Microscopical Evaluation

MICROSCOPICAL EVALUATION

Microscopic evaluation is indispensable in the initial iden-tification of herbs, as well as in identifying small fragments of crude or powdered herbs, and in the detection of adulterants (e.g. insects, animal faeces, mold, fungi, etc.) as well as identifying the plant by characteristic tissue features. Every plant possesses a characteristic tissue structure, which can be demonstrated through study of tissue arrangement, cell walls, and configuration when properly mounted in stains, reagents, and media. Lignin stains red or pink with a drop of phloroglucinol and concentrated hydrochloric acid. Mucilage is stained pink with rhuthenium red, and N/50 iodine solution stains starch and hemicellulose blue.

The characteristic features of cell walls, cell contents, starch grains, calcium oxalate crystals, trichomes, fibres, vessels, etc. have been studied in details. Surinam quassia is recognized by the absence of calcium oxalate and pres-ence of uniseriate medullary rays, crystal fibres, and wavy medullary rays of cascara bark, lignified trichomes, and plasmodesma in nux vomica. Stone cells are absent in the frangula bark, whereas they are present in cascara. Presence of pith in rhizomes and absence in roots, warty trichomes of senna, and presence or absence of crystals of aloin indicates different varieties of aloes, glandular trichomes of mint, etc. The powder of clove stalks contains sclereids and calcium oxalate crystals, but cloves do not contain these two. Rauwolfia micrantho, R. densiflora, and R. perokensis are found to serve as an adulterant for R. serpentine. The roots of these species can be

 differentiated from R. serpentine by the presence of sclerenchyma in the above species which is absent in R. serpentine.

The techniques like microscopic linear measurements, determination of leaf constants and quantitative microscopy are also used in this evaluation.

Linear measurements include size of starch grains, length and width of fibres, trichomes, etc. The diameter of starch grains present in ipecacuanha assists in distinguishing its varieties. The diameter of starch grains in cassia bark distinguishes from cinnamon and detects senna stalk in powdered senna leaf. The size of the stomata in leaves of Barosma betulina distinguishes it from other species of Barosma. The diameter of phloem fibres aids the detection of cassia in cinnamon, and the width of the vessel helps to detect clove stalks in powdered cloves. Measurements of diameter for the identification of commercial starches and for the detection in them of foreign starch are few examples of linear measurements.

Determination of leaf constants include: stomatal number, stomatal index, vein islet, vein termination number, and palisade ratios. Stomatal number is average number of stomata per sq. mm of epidermis of the leaf.

Stomatal index: It is the percentage which the numbers of stomata form to the total number of epidermal cells, each stoma being counted as one cell. Stomatal index can be calculated by using the following formula:

Stomatal Index (S.I.) =    S / E + S × 100

where,

S = number of stomata per unit area and

E = number of epidermal cells in the same unit area.

Timmerman (1927) and Rowson (1943) were amongst the first few to investigate leaf drugs for stomatal number and stomatal index.

Vein-islet number: It is defined as the number of vein islets per sq. mm of the leaf surface midway between the midrib and the margin. It is a constant for a given species of the plant and is used as a characteristic for the identification of the allied species. Levin in 1929 determined vein-islet numbers of several dicot leaves.

Veinlet termination number: It is defined as the number of veinlet termination per sq. mm of the leaf surface midway between midrib and margin. A vein termination is the ultimate free termination of veinlet. Hall and Melville in 1951 determined veinlet termination number of distinguishing between Indian and Alexandrian Senna.

Palisade ratio: It is defined as the average number of palisade cells beneath each epidermal cell. Unlike vein-islet number for the determination of which an unbroken portion of the leaf is required, palisade ratio can be deter-mined with the powdered drug. The technique of palisade ratio determination was introduced by Zorning and Weiss (1925) in their studies on Compositae.

One example is vein-islet number of Alexandrian senna is 25–29.5, whereas Indian senna is 19.5–22.5. Stomatal index of Alexandrian senna is 10–15, whereas that of Indian Senna is 14–20.

Quantitative Microscopy (Lycopodium Spore Method)

This is an important technique employed in identification of crude drug when chemical and physical methods are inapplicable. Using this, one can determine the proportions of the substances present by means of the microscope, using the Lycopodium spore method.

The powdered drugs with well-defined particles which may be counted—for example, starch grains or single-layered cells or tissues—the area of which may be traced under suitable magnification or the objects of uniform thickness, and the length of which, can be measured under suitable magnification and actual area calculated are usually evaluated using this method.

Adulterated starchy drugs can be determined by counting the number of starch grains per mg and calculating the amount from the known number of starch grains per mg of the pure starch or starchy material.

Thus, if spent ginger is the adulterant, one knows that ginger contains 286,000 starch grains per mg, and the amount used as an adulterant can be calculated by using this figure. The percentage purity of an authentic powdered ginger is calculated using the following equation:

[ N × W × 94,000 × 100 ]/ [ S × M × P ] = % purity of drugs

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 basis of sample dried at 105°C; and

P = 2,86,000 in case of ginger starch grains powder.

If the material is one for which a constant is not available, it is necessary to determine one by a preliminary experiment.