem or fruits. Emergences are not microscopic structures. They are very hard, stout in nature and meant for plant protection, e.g., Rose. **Figure 15.17** Emergence of rose ## **Observation of Powdered Drugs** The preliminary tests for the observation: * Colour observation and its description. * Mixing one quantity of powder with a few drops of water and then leaving it for softening for some time. The water extract and concentrated liquid are dissolved and the gummy and mucilaginous nature may be manifested. * Presence of calcium: A small portion of powder is mixed with dilute sulphuric acid; this mixture will give an effervescence if calcium is present. * Observation of fixed oils: One quantity of powder is pressed on a piece of filter paper, to form an oily spot, which is extended and persists when the paper is heated. * Presence of saponins: One quantity of powder is stirred in a test tube with water; If abundant foam appears, the result is positive; boil softly and a smell is emitted then it is divided in two portions and each portion may be tested for tannins and derived anthraquinone. ## **Lycopodium Spore Method** It is an important analytical technique for powdered drugs, especially when chemical and other methods of evaluation of crude drugs fail as accurate measures of quality. Wallis demonstrated that the spores of Lycopodium are exceptionally uniform in size (25 microns) and that 1 mg of lycopodium contains 94,000 spores. The number of spores per milligram is determined by direct counting and by calculation based on specific gravity and dimensions of the spores. With this valuable technique, it is possible to evaluate powdered drugs with the following characteristics: * Well-defined fragments/particles which may be counted (pollen or starch). * Single-layered tissues or cells whose area may be traced under suitable magnification and the actual area calculated or the objects of uniform thickness, the length of which can be measured at a definite magnification and their real size calculated. Mounts containing a definite proportion of the powder and lycopodium are used and the lycopodium spores counted in each of the fields in which the number or area of the particles in the powder is determined. In this method the moisture content of the powdered material is determined. A mixture of weighed quantities of the powder and lycopodium spores are suspended in a suitable viscous liquid. A drop of this suspension is mounted and examined with a 4 mm objective. The number of lycopodium spores and the number of characteristic particles are counted in 25 various fields. The same experiment is repeated with a second similar suspension. From the mean of these results and a knowledge of the weights of Lycopodium and powder in the mixture, the number of characteristic particles in 1 mg of the powder may be determined. The percentage of an authentic powdered ginger is calculated using the following equation: $percentage = \frac{N \times W \times 94,000 \times 100}{S \times M \times P}$ where, * N = Number of characteristic structures (e.g., starch grains) in 25 fields * W = Weight in mg of Lycopodium taken * S = Number of Lycopodium spores in the same 25 fields * M = Weight in mg of the sample calculated on the basis of sample dried at 105°C * P = 2,86,000 in case of starch grains in ginger powder. Lycopodium spore method can be used for evaluation of powdered clove, ginger, cardamom, nutmeg, umbelliferous fruits, number of pollen grains in pyrethrum powder, etc.