WHO & ICH GUIDELINES FOR ASSESSMENT OF HERBAL DRUGS (PART - 2) T.Y B.PHARM | SEM VI | HERBAL DRUG TECHNOLOGY | PCI SYLLABUS

WHO & ICH GUIDELINES FOR ASSESSMENT OF HERBAL DRUGS (PART - 2) T.Y B.PHARM | SEM VI | HERBAL DRUG TECHNOLOGY | PCI SYLLABUS | UNIT IV.

MICROSCOPIC EVALUATION OF CRUDE DRUGS

Microscopic evaluation help in the identification of right variety & search for adulteration.

Microscopic evaluation allows more detailed examination & mostly used for qualitative

evaluation of organized drugs by their known histological characters in entire & in powder

form.

Microscopic evaluation include…..

• Qualitative microscopy.

• Linear measurement.

• Determination of leaf constant.

• Quantitative microscopy.

QUALITATIVE MICROSCOPY:-

Every plant posses a characteristics tissue structure that can be demonstrated through study

of tissue arrangements, cell wall structure, cell content (calcium oxalate crystals, fibers,

vessels, parenchyma, trichomes etc). For the effective result , various reagents and stain can

be used to distinguished cellular structure.

Eg. A drop of phloroglucinol and concentrated HCL give red stain with lignin. Mucilage are

stained pink with ruthenium red. Starches stained blue with iodine solution.

LINEAR MEASUREMENT:-

These include identification of crude drugs by measurement:-

• Size of starch grains. (e.g.. Diameter of starch grains in cassia bark distinguishes from

cinnamon bark)

• Size of the stomata

• Diameters of phloem fibers(detection of cassia in cinnamon)

• Width of the vessel (detection of clove stalks in powdered cloves)

• Length and width of fibers.

DETERMINATION OF LEAF CONSTANT:-

Various parameters studied here are…..

1.Palisade ratio

2.Vein islet numbers

3.Vein termination numbers

4. Stomata

5. Stomatal number

6. Stomatal index

7. Trichomes & plant hairs

8.Calcium oxalate crystals.

1.PALISADE RATIO:-

It represents the average no of palisade cells beneath each epidermal cell. (Palisade

cells are plant cells located on the leaves, right below the epidermis and cuticle. Palisade

cells contain the largest number of chloroplasts per cell,

Eg. Adhatoda vasica – 5.5 – 6.5

Azadiracta indica – 3 - 3.5

2. VEIN ISLET NUMBERS:-

The no of vein-islet per square mm of leaf surface midway between midrib and margin.

This value is constant for a given species of a plant and used as a characteristics for the

identification of allied species.

Eg: Atropa acuminate 8 - 11,

Cannabis sativa- 20 - 30

3. VEIN TERMINATION NUMBERS:-

The no of vein let termination per square mm of leaf surface midway between midrib and

margin.(unbroken leaf part is required)

E.g. Veinlet termination of Indian senna and Alexandrian senna is quite different for each

other.

4. STOMATA:-

A stoma is a minute epidermal opening with following Characteristics.......

- A central pore

- Two kidney shaped similar cells containing chloroplasts known as Guard cells and

- Varying number of subsidiary (epidermal) cells covering the guard cells.

A stoma is made up of a pair of identical guard cells, forming a pore in centre through which

gaseous exchange takes place.

TYPES OF STOMATA:- Four types

1.Moss type

2.Gymnospermous type

3.Gramineous type

4.Dicotyledonous:- There are 4 basic types of stomata among the dicotyledonus, these

types are distinguished on the basis of the subsidiary cells surrounding the stomata & their

arrangements, The four types are as the following, Paracytic, Diacytic, Anisocytic,

Anomocytic, Actinocytic.

1.Paracytic or Rubiaceous or Parallel cell stomata:-

In this stomata two guard cells covered by two subsidiary cells. 2 of them have their long

axes parallel to the pore, which means that the axes of the subsidiary cells are parallel to the

axes of the pore. E.g. Senna

2. Diacytic or Caryophyllaceous or cross – celled stomata:-

In this stomata two guard cells covered by two subsidiary cells having their long axes

perpendicular to the pore. E.g. Peppermint.

3. Anisocytic or Cruciferous or unequal – celled stomata:-

In this stomata number of guard cells is two but covered by 3 or more subsidiary cells, one

of them is distinctly smaller than the others. E.g. Hyoscymus niger.

4. Anomocytic or Raunculaceous or Irregular celled:-

In this stomata the number of guard cell is two but surrounded by varying number of

subsidiary cell. E.g. Digitalis

5. Actinocytic or Radiate celled stomata:-

Two guard ells are surrounded by radiating subsidiary cells.

5. STOMATAL NUMBER:-

The average number of stomata present per square millimeter of the epidermis is know as stomatal

number.

6. STOMATAL INDEX:-
It is the percentage proportion which the no of stomata form to the total no of epidermal
cell.(each stomata being counted as one cell).
Stomatal number varies considerably with the age of the leaf but stomatal index is relatively
constant for a given species.

Stomatal index can be calculated by using following equation......

S.I= S/E+S*100
Where,
SI – Stomatal index
S = No of stomata per unit area
E = No of epidermal cell in the same unit area.

7. TRICHOMES OR PLANT HAIRS:-

These are warty outgrowth of epidermal cells. A trichomes consists of two parts…..

roots which is based in the epidermal lining and

Body which is outside the epidermal lining.

TYPES OF TRICHOMES:- Trichomes are of three types…

- Covering Trichomes
- Glandular Trichomes
- Hydatodes.

1.COVERING TRICHOMES:-
These are long, slender, in shape and are simple. E.g. Nux vomica.

2.GLANDULAR TRICHOMES:-
Some hairs have multicellular stalk and head the latter
is composed of glandular cells. Stick exudation present on the surface of certain leaves and
buds are secreted by colleters. Exception like salt secreting glands as found in tamarix and
calcium secreting glands of plumbago. E.g. Vasaka, Digitalis.

3. 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.

8. CALCIUM OXALATE CRYSTALS:-
This are organic crystalline compound by virtue of their specific shapes can be utilized for
the identification of herbal drug. Due to this reason they are called as diagnostic characters'
of the plant.

TYPES OF CALCIUM OXALATE CRYSTALS:-
1.Cubical (Cube shape):- All sides are equal. E.g. Senna, Glycyrrhiza.

2. Rhombic (Diamond shape): E.g. Kurchi, Mimosa.

3. Tetragonal:- Two axes same one different. E.g. Onion

4. Monoclinic:- All three axes are un – equal. E.g. Oak gall, Veratrum

5. Acicular:- Long, slender, pointed bundles. E.g. Cinnamon, Squill

6. Rosettes clusters:- (aggregation of crystals) similar to expanded rose flower.
E.g. Clove, Arjuna

7. Microsphenoidal (Minute in structure):- Very minute, deltoid or arrow shaped
crystals occupying full cells in which they occour. E.g. Henbane.

QUANTITATIVE MICROSCOPY:-
LYCOPODIUM SPORE METHOD:-
It is an important analytical technique for powdered crude drugs especially when chemical
and other evaluation of crude drugs fail as accurate measures of quality.
Lycopodium spores are much characterized in shape and appearance & uniform in size (25
um) on average, 94000 spores present per mg of lycopodium powder.

A powder drug is evaluated by this technique if it contains…
- Well defined particles which may be counted eg. Starch grains or pollen grains.
- 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 under suitable
magnification and actual area calculated.

The percentage purity of an authentic drug is calculated using the following
equation….

percent purity of drug = N*w*94000*100/S*M*P

Where, N = No of characteristics structures in 26 fields

W = weight in mg of lycopodium taken

S = no of lycopodium spores in the same 25 fields

M = weight in mg of the sample, calculated on basis of sample dried at 105c

P = Standard value of characteristics structure