Evaluation /Quality Control / assessment of crude drug

 according to WHO guidelines 

(Part-II)

 Microscopic evaluation (Leaf Constant)

(S.Y. B. Pharm Unit -IV ) Unit -I


(T.Y. B. Pharm Sem -VI ) -Unit - IV


(Final Yr. B. Pharm) Section -II

As per PCI Syllabus 

METHODS OF DRUG EVALUATION

The evaluation of a drug is done by following methods.
1. Organoleptic evaluation
2. Morphological evaluation
3. Microscopic evaluation
4. Physical evaluation
5. Chemical evaluation
6. Analytical evaluation
7. Biological evaluation

Microscopic evaluation 


Helps in the  study of the presence of adulterants & correct identification of the medicinal plants. Drug is soaked in water if it is not fresh, then fine T.S. is taken and stained for  study of the arrangement of the cells important staining liquids used are Phloroglucinol, and HCL for lignified tissues, Chlor-zinc iodide for Cellulose tissues, Ruthenium red for gums & mucilage containing cells.

The slides of this test drug are compared with slides of the authentic crude drugs. This helps in the study of substances like starch, fixed oils, alleurone grains, calcium oxalate, mucilage, etc. E.g. P. amaraus shows Wavy walled epidermal parenchyma whereas P. madraspatensis shows straight walled epidermal parenchyma.

i. Palisade Ratio
ii. Stomata
iii. Stomatal Number
iV. Stomatal Index
v. Vein-islet Number
vi. Vein-termination Number
vii. Trichomes or plant hairs
viii. Calcium oxalate crystals
ix. Quantitative Microscopy: Lycopodium spore method

I. Palisade ratio
It represents the average number of palisade cells beneath one epidermal Cell, using four continuous epidermal cells for the count.
It is determined from powdered drug with the help of camera-Lucida 

Examples:
Adhatoda vasica: 5.5-6.5
Cassia angustifolia: 5.5-10.0
Digitalis lanata: 2.5-6.5


II. Stomata:

(primary and important function is gaseous exchange)
A minute epidermal opening present on Arial parts of plants, Stomata consists of central pore, two kidney shaped similar cells (guard cells) and varying number of subsidiary cells.
Epidermis of leaf shows different characteristics e.g. cuticle, stomata, trichomes.

Types of stomata (Four types)
•Moss type 
• Gymnospermous type
•Gramineous type
Dicotyledonous---it is having diagnostic significance and classified based on form of arrangement of subsidiary cells. 

a.Paracytic or rubiaceous or parallel-cell stomata:
In this stomata two guard cells covered by two subsidiary cells, e.g. Senna

b. Diacytic or caryophyllaceous or cross-celled stomata: In these stomata the guard cells are covered by two subsidiary cells on right angle to that of stomata. e.g. peppermint .

c. Anisocytic or cruciferous or unequal-celled stomata: In this stomata number of guard cells is two but covered by three subsidiary cells and in that one is small in size with other two e.g. Datura.

d. Anomocytic or ranunculaceous or irregular celled: In this type stoma is surrounded by varying number of subsidiary cells. e.g. digitalis.

e. Actinocytic or radiate celled stomata: two guard cells are surrounded by radiating subsidiary cells.




III. Stomatal Number:
The average number of stomata present per square millimeter of the epidermis is known as stomatal number.

lV. Stomatal index

 It is the percentage proportion of the number of stomata to the total number of epidermal cells.

Stomatal number varies considerably with the age of the leaf but stomatal index is relatively constant for a given species.
Stomatal index calculated by:

            S.I = S/E+S   

Where,
S.I. = Stomatal index
S = Number of stomata per unit area
E=N umber of epidermal cells in the same unit area 

V. Vein-islet Number

Vein-islet number is defined as the number of vein-islets per sq.mm. of leaf surface

VI. Vein-termination Number

It is defined as the number of veinlet terminations per. sq. mm of the leaf surface between midrib and margin. 

VII. Trichomes or plant hairs

These may be referred to as plant hairs. These are warty outgrowth of epidermal cells. A trichome consists of two parts, root which is based in the epidermal lining and body which is outside the epidermal lining.

Trichomes are of three types

covering trichomes, 
Glandular trichomes, 
Hydatodes.

They may be unicellular or multicellular

Covering trichomes:

These are long, slender in shape and are simple, e.g. Nux Vomica.

Glandular trichomes (Colleters)

Some hairs have multicellular stalk and head the latter is composed of glandular cells. Sticks exudations present on the surface of certain leaves and buds are secreted by colleters. Exceptions like salt secreting glands as found in Tamarix and calcium secteting glands of Plumbago, e.g Vasaka, digitalis.

Hydatodes:

These are of special kind and their examples are London Pride, Piper betel. They are developed for water absorption or secretion.
Vesiculate hairs in which the surface of leaves and young stem appear to be covered by ice beads.



VIII. Calcium oxalate crystals

Several cell contents present in vegetable drugs. The inorganic crystalline compound by virtue of their specific shapes can be utilized for the identification of herbal drugs. Due to this reason they are called as diagnostic characters of the Plant.
i. Cubical (cube shape) e.g., Senna, Glycyrrhiza.
ii. Rhombic (diamond shape) .
iii. T etragonal e.g., Onion.
iv. Mono clinic (all three axes are un-equal) e.g., Gall.
v.  Acicular (long slender, Pointed, bundles) e.g. Squill, Cinnamon
vi. Rosettes clusters (aggregation 0f crystals) e.g. Clove‘ Arjuna
vii. Microsphenoidal (minute in structures) e.g., Henbane.

IX, Quantitative microscopy

Lycopodium spore method: 
it is used when especially chemical and other methods of evaluation of drugs fail to determine quality. Lycopodium spores are much characterized in shape and appearance and uniform in size (25 pm) on average, 94000 Spores present/ mg of Lycopodium powder.  
It consists of:
•Well defined particles which may be counted.
•Single layered cells or tissues the area of which may be traced under suitable magnification and actual area calculated
•The objects of uniform thickness, the length of which can be measured, and actual area calculated.