My dear, I have
taken this all material from Quality control methods for medicinal plant
materials, WHO Library Cataloguing in Publication Data, World Health
Organization 1998
Precision of measurement
Temperature
Solubility
Unless otherwise specified in the test procedure for the plant material
concerned, the approximate solubility of medicinal plant materials should be
determined at 20°C. Solubility is expressed in terms of "parts",
representing the number of millilitres (ml) of the solvent, in which 1 g of the
solid is soluble. Descriptive terms are sometimes used to indicate the
solubility of a substance, with the following meanings:
Containers
The container and its closure must not interact physically or chemically with
the material within in any way that would alter its quality. The following
descriptive terms are used to indicate general requirements for the
permeability of containers:
A well-closed container
must protect the contents from extraneous matter or from loss of the material
under normal conditions of handling, shipment, or storage.
A tightly closed container must protect the contents from extraneous matter,
from loss of the material, and from efflorescence, deliquescence, or evaporation
under normal conditions of handling, shipment, or storage. If the container is
intended to be opened on several occasions, it must be designed to be airtight
after reclosure.
Protection from light
Medicinal plant
materials requiring protection from light should be kept in a light-resistant
container that - either by reason of the inherent properties of the material
from which it is made or because a special coating has been applied to it -
shields the contents from the effects of light. Alternatively, the container
may be placed inside a suitable light-resistant (opaque) covering and/or stored
in a dark place.
Temperature
Materials that need to be stored at temperatures other than room temperature
should be labelled accordingly.
Humidity
Low humidity may be maintained, if necessary, by the use of a desiccant in the container provided that direct contact with the product is avoided. Care must be taken when the container is opened in damp or humid conditions.
Size of cut
Medicinal plant
materials are used either whole, or in cut or powdered form. Cut medicinal
plant materials are prepared by cutting or crushing the plant into small
pieces. The cut is graded according to the aperture size of the mesh of the
sieve through which the material will pass, and is indicated as follows:
Powder fineness and
sieve size
Powders
The coarseness or fineness of a powder is classed according to the nominal
aperture size expressed in hum of the mesh of the sieve through which the
powder will pass, and is indicated as follows:
Descriptive term
Particle size
Coarse (2000/355) All the particles will pass through a
No. 2000 sieve, and not more than 40% through a No. 355 sieve
Moderately coarse (710/250) All the particles will pass
through a No. 710 sieve, and not more than 40% through a No. 250 sieve
Moderately fine (355/180) All the particles will pass through a
No. 355 sieve, and not more than 40% through a No. 180 sieve
Fine (180) All the particles will pass through
a No. 180 sieve
Very fine (125) All the particles will pass through a No.
125 sieve
Sieves
The wire sieves used to sift powdered medicinal plant materials are classified
by numbers that indicate their nominal aperture size expressed in μm. The
sieves are made of wire of uniform circular cross-section, and have the
following specifications:
Number of sieve Nominal
size of aperture Nominal diameter of wire.
Determination of foreign matter
Medicinal plant
materials should be entirely free from visible signs of contamination by moulds
or insects, and other animal contamination, including animal excreta. No
abnormal odour, discoloration, slime or signs of deterioration should be
detected. It is seldom possible to obtain marketed plant materials that are
entirely free from some form of innocuous foreign matter.
However, no poisonous,
dangerous or otherwise harmful foreign matter or residue should be allowed.
During storage, products should be kept in a clean and hygienic place, so that
no contamination occurs. Special care should be taken to avoid formation of
moulds, since they may produce aflatoxins.
Macroscopic examination
can conveniently be employed for determining the presence of foreign matter in
whole or cut plant materials. However, microscopy is indispensable for powdered
materials.
Any soil, stones, sand,
dust and other foreign inorganic matter must be removed before medicinal plant
materials are cut or ground for testing.
Foreign matter is material consisting of any or all of the
following:
- 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 not
adhering to the medicinal plant materials, such as soil, stones, sand, and
dust.
Sample size
It is difficult to
prepare a pooled sample of foreign matter since most of it adheres to the
medicinal plant materials which are intrinsically non-uniform.
Special procedures
requiring considerable practice are therefore necessary. The problem is
especially difficult when the samples of unbroken crude medicinal plant
materials selected are small; they should be sufficiently large to be
representative.
The following quantities
constitute a sample, unless otherwise specified in the test procedure for the
plant material concerned.
Visual examination and odour
Wrinkled and contracted
leaves, herbs or flowers should be softened and stretched flat ("Preliminary
treatment"). Certain fruits and seeds may also require softening before
dissection and observation of internal characteristics. No other preliminary
treatment is required.
Recommended procedures
Size
A graduated ruler in millimetres is adequate for the measurement of the length,
width and thickness of crude materials. Small seeds and fruits may be measured
by aligning 10 of them on a sheet of calibrated paper, with 1 mm spacing
between lines, and dividing the result by 10.
Colour
Examine the untreated sample under diffuse daylight. If necessary, an
artificial light source with wavelengths similar to those of daylight may be
used. The colour of the sample should be compared with that of a reference
sample.
Surface characteristics,
texture and fracture characteristics Examine the untreated sample. If
necessary, a magnifying lens (6x to 10x) may be used. Wetting with water or
reagents, as required, may be necessary to observe the characteristics of a cut
surface. Touch the material to determine if it is soft or hard; bend and
rupture it to obtain information on brittleness and the appearance of the
fracture plane - whether it is fibrous, smooth, rough, granular, etc.
Odour
If the material is expected to be innocuous, place a small portion of the sample
in the palm of the hand or a beaker of suitable size, and slowly and repeatedly
inhale the air over the material. If no distinct odour is perceptible, crush
the sample between the thumb and index finger or between the palms of the hands
using gentle pressure. If the material is known to be dangerous, crush by mechanical
means and then pour a small quantity of boiling water onto the crushed sample
in a beaker. First, determine the strength of the odour (none, weak, distinct,
strong) and then the odour sensation (aromatic, fruity, musty, mouldy, rancid,
etc.). A direct comparison of the odour with a defined substance is advisable
(e.g. peppermint should have an odour similar to menthol, cloves an odour
similar to eugenol).
Taste
N.B. This test should be applied only if specifically required for a given
plant material.
Inspection by microscopy
Once the material has
been examined and classified according to external characteristics, inspection
by microscopy can be carried out as the next step.
Equipment
The following are required: - a microscope equipped with lenses providing a
wide range of
magnification and a substage condenser, a graduated mechanical stage,
objectives with a magnification of 4x, 10x, and 40x, and colour filters of
ground glass, blue-green; high eyepoint eyepieces are preferred for wearers of
spectacles;
- a lamp, either
separate or incorporated into the microscope;
- a set of polarizing
filters;
- a stage micrometer and
an ocular micrometer to be inserted into a 6x eyepiece and placed
on the diaphragm or,
preferably, a micrometer eyepiece;
- a set of drawing
attachments for the microscope;
- a microburner (Bunsen
type);
- slides and
cover-glasses of standard size;
- a set of botanical
dissecting instruments.
Recommended procedures
Preliminary treatment
Select a representative
sample of the material. Dried parts of a plant may require softening before
preparation for microscopy, preferably by being placed in a moist atmosphere,
or by soaking in water. For small quantities of material, place a wad of
cotton-wool moistened with water into the bottom of a test-tube and cover with
a piece of filter-paper. Place the material being examined on the paper,
stopper the tube and allow to stand overnight or until the material is soft and
suitable for cutting. Use a desiccator for larger quantities of material,
placing water into the lower part instead of the drying agent. Bark, wood and
other dense and hard materials usually need to be soaked in water or equal
parts of water, ethanol and glycerol for a few hours or overnight until they
are soft enough to be cut. Boiling in water for a few minutes may sometimes be
necessary.
Any water-soluble
contents can be removed from the cells by soaking in water. Starch grains can
be gelatinized by heating in water. In certain cases, material can be moistened
with water for a few minutes to soften the surfaces and allow sections to be
cut.
Preparation of specimens
Powdered materials
Place 1 or 2 drops of
water, glycerol/ethanol TS or chloral hydrate TS on a glass slide. Moisten the
tip of a needle with water and dip into the powder. Transfer a small quantity
of the material that adheres to the needle tip into the drop of fluid on the
slide. Stir thoroughly, but carefully, and apply a cover-glass. Press lightly
on the cover-glass with the handle of the needle, and remove excess fluid from
the margin of the cover-glass with a strip of filter-paper. Other fluids may be
used, if necessary, in the same manner.
If the specimen is to be
freed from air bubbles, boil carefully over a small flame of a microburner
until the air is completely removed. Care should be taken to replace the fluid
that evaporates so that the space beneath the cover-glass is completely filled
with fluid at the conclusion of the operation.
Surface tissues of leaves and flowers
To render pieces of thin
leaves transparent, boil them directly on a slide. Cut a piece of leaf into two
portions, turn one piece upper side down and add chloral hydrate TS. Boil the
specimen carefully over a small flame of a microburner and, as soon as bubbles
escape, remove the slide from the flame. When the bubbles have ceased to
appear, boil again until the fragments are transparent.
For slightly thicker but
still papery leaves, cut square pieces, about 6 mm from the edge of the leaf,
if not otherwise specified. The pieces should be taken onethird to one-half of
the way from the leaf-base and should include a midrib or large vein. In
addition, cut 1 or 2 pieces from the edge including 1 or 2 indentations, where
appropriate. For broken or cut leaves take suitable fragments as described
above. Place the fragments in a test-tube containing chloral hydrate TS and
boil for a few minutes until they become transparent. Transfer a fragment to a
slide and cut it into two equal portions. Turn one piece upper side down and
align the two pieces so that both upper and lower surfaces can be observed
under the microscope. Add 1-2 drops of chloral hydrate TS and apply a
cover-glass.
For thicker leaves, that do not become transparent enough when prepared by the
method described above, clarify fragments by boiling with chloral hydrate TS in
a test-tube. Transfer a fragment onto a slide, cut it into two equal portions
and turn one portion upper side down. Scrape the surface of the two portions
using a scalpel until only a single layer of epidermis remains. Wash the
epidermis with drops of chloral hydrate TS or glycerol /ethanol TS to remove
any residues. If possible, turn both parts of the epidermis upper side down,
and add one of the above fluids.
For very thick or fleshy
leaves, pull off the upper and lower parts of epidermis by winding the softened
leaf around the index finger, pressing with the thumb and the middle finger
against the index finger and carefully incising, catching the incised part with
forceps, and bending the epidermis backwards carefully.
Petals and sepals of
flowers may be treated in a similar manner.
Sections
Select representative pieces of the material being examined and cut into
suitable lengths, one end of which is softened and smoothed. Prepare cross or
transverse sections by cutting with a razor blade or microtome at a right angle
to the longitudinal axis of the material. Prepare longitudinal sections by
cutting in parallel with the longitudinal axis, either in a radial direction (radial section) or in a tangential
direction (tangential section).
Thick materials, such as
wood, woody stems, rhizomes and roots can be cut by holding the softened
material between the thumb and index finger, supported by the middle finger or
by holding it in the central hole of a hand microtome.
Thin materials such as
leaves, petals and slender stems should be bound between two halves of a piece
of elder-pith or other suitable support. If necessary, moisten the surface to
be cut and the blade with ethanol (∼375 g/l) TS. Cut the sections as thinly and
evenly as possible. Transfer the sections with a brush moistened with ethanol (∼150g/l) TS to a dish containing ethanol (∼150g/l) TS. Select satisfactory sections for the
preparation of the slides. For certain materials a sliding microtome may be
used. Seeds and fruits that are very flat, or that are small and spherical, and
cannot be held in the manner described above may be inserted into a notch cut
into a small rubber stopper or embedded in hard paraffin (paraffin wax) as
follows. Prepare a hard paraffin block, rectangular in shape, measuring about 7
x 7 x 15 mm, and melt a small hole in the centre of one end using a heated
needle or thin glass rod. Press the material, which should be dry or softened
by exposure to moisture, into this hole. Then prepare sections with a
microtome. For the examination of mucilage, aleurone grains or spherical
aggregations of inulin, cut the material without using water.
Clarification of microscopic particles
The presence of certain
cell contents, such as starch grains, aleurone grains, plastids, fats and oils,
may render sections non-translucent and obscure certain characteristics.
Reagents that dissolve some of these contents can be used in order to make the
remaining parts stand out clearly or produce a penetrating effect. This renders
the section more transparent and reveals details of the structures.
If the refractive index
of the clarifying agent is close to that of the cell structure, the material
being tested becomes almost invisible; if it differs appreciably, the material
becomes markedly evident.
The most frequently used
clarifying agents are described below (for the methods of preparation, see
section 22, "Reagents and solutions").
Measurement of specimens
Equipment
Use a microscope with an ocular micrometer to measure the size of small
objects. The scales should be calibrated using a stage micrometer, consisting
of a glass slide of usual size, upon which a scale is engraved or photographed,
usually 1 or 2 mm long, in 0.1 and 0.01 mm divisions. The ocular micrometer
consists of a small disc of glass, across the diameter of which a 100-line scale
is engraved or photographed. The disc is placed into the eyepiece.
Place the ocular micrometer scale in the eyepiece of the microscope. Put the stage micrometer on the microscope stage and focus on the scale divisions. Both scales should be sharply defined in the field of view. Turn the eyepiece to place the scales in a parallel position and, if necessary, move the stage micrometer until the starting lines of both scales coincide (Fig. 1). Now find another point, as far along the scale as possible, where two other division lines are exactly superimposed. Count the number of divisions on the ocular micrometer and the corresponding length on the stage micrometer scale, in order to determine the length that is equivalent to one division on the ocular micrometer scale; for example, if 100 divisions on the ocular micrometer scale are equal to 30 divisions on the stage micrometer scale, since the divisions on the stage micrometer scale are 0.01 mm apart, 100 ocular micrometer divisions are equivalent to 0.30 mm, therefore each division on the ocular micro-meter scale represents 3.0 μm. Since the calibrations apply only for a particular lens combination, it is advisable to determine and record the ocular micrometer values for the most frequently used combinations.
Alignment of the stage micrometer and the ocular micrometer
Method
Place the specimen on the microscope stage and focus on the object to be
measured. Superimpose the ocular micrometer scale and read off the dimensions
of the object. Multiply the number of scale divisions by the micrometer value
to give the actual dimension in micrometres. By this method, using a 40x
objective and a 6x eyepiece, measurements are correct to the nearest 2 μm, i.e.
a dimension of 20 μm is liable to an error of 2 μm, or 10%, or ±5%; a dimension
of 100 μm is liable to an error of 2% or ±1%.
For curved and elongated
objects, the measurement of lengths may be made using a microscope equipped
with a drawing apparatus or a camera lucida. The instrument should be set up so
that the image of the object, the drawing paper and the pencil are in focus
simultaneously. With the stage micrometer in place, trace the lines of the
stage micrometer scale upon the paper fastened on a drawing board. Tilt the
drawing board, if necessary, until the divisions drawn upon the paper are
equally spaced. The magnification is determined by measuring the distance
between selected lines on the drawing paper and dividing by the distance
between the corresponding lines on the stage micrometer. Place the specimen on
the microscope stage and trace the image of the object on the paper.
Superimpose a coloured thread along the length of the object drawn on the paper
and after straightening it, measure the length of the thread using a
ruler graduated in millimeters; divide the measured length by the magnification
to give the actual length of the object.
Leaf stomata
Types of stoma
In the mature leaf, four
significantly different types of stoma are distinguished by their form and the
arrangement of the surrounding cells, especially the subsidiary cells:
• The anomocytic or ranunculaceous (irregular-celled)
type; the stoma is surrounded by a varying number of cells, generally not
different from those of the epidermis.
• The anisocytic or cruciferous (unequal-celled) type; the stoma is usually surrounded by three or
four subsidiary cells, one of which is markedly smaller than the others.
• The diacytic or caryophyllaceous (cross-celled) type; the stoma is accompanied by two subsidiary
cells, the common wall of which is at right angles to the stoma.
• The paracytic or rubiaceous (parallel-celled) type; the stoma has two subsidiary cells, of which
the long axes are parallel to the axis of the stoma.
In describing an epidermis where certain stomata differ from the predominant
type, the term applying to the majority of stomata is used.
Determination of the stomatal index
Place fragments of
leaves, about 5 x 5 mm in size, in a test tube containing about 5 ml of chloral
hydrate TS and heat on a water bath for about 15 minutes or until the fragments
are transparent. Transfer a fragment to a slide and prepare it as described
earlier, the lower epidermis uppermost, in chloral hydrate TS; place a small
drop of glycerol/ethanol TS at one side of the cover-glass to prevent the
material from drying. Examine under a microscope with a 40x objective and a 6x
eyepiece, equipped with a drawing apparatus. Mark on the drawing paper a cross
(χ) for each epidermal cell and a circle (o) for each stoma. Calculate the
stomatal index as follows:
stomatal index = S x100
-------------------
E+ S
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