Herbal Pharmacognosy 312
Practical manual
Coordinators: Miss M. Hess and MissS. Nyati
Pharmacognosy Practicals
List of plants
1. Senna
2. Digitalis
3. Belladonna
4. Stramonium
5. Ginger
6. Liquorice
7. Lobelia
8. Cascara
9. Clove
10. Rhubarb
11. Fennel
12. Nux vomica
13. Cardamom
SENNA
Consists of dried leaflets of the paripinnate leaves of Tinnevelly senna
(C. angustifolia)
DIAGNOSTIC MICROSCOPICAL CHARACTERS:
(i) Thin-walled polyhedral cells (straight or slightly wavy walls) and
paracytic stomata of the epidermis. Mucilage is present in the inner
half of many epidermis cells. (Stain with Ruthenium Red solution?)
(ii) Epidermal covering trichome: conical, unicellular, thick-walled,
warty, frequently curves near the base. May occur scattered or
attached to fragments of epidermis. Scars frequently visible on
both epidermises, showing the point of attachment of the
trichomes.
(iii) Palisade tissue, occurring beneath both epidermal surfaces,
those below the upper epidermis being elongated and fairly
straight-walled, while those beneath the lower epidermis are
shorter, with serous walls.
(iv) Rounded spongy cells of the mesophyll between the two
palisade layers frequently containing cluster crystals of calcium
oxalate.
(v)Pericyclic fibres, occurring in groups: those are thick-walled,
lignified, (stain with Phloroglucinol/HCL) with a few pits, flanked by a
parenchymous sheath, the cells of which frequently contain single
prisms of calcium oxalate.
CONSTITUENTS OF SENNA LEAF
Chiefly anthraquinone-type compounds both free and combined as
glycosides. Their presence may be detected by means of Bornträger’s
test. (Positive)
See monograph of Senna Fruit and carry out the test as described.
MICROSCOPICAL EXAMINATION:
(i) Warm 100mg of powdered Senna leaf in a test tube with 5ml of
70% alcohol and shake well. Filter, using a further 5ml of 70%
alcohol to rinse the tube and residue
(ii) Reserving a little of the residue obtained, place the remainder in
a small test tube with 2ml of chlorohydrate solution. Heat, with
shaking in a beaker of boiling water until the fragments are
sufficiently transparent, completing the process by boiling the
test tube gently in a low flame.
(iii) On a slide place one drop of the cleared (filtered) material, add
one drop of glycerine (50%) and examine microscopically after
covering with a cover slip. Use both L.P and H.P and make
sketches of the diagnostic features mentioned above.
(iv) On a slide place one drop of cleared material, add one drop of
Phloroglucinol, one drop of conc. HCL and one drop of Glycerine
(50%). Examine using L.P. and H.P.
DIGITALIS
Consists of the dried leaves of Digitalis purpurea
DIAGNOSTIC MICROSCOPICAL CHARACTERS:
(i) Epidermal cells in surface view:
Upper epidermis: irregularly shaped cells with slightly thickened
walls, which may show beading or pitting. The underlying
palisade cells are fairly large and loosely packed.
Lower epidermis: smaller cells having thin, sinuous walls
Both epidermises show occasional scars (cicatrices) where
trichomes were attached.
(ii) Stomata: circular, anomocytic, abundant on lower epidermis,
absent or infrequent on upper epidermis.
(iii) Trichomes, scattered or attached to fragments of epidermis,
both covering and glandular types. Former are numerous,
unistriate, 3-5 cells I length, conical and bluntly pointed, with
thin faintly wary walls. Frequently one or more cells may be
collapsed. Latter are two types, those which are more numerous
having single-celled stalk and a bicellular head (rarely
unicellular): others, less numerous, having unistriate
multicellular stalk and a unicellular head.
(iv) Occasional fragments of thin-walled parenchyma from the cortex
of the midrib and larger veins, composed of longitudinally
elongated cells
(v) The absence of calcium oxalate crystals, sclerenchymatous
elements and fibres.
(vi) The large water-pores, one or rarely two, situated at the apex of
most of the teeth.
CONSTITUENTS OF DIGITALIS LEAF
Digitalis is chiefly a mixture of cardiac glycosides, together with
hydrolytic enzymes and saponins. Digitalis leaf responds to the Keller-
Kiliani test (for digitoxose).
MICROSCOPICAL EXAMINATION:
Clear some of the powdered drug by heating in a test tube with chloral
hydrate solution, in a beaker of boiling water. Make mounts of the
cleared material I chloral hydrae solution and glycerine (50%0, making
sketches of the diagnostic features given above.
Note in each case the physical characteristics of the powdered drug
i.e. colour, odour and taste.
BELLADONNA
Belladonna herb consists of the leaves, or leaves with other aerial
parts of Atropa belladonna. These are collected when the plants are in
flower and dried. The plant contains no less than 0.3% of the alkaloids
of Belladonna herb, calculated hyoscomine.
DIAGNOSTIC MICROSCOPICAL CHARACTERISTICS
1. The anomocytic stomata on both epidermal surfaces, more
numerous on the lower epidermis
2. The epidermal cells of the upper epidermis have a slightly wavy
outline and strongly striated cuticle while whereas those of the
lower epidermis have more sinuous walls. In the regions over the
veins these cells are straight-walled and elongated.
3. Trichomes, more numerous on younger leaves, either uniseriate
covering trichomes, short clavate glandular (multi cellular
heads) or long glandular (uniseriate stalks and unicellular heads)
(conical 4-5 celled stalk, smooth thin walls).
4. The single layer of palisade parenchyma is below upper
epidermis.
5. Conspicuous idioblasts composed of parenchymatous cells filled
with microspheroidal crystals of calcium oxalate. These occur in
the spongy mesophyll in the palisade mesophyll of the leaves
and in the stem parenchyma. In the powdered drug individual
crystals may be found scattered or broken.
6. The occasional stem fragments showing large, articulately
thickened lignified xylem vessels, associated with thin-walled
lignified fibres and xylem parenchyma. Fragments of unlignified
parenchyma from the pith and cortex frequently include
idioblasts containing microspheroidal crystals of calcium oxalate.
7. The premedullary phloem of the midribs and stem
8. The pollen grains mounted in chloral hydrate solution, sub
spherical with three pores and three furrows, the exine is
marked with numerous fine pits in a radiating arrangement;
contents…. minute starch granules and oil droplets
9. Fragments of endothelium
CONSTITUENTS OF BELLADONA
Alkaloids of the tropane type, chiefly (-) hyoscyamine, together with
volatile bases and B methylaesculetin, which gives a blue fluorescence
in ultra violet light
Preliminary tests
i) Note colour, odour, taste…
ii) Mix a small quantity of the powder with a few drops of water,
allow standing. Note any changes
iii) Shake a little powder in half-a-tube-full of water, note frothing
GENERAL TEST FOR PRESENCE OF ALKALOIDS AND NOTE THE
RESULTS:
Shake 300 mg of powdered drug with 6 ml 70% alcohol in a large test
tube warming slightly. Filter and evaporate in a small dish over boiling
water (do not completely dry). To the residue, add 2 ml of diluted HCL
to dissolve the alkaloids. Filter and pass more diluted. HCL through the
filter to produce 2 ml of filtrate. Divide filtrate into four equal portions
and treat as follows:
(i) 0.5 ml of filtrate – control
(ii) 0.5 ml filtrate plus 1 drop of Meyer’s Reagent
(iii) 0.5 ml filtrate plus 1 drop N/50 Iodine solution
(iv) 0.5 ml of filtrate plus 1 drop of 5% Tannic acid solution
MICROSCOPICAL EXAMINATION
A. Clear 100 mg of powdered drug by boiling with 4 ml chloral
hydrate solution in a test tube until sufficiently transparent.
Prepare two mounts as follows:
(i) In chloral hydrate and 50% glycerine
(ii) In phloroglucinol and HCL and 50% glycerine
Examine and sketch the diagnostic features mentioned
B. Make mounts of the powdered drug in the following reagents,
examining for cells contents:
(a) Iodine water
(b) Phloroglucinol and HCL
(c) Soudan IV solution
Examine and sketch the diagnostic features mentioned
STRAMONIUM
Stramonium consists of the dried leaves and flowering tops of Datura
stramonium and Datura tatula, or a mixture of both species,
containing not less than 0.25% of the alkaloids of stramonium
calculated as hyoscyamine.
MICROSCOPICAL FEATURES
A. Leaf:
1. Trichomes, more numerous on younger leaves and are
either simple or uniserate conical covering trichomes. (3-5
celled, warty, short, clevate, glandular trichomes, 2-7
celled head).
3. Epidermal cells with more or less sinuous walls and smooth
cuticle.
4. Stomata:Anisocytic
5. Mesophyll with single palisade layer, beneath which is
found a crystal layer with cluster crystals, occasional
prisms or micro spheroids of calcium oxalate.
6. Midribs containing an arc of several vascular bundles, with
collenchymas beneath both epidermal surfaces.
B. Stem:
1. Trichomes, few, uniseriate, up to 800 microns In length.
2. Few primary phloem fibres
3. Xylem: numerous fibres and large spiral, annular and
reticulate vessels
4. Perimedullary phloem present
5. Pith parenchyma with calcium oxalate crystals as I leaf.
C. Calyx:
Outer epidermis with scattered simple uniserate trichomes (20-
40 microns long) anomocytic stomata, inner epidermis with
short glandular trichomes and rare stomata, cells of both
epidermises having wavy anticlinal walls, mesophyll containing
many idioblasts with crystals or prisms.
D. Corolla:
It has simple uniserate trichomes (lower half of inner epidermis),
mesophyll with calcium oxalate crystals, anthers with
endothelium cells supported by anatomising bands of
thickening, pollen grains, in chloral hydrate solution; sub
spherical 60-80 microns in diameter, with three pores and
coarsely granular surface.
E. Seeds:
Outer wall of epidermis of young seeds is mucilaginous.
DIAGNOSTIC MICROSCOPICAL CHARACTERISTICS
1. Epidermal cells as described in A2 above, these of the lower
epidermis having more sinuous walls.
2. Anomocytic stomata, more numerous on lower than upper
epidermis
3. Epidermal trichomes as in A1 above.
4. Crystal layer A4 above, below the palisade layer, most cells
containing a large cluster crystal of calcium oxalate, absent from
the cells adjoining the veins
5. Prisms and micro spheroids of calcium oxalate in the
parenchyma of the midrib and stem
6. The trichomes, numerous xylem fibres and vessels of the stem,
B3 above
7. Pollen grains as in D above.
CONSTITUENTS OF STRAMONIUM
Alkaloids of the tropane type, chiefly hyoscyamine and hyoscine, Using
300mg of powdered drug, carry out the general test for alkaloids.
General test for prescence of alkaloids and note the results:
Shake 300 mg of powdered drug with 6 ml 70% alcohol in a large test
tube warming slightly. Filter and evaporate in a small dish over boiling
water (do not completely dry). To the residue, add 2 ml of diluted. HCL
to dissolve the alkaloids. Filter and pass more diluted. HCL through the
filter to produce 2 ml of filtrate. Divide filtrate into four equal portions
and treat as follows:
(v) 0.5 ml of filtrate – control
(vi) 0.5 ml filtrate plus 1 drop of Meyer’s Reagent
(vii) 0.5 ml filtrate plus 1 drop N/50 Iodine solution
(viii) 0.5 ml of filtrate plus 1 drop of 5% Tannic acid solution
MICROSCOPICAL EXAMINATION
1. Examine suitably cleared portions of leaf and powdered drug in
each case preparing mounts in:
(a) Chloral hydrate solution and 50% glycerine
(b) Phloroglucinol solution, HCL and 50% glycerol
Make annotated sketches of the diagnostic features seen
2. Make mounts of dry powdered drug
(d) Iodine water
(e) Phloroglucinol and HCL
(f) Soudan IV solution
Examine for cell contents
GINGER
Ginger is the rhizome of Zingiber officinale scraped to remove the dark
outer skin, dried in the sun and is commercially known as unbleached
Jamaica ginger.
MICROSCOPICAL FEATURES
i. Cork: Absent in the official drug (scraped)
ii. Cortex: Of thin-walled, rounded or oval parenchyma with
scattered vascular bundles and numerous sub spherical oleorein
cells (idioblasts of about 40 -80 microns in diameter), with
cuticularised walls containing yellow or red-brown oleoresin.
iii. Endodermis: Free from starch, is a single layer of somewhat
collapsed cells with slightly thickened radial walls.
iv. Vascular bundles: Collateral scattered throughout the cortex and
stele. Consists of 1-14 reticulate or spiral vessels (up to 70
microns in diameter) giving no characteristic lignin reaction, and
phloem tissue showing well-marked septate fibres (up to 30
microns wide and 600 microns long), with oblique slit-shaped
pits.Immediately inside the endodermis is a row of early
contiguous collateral bundles, usually without accompanying
fibres.
v. Stele: Of thin walled parenchyma containing scattered vascular
bundles (as in iv above), idioblasts (as in the cortex), and a
second type of secretory cells (idioblasts about 8-0 microns wide
and 130 microns long). The latter are found singly or in axial
rows adjacent t the vessels, and are filled with dark reddish-
brown contents.
vi. Starch granules: Filling the parenchyma of cortex and stele. Are
flattened, ovate to sack-shaped with markedly eccentric hilum
(5-60 microns long, up to 25 microns wide and 7 microns thick),
markedly fine transverse striations.
DIAGNOSTIC MICROSCOPICAL FEATURES OF THE POWDERED
DRUG
1. Abundant thin-walled parenchyma of the ground tissue
containing grains
2. Starch grains: as in (vi) above
3. Vessels: spiral or reticulate, giving no lignin reaction, often
accompanied by pigment cells as in (v) above.
4. Fibres: as in (iv) above
5. Oleo-resin cells: as in (ii) above, bright yellow in the uncleared
drug. Oleo-resin may also be seen on fragments or droplets.
6. Cork: sclereids, calcium oxalate and trichomes are absent.
LABORATORY TESTS
Preliminary tests
1. Note the colour, odour and taste.
2. Mix a small quantity of the powder with a few drops of the water
and allow to stand. Note any changes.
MICROSCOPICAL EXAMINATION
A. Cut transverse sections of fresh ginger rhizome and mount in the
following regents:
1. Glycerine 50%
2. Choral hydrate solution + 50% glycerine – warm
3. Iodine water + glycerine 50%
4. KOH 5% + glycerine 50% - warm
5. Phloroglucinol/HCL + glycerine 50%
B. Make mounts of uncleared powdered drug in the following
reagents:
1. Glycerine 50%
2. Water + glycerine 50%
3. Phloroglucinol/HCL + glycerine 50%
4. Soudan IV solution + glycerine 50%
5. KOH 5% + glycerine 50%
C. Clear 50- 100 mg of powdered drug with about 4 ml chloral
hydrate solution:
1. Glycerine 50%
2. Phloroglucinol/HCL + glycerine
Examine and sketch features seen.
LIQUORICE
Liquorice consists of the dried peeled or unpeeled root and stolon of
various species of Glycyrrhiza, yielding a drug having a sweet taste
almost free from bitterness.
MICROSCOPICAL FEATURES
Stolon:
1. Cork of 10 -20 or more layers of brick-shaped cells having red-
brown amorphous contents. The inner layers having thicker,
colourless walls.
2. Phelloderm: Usually 1-3 layers of radially arranged
parenchymatous cells containing isolated calcium oxalate prisms.
3. Secondary phloem: Broad with wide parenchymatous medullary
rays
4. Phloem fibres: With thickened yellow cellulosic ( in the inner part)
and lignified (in the outer part) walls in groups of 10-50 radially
arranged fibres, each group surrounded by a parenchymatous
sheath of small cells containing a prism of calcium oxalate (10-35
microns long)
5. Cambium: Incomplete, three or more rows of flattened cells
6. Secondary xylem divided by radial medullary rays (3-5 cells
wide), and consisting of
(a) Large vessels (80-200 microns in diameter) with thick
yellow pitted or reticulately thickened walls with numerous
bordered pits having a slit-shaped opening
(b) Groups of lignified fibre with associated crystal sheaths
similar to those of the phloem.
(c) Xylem parenchyma of two kinds, ones with thickened
pitted walls between the vessels and those with thin walls.
7. Pith parenchyma: Longitudinal rows.
Root:
Closely resembling stolon but with pith absent. Xylem is tetrarch
consisting usually of 4 principal medullary rays at right angles to each
other.
Abundant starch grains are present in all parenchymatous tissues.
Mostly simple rounded to ovoid, with the large granules showing slit-
shaped hilum. Cork, phelloderm (secondary cortex) and part of the
secondary phloem are absent from the peeled drug.
DIAGNOSTIC MICROSCOPICAL FEATURES OF POWDERED DRUG
Powder prepared from peeled drug is usually more yellow in colour
than that from unpeeled drug. Both have a sweet taste and a faint,
characteristic colour. Unless otherwise specified, powdered liquorice is
always prepared from peeled drug.
I. Cork cells as in cork, in the unpeeled drug
II. Starch granules as above
III. Thick-walled lignified or partially lignified fibres from both xylem
and phloem, groups of 10-50 with accompanying
parenchymatous sheath of small rectangular cells containing a
prism of calcium oxalate.
IV. Large xylem vessels as in above, usually accompanied by
lignified xylem parenchyma.
V. Calcium oxalate prisms, both in crystal sheath and scattered in
the parenchyma of the pith and medullary rays.
VI. Thin-walled parenchymatous tissue from cortex, medullary rays
and pith. (cells usually filled with starch).
LABORATORY TESTS
Preliminary tests
1. Note the colour, odour and taste
2. Mix a small quantity of the powder with a few drops of water and
allow to stand. Note any changes
3. Shake a little powder in half a test-tube-full of water, note
frothing
Chemical test
a. For the presence of free glucose in Liquorice (reducing sugar)
Macerate 100 mg of powder with 5 ml of water for 10 minutes,
shaking at frequent intervals (Note frothing – presence of
saponin-like glycoside). Warm gently, filter and to the filtrate add
an equal volume of Fehling’s solution. Place the test tube in a
beaker of boiling water and note results.
b. Colour reaction with 66% w/w H 2SO4
To a small amount of powder (on a white tile), add 2 drops of
66% w/w
H2SO4. Note staining.
c. Colour reaction with iodine.
To a small quantity of powder, add 2 drops of iodine-solution.
Note staining. Compare the colour with that obtained when 2
drops of water are added to a similar quantity of powder on a
tile.
MICROSCOPICAL EXAMINATION
Prepare mounts of the powdered (uncleared drug) in the following
reagents for examining cell contents, e.g. starch, calcium oxalate etc.,
and for modified cell walls (lignification, etc.)
1. Water
2. Glycerine 50%
3. Iodine water + glycerine 50%
4. Phloroglucinol/HCL + glycerine 50%
5. Soudan IV + glycerine 50%
6. 1 drop of 66% w/w H 2SO4 (Care should be taken with the
mounting and viewing of this slide.)
Examination of cleared material
Clear about 100mg of powder by heating with 6 ml of choral hydrate
solution in a beaker of boiling water, until fairly clear. (Shake
frequently). Dilute with an equal volume of water, mix and centrifuge
using the suspension that remains after the supernatant liquid has
been decanted to prepare mounts in:
1. Chloral hydrate solution + glycerine 50%
2. Phloroglucinol/HCL + glycerine 50%
Examine and make sketches of diagnostic feature mentioned in I-VI
above.
LOBELIA
Lobelia consists of the dried aerial part of Lobelia inflate, cut down
when the lower fruits are nearly ripe, and dried.
DIAGNOSTIC MICROSCOPICAL FEATURES
1. Epidermal cells of the leaf; those of the upper epidermis having
slightly sinuous, irregularly thickened walls and, occasionally,
cuticular striations marking the position of small papillae. Those of
the lower leaf epidermis have thin distinctly wavy walls.
2. Stomata, anomocytic, present on the lower epidermis only.
3. Covering trichomes, scattered or attached to epidermal cells, large
(about 300 microns), unicellular, conical, thin walled and warty.
4. Mesophyll cells, some containing abundant fat crystals, in the form
o prisms, often arranged in fan shaped groups; on warming, these
form droplets of oil (stain with Soudan IV solution).
5. Epidermal cells of the stem, with striated cuticle and beaded walls.
6. Anastomosing latex vessels of the stem and leaf phloem (stain
with iodine solution).
7. Lignified and pitted parenchyma of the stem; composed of xylem
parenchyma, fibrous cells and pith parenchyma.
8. Occasional pollen grains, small (24-30 microns) spherical, having
three pores and a smooth exine.
9. Epidermal cells of the seed (about 100 by 25 microns), elongated,
polygonal, with thickened and lignified walls.
10. Occasional groups of sclereids from the fruit Pericarp, having
unevenly thickened, sinuous, and lignified walls.
CONSITUENTS OF LOBELIA HERB
Alkaloids of the piperidine group, of which lobeline is the most
important. Carry out a certain test to ascertain their presence.
MICROSCOPICAL EXAMINATION
Examine suitably cleared mounts of leaf and powdered drugs, in
chloral hydrated/glycerine; and Phloroglucinol/HCL and
glycerine.
Prepare mounts of dry powdered drug in reagents (1), (B), (C)
and (D) under stramonium, examining for cell contents.
CASCARA (Cascara sagrada)
Cascara is the dried bark of Rhamnus purshiana (fam. Rhamnaceae)
collected at least one year before use.
MICROSCOPICAL FEATURES
1. A narrow cork consisting of several layers of brick-shaped, thin-
walled cells containing amorphous yellowish brown masses.
2. A narrow cortex consisting of a few layers of collenchymatous
cells and several layers of rounded or oval parenchyma.
3. Primary phloem containing small groups of fibres.
4. A wide phloem consisting of bands of sieve tissue, alternating
with bands of parenchyma, in each of which is embedded a band
or smaller group of up to 30 phloem fibres, each fibre being 8-15
microns wide. Sieve tubes, with abundant sieve area on both
oblique ends and side wall, are present; companion cells are
absent.
5. Sclereids, in the cortex and phloem parenchyma, in ovoid groups
of up to 2 000 sclereids.
6. Parenchymatous sheaths, surrounding the groups of sclereids
and phloem fibres, with calcium oxalate prisms in many of the
cell. The remaining parenchyma cells often contain cluster
crystals of calcium oxalate10-25-40 microns in diameter, starch
grains about 6 microns in diameter, and yellow colouring matter
which is coloured violet by alkali.
7. Medullary rays 1-5 cells wide.
DIAGNOSTIC MICROSCOPICAL FEATURES OF POWDERED DRUGS
1. Fragments of cork as in 1. above. (Powdered Cascara bark is a
yellow-brown to red-brown with a characteristic odour and an
intensely bitter and nauseous taste.)
2. Groups of sclereids composed of tightly packed, irregularly ovoid
cells with thick lignified walls and funnel-shaped pits. The
parenchyma cells abutting on these groups contain prisms of
calcium oxalate, forming a crystal ‘sheath’.
3. Numerous bundles of slender lignified phloem fibres, sometimes
pitted, surrounded by a calcium oxalate prism sheath.
4. Thin-walled sieve-tubes with well defined sieve plates on oblique
end walls.
5. Medullary rays within phloem tissue, transversing it at right
angles; cells are thin-walled and filled with yellow-brown
contents, walls may be pitted.
6. Parenchyma, thin walled, the cells containing small spherical
starch granules, cluster crystals of calcium oxalate and yellow-
brown colouring matter.
7. Calcium oxalate crystals, prisms and cluster crystals, scattered
in powder, or in the parenchymous cells.
8. Occasional fragments of yellow-green liverworts and mosses;
the former composed of a single layer of rounded cell, with
unevenly thickened walls (no midrib); the latter of small thin-
walled elongated cells, usually in a single layer, possessing a
midrib several cells thick.
9. Occasional groups of cortical collenchyma, the walls showing
large oval pits, and cells frequently containing starch grains and
calcium oxalate clusters.
CHEMICAL CONSTITUENTS
A. Anthraquinone derivatives
1. C-glycosides: (aloin-like) 80 – 90 %
(a) Cascarosides A, B, C, D: primary glycosides based on
optical isomers of barbarian and chrysaloin (actually
both O and C glycosides i.e. having sugar portions
attached through both oxygen and carbon atoms).
(b)Aloins:
Barbaloins, derived from aloe emodin anthrone.
Chrysaloin, derived from chrysophanol anthrone.
2. O-glycosides (based on emodin) 10 – 20 %
B. Free anthraquinones, aloe-emodin, chrysophanol,
emodin
The purgative activity of Cascara depends on its glycoside content, the
cacaosides being most active. The free anthraquinones have little
therapeutic use, the regular sugar being essential as a means of
transporting the aglycone to its site of action in the large intestine.
Therefore, the hydrolytic action of the enzyme system also present in
the bark should be guarded against during the production of galenicals
of Cascara.
It is interesting to note that, in the current BP methods for the
preparation of Cascara liquid and dry extracts, by percolation with cold
water, significant hydrolysis of the glycosides take place, giving final
products which contain only about half the percolation process,
sufficient appreciable breakdown of especially O-glycoside links
(conversion of Cascarosides to less active aloins and hydrolysis of O-
glycosides to inactive aglycones).
By contrast, the preparation of Cascara elixir involves percolation with
boiling water, and the product has glycosidal content approaching that
of original bark. It seems that the use of boiling water destroys
enzyme activity with little effect on the cascarosides and far less O-
glycoside breakdown – perhaps an indication that the BP methods or
preparation of liquid and dry extracts could be improved upon.
CHEMICAL TESTS
1. For anthraquinone derivatives
See BP 1968 “Identification”. Note that benzene may be used in place
of carbon tetrachloride, and that the final solution may need to stand
for 5 minutes before the characteristic pink colour develops.
2. Colour reaction with alkali
To a small quantity of powdered drug on a white recessed tile add 2
drops of KOH solution (5%). The yellowish-brown contents of cells of
cortex, medullary rays and phloem parenchyma change to purplish-
red. (See also tests for cell content below.)
3. For the presence of substances yielding reducing sugars
on hydrolysis
E.g. starch and glycosides
Place 100mg of powdered drug in a large test tube with 5ml of diluted
H2SO4 and heat in a beaker of boiling water for 5 minutes.
Filter hot, cool, and add an equal volume of Fehling’s solution, after
neutralizing with NaOH solution. Replace the tube in a beaker of
boiling water for a few minutes and note the appearance or non-
appearance of a brick-red precipitate.
4. Cell contents
Prepare mounts of the powdered (uncleared) drug in the following
reagents, examining the cell contents, and making sketches where
necessary.
i. Water + glycerine 50%
ii. Glycerine 50%
iii. Iodine water + glycerine 50%
iv. Phloroglucinol / HCL + glycerine 50%
v. Ruthenium Red + glycerine 50%
vi. Soudan IV + glycerine 50%
vii. KOH 5% solution + glycerine 50% (see 2. above)
5. Examination of cleared material
Clear about 100mg of powdered drug by heating with about 6ml of
choral hydrate solution in a beaker of boiling water, until fairly clear.
(Shake frequently.) Dilute with an equal amount of water to reduce
viscosity, mix and centrifuge. Remove the clear supernatant liquid
and use the remaining suspension to prepare mounts in:
(a) Chloral hydrate solution + glycerine 50%
(b) Phloroglucinol / HCL + glycerine 50%
Examine and make sketches of diagnostic features (i) - (ix)
CLOVE (Caryophyllum)
Clove consists of the dried flower buds of Eugenia caryophyllus (fam.
Myrtaceae)
MICROSCOPICAL FEATURES
A. A transverse section of the hypanthium, below the ovary,
shows:
1. Epidermis: Small, polygonal cells with thick cuticle; large
circular anomocytic stomata.
2. A zone of 2-3 rows of large, ovoid schizo-lysigenous oil
ducts, up to 200 microns in length, lined with secretory
epithelium embedded in a parenchymatous ground tissue,
the cells frequently contains calcium oxalate cluster
crystals.
3. A zone of bicollateral fibro-vascular bundles with
accompanying thick-walled, lignified fibres. The latter how
few pits, some have yellow-brown contents; usually seen
isolated in the powder, or in the small groups of one or
two, associated with vessels or parenchymatous cells.
4. A zone of aerenchyma consisting of air spaces surrounded
by chains of parenchymatous cells.
5. An inner zone of fibro-vascular tissue.
6. Colomella - central zone of parenchyma, the cells
containing abundant calcium oxalate cluster crystals.
B. Sepals and petals
Show simplified leaf structure, the mesophyll parenchyma
containing calcium oxalate clusters and embedded oil ducts.
Epidermis of sepals resembles that of the hypanthium; the
epidermis of the petals shows no stomata and irregular cells.
C. Stamens
Composed of a filament, a connective and an anther. Filament
shows an epidermis of longitudinally elongated cells, ground
parenchyma with calcium oxalate clusters, oil ducts and a
central vascular strand. The anther wall shows a typical fibrous
layer; small cells with bands of lignified thickening on the side
walls (sectional view). If seen in surface view, the cell walls
have a beaded appearance.
Pollen grains are abundant, small triangular, 15 -20 microns in
diameter, seen scattered in the powder, or enclosed in pollen
sacs.
DIAGNOSTIC MICROSCOPICAL FEATURES OF POWDERED DRUG
(Note colour, odour and taste)
i. Epidermis of hypanthium and sepals as in (A) and (B) above.
ii. Fragments of aerenchyma from hypanthium.
iii. Calcium oxalate cluster crystals, abundant in the
parenchymatous tissue, occasionally scattered in the powder.
iv. Abundant yellowish brown parenchyma from the hypanthium,
the cells often unevenly thickened and collenchymatous.
v. Fragments of oil ducts as in (A2) above.
vi. Isolated pericyclic fibres, as in (A3) above.
vii. Stamens, each with an oil gland in the apex of the connective.
viii. Fibrous layer of the anther walls.
ix. Pollen grains
x. The small number of sclereids, calcium oxalate prisms and
reticulate vessels from the stalk. (BPC: not more than 5% of
stalk)
xi. Absence of starch; derived from clove fruits (BPC not more than
1.0% of foreign organic matter, i.e. no clove fruits, blown cloves
or clove dust, and not more than the official limit of stalk.)
CHEMICAL CONSTITUENTS
Volatile oil, 15-20% (BPC: not less than 15% v/w)
Tannins, about 13%, chiefly gallotannic acid
Fatty oil, up to 10%
Resin
CHEMICAL TESTS
1. To a little powder on a recessed tile, add 1-2 drops of 5% FeCl3
solution. Note colour; compare with that obtained with a little
powder + water.
2. To about 250mg of powdered drug, add 10ml boiling water;
shake well and filter. Test for the presence of tannins as follows:
i. To 5ml of filtrate, add 1-2 drops of 5% FeCL3 solution. Note
colour.
ii. To 5ml filtrate, add 5ml Mitchell’s reagent, followed by 1.0g of
ammonium acetate. Note colour.
3. To 1 drop of clove oil in 3ml of 70% alcohol, add 1-2 drops of 5%
FeCl3 solution, noting the colour produced. Explain results.
MICROSCOPICAL EXAMINATION
1. Cut transverse sections of softened clove (soaked in water for 48
hours) just below the ovary and mount in:
i. 50% glycerine
ii. Chloral hydrate solution + 50% glycerine; warm.
iii. Phloroglucinol / HCL + 50% glycerine
iv. Sudan IV solution + 50% glycerine
v. 50% KOH solution – examine for needle-like crystals of
potassium eugenate in the oil glands.
vi. 50% FeCl3 solution + glycerine – note colouration of contents
of oil glands and parenchymatous ground tissue. Draw a
schematic T/S of the hypanthium.
2. Mount a stamen in chloral hydrate solution + glycerine, warm,
and sketch features seen.
3. Make mounts of the powdered drug in reagents (i)-(iv) above,
and also in ruthenium red solution and iodine water (plus
glycerine).
4. Clear about 5mg of powder and mount in:
i. Chloral hydrate solution + glycerine 50%
ii. Phloroglucinol / HCL +glycerine 50%
Sketch diagnostic features seen in procedures 3. and 4. above.
RHUBARB
Rhubarb is the rhizome of Rheum palmatum (and possible other
species and hybrids of Theum, except R. rhaponticum), deprived of
most of its bark and dried.
MICROSCOPICAL FEATURES
Note the colour of the powdered drug (yellow-brown to orange-brown),
the characteristic odour, gritty texture and astringent, bitter taste.
i. Phloem: absent or scanty, parenchymatous with scattered
groups of sieve tissue.
ii. Cambium: at or near periphery
iii. Xylem: mostly reticulate vessels ( up to 100 microns wide) with
some spiral or annular vessels, none of these are lignified
iv. Medullary rays: parenchymatous (usually 2-3 cells wide), with
yellow amorphous contents that are insoluble in alcohol (55%)
and soluble in water and in choral hydrate solution. Give a pink-
toned colour with alkalis (Pink with dilute ammonia solution,
deep red with KOH solution).
v. Parenchymatous tissue: the cells are thin-walled and polyhedral
containing starch grains (simple or compound, 4-30 in diameter).
Numerous idioblasts are also seen containing cluster crystals of
calcium oxalate.
DIAGNOSTIC FEATURES OF THE POWDRED DRUG
1. Abundant parenchyma of the medullary rays and ground tissue.
Cells of the medullary rays having slightly thickened brownish-
yellow walls and yellow cell contents giving reactions as
described in (iv0) above. The cells of the ground tissue may be
filled with starch granules or calcium oxalate cluster crystals as
in (v) above. These cells may be thin-walled and elongated,
rounded or rectangular and the walls may show irregular
thickening.
2. Calcium oxalate crystals: in the cells of the parenchyma
(idioblast) or scattered in the powder, often fragmented.
3. The vessels are usually in fragments as in (iii) above.
4. Starch granules: in the cells of the parenchyma or loose in the
powder.
5. The absence of fibres, cork and sclerenchymatous cells.
LABORATORY
Preliminary tests
Note the colour, odour and taste
Mix a small quantity of the powder with a few drops of water and allow
to stand. Note any changes.\Shake a little powder in a half a test-tube-
full of water, note frothing.
CHEMICAL TESTS
1. For the presence of antraquionone derivatives
Shake 100 mg of powder with 5 ml diluted H2 SO4 in a test tube.
Heat in boiling water for at least 2 minutes, filter while hot and
cool. Shake the cooled filtrate with an equal volume of benzene.
Separate benzene layer and add half its volume of diluted
ammonia solution to it. A pink or red colour is produced in the
ammoniacal layer. This layer indicates the presence of
anthraquinone glycosides.
2. Colour reaction with alkali
To a little powder on a white tile, add 2 drops of KOH solution
(5%). The yellow contents of the cells pf the medullary rays
become deep red. Repeat the test with dilute ammonia solution.
3. For the presence of tannins
To a little powder on a white tile, add 2 drops of FeCl3 solution.
Note colour, repeat the test using water and compare.
MICROSCOPICAL EXAMINATION
Prepare mounts of the powdered (uncleared) drug in the following
reagents:
1) Water
2) Glycerine 50%
3) Alcohol 95% + glycerine 50%
4) Phloroglucinol/HCL + glycerine 50%
5) Soudan IV + glycerine 50%
6) KOH solution 5% + glycerine 50%
Examine and make sketch where necessary
EXAMINATION OF CLEARED MATERIAL
Clear 100 mg of powder in the usual manner, centrifuge and make
mounts in:
Choral hydrate+ glycerine 50%
Phloroglucinol/HCL+ glycerine 50%
Examine and draw diagnostic features (i-v)
FENNEL (Fennel fruit, Foeniculum) BPC, 1968
Fennel consists of the dried fruits of the cultivated plants of
Foeniculum vulgare (fam. Umbelliferae), containing not less than 1.2%
v/w of volatile oil.
MICROSCOPICAL FEATURES
i. Epicarp: Composed of polygonal, tabular cells, with unstriated
cuticle and occasional aromocytic stomata.
ii. Mesocarp: Containing much thickened and lignified parenchyma
in the region of the vascular strands of ribs, the bands of the
thickening giving a reticulate appearance to the walls; large oval
or rounded pits are frequently present in the thickened walls. In
the powdered drug, these cells are often found in groups
associated with the fibro-vascular tissue or with fragments of the
endocarp.
iii. Endocarp: Composed of a layer of thin-walled, lignified cells,
elongated in surface view and arranged in groups of 5-8 cells
with their long axes parallel to one another-termed a parquetry
arrangement.
iv. Brown vittae: Running through the mericarp, about 250microns
wide.
v. Fibro-vascular tissue: Consisting of small amounts of lignified
vessels, tracheids and fibres, the walls of the former may be
thickened or pitted.
vi. Endosperm: Of thick-walled polygonal cells
vii. Testa: Usually a single layer of thin-walled, brown cells.
DIAGNOSTIC MICROSCOPICAL FEATURES OF THE POWDERED
DRUG
i. Epicarp with stomata as in (i) above.
ii. Parenchyma of the mesocarp as in (ii) above.
iii. Fragments of brown vittae.
iv. Endocarp cells, as in (iii) above
v. Endosperm cells, as in (vi) above.
vi. Fibro-vascular tissues as in (v) above.
vii. Absence of starch and trichomes.
Note the colour and odour of the powdered drug.
CHEMICAL CONSTITUENTS
Volatile oil 0.8-4% (chiefly fenchore and anethole)
Fixed oil, aleurone grains, calcium oxalate
MICROSCOPICAL EXAMINATION
A. Cut transverse sections of a softened fruit, mounting in:
i. Glycerine 50%
ii. Chloral hydrate solution + glycerine 50%
iii. Phloroglucinol / HCL and glycerine 50%
iv. Sudan iv + glycerine 50%
v. Iodine water + glycerine 50% (defat with ether first, and
examine for aleurone grains)
B. Make mount of powdered drug, in reagents (i)-(v) above.
C. Clear about 50mg of powdered drug, centrifuge, and mount in:
i. Chloral hydrate solution + glycerine 50%
ii. Phloroglucinol / HCL + glycerine 50%
NUX VOMICA
Nux vomica consists of the dried ripe seeds of Strychnos nux vomica
(fam. Loganiaceae), containing not less than 1, 2% of strychnine.
MICROSCOPICAL FEATURES
i. Testa: Consisting of one integument only; the outer epidermis of
thick-walled lignified cells with sinuous polygonal outlines (surface
view), small branching lumina and oblique linear pits, each cell
prolonged externally into a close oppressed trichome up to 1mm
long. The walls of the trichomes are strengthened by about 10
strongly lignified ribs (often seen in the powder as small rod like
fragments).
Note: The length of lignified rib per mg seed has been measured,
and constitutes a diagnostic feature of the drug (BPC “The length of
the lignified rib per mg of seed is 167 to 184 to 206cm”). This
value can be used to determine the amount of powdered nux
vomica in mixtures containing it.
The remainder of the testa consists of a flattened parenchyma,
appearing in section as a brown band (presence of pigment).
ii. Small spiral vessels from the region of the hilum –
components of a short vascular strand.
iii.Endosperm: With very thick-walled hemicellulosic polyhedral
cells showing no obvious pits, but connected by fine
protoplasmic threads (plasmo desmata) and containing in the
protoplasm fixed oil and aleurone grains up to about 30 microns
in diameter. The alkoloids strychnine and brucine occur in the
endosperm, strychnine in the inner part mainly, brucine in the
outer layers – these give characteristic colour reactions. (See
below)
iv. Embryo: Of small parenchymatous cells with oil and small,
irregular aleurone grains.
DIAGNOSTIC MICROSCOPICAL FEATURES OF THE POWDERED
DRUG
i. Epidermal trichomes as in (i) above, usually seen in side view; or
rod like fragments from the trichome walls, or trichome bases.
ii. Cells of the endosperm as in (iii) above, aleurone grains, plasmo
desmata and oil.
iii. Absence of starch and calcium oxalate.
NB: Note colour of powder.
CHEMICAL CONSTITUENTS
i. Indole alkoloids: Strychnine (violet colour with sulphovanadia
acid) Brucine (crimson colour with nitric acid)
Also: strychnine and vomicine (traces)
ii. A glycoside, loganin (meliatin)
iii. Fatty matter 3 – 4%
iv. Chlorogenic acid – pseudotannin
TEST FOR THE PRESENCE OF ALKALOIDS
Shake about 50mg of powdered drug with 1-2 ml of ether to defat;
allow ether to evaporate. Warm powder with 5 ml of 70% alcohol to
extract the alkaloids, shake and centrifuge. Evaporate the
supernatant liquid in a small dish to near dryness and add to the
residue about 2 ml dilute HCL. Fill and pass more dilute HCL through
the filter, if necessary, to give 2 ml of filtrate, which should be divided
into 4 equal portions:
i. +1 drop Mayer’s reagent
ii. +1 drop Tannic acid solution
iii. +1 drop Iodine solution
iv. Control
Divide (iv) into 2 portions, adding 1 drop of sulphovanadic acid to one,
and 1 drop of 50% nitric acid to the other. Compare the colour
reactions obtained by treating a small amount of strychnine HCL and
brucine alkaloid on a white tile, with sulphovanadic acid and 50% nitric
acid, respectively.
Compare also the colour reactions given by a little defatted powder on
a white tile, with the same two reagents.
MICROSCOPICAL EXAMINATION
1. Cut thin sections of softened seed and mount in:
i. 50% glycerine
ii. Chloral hydrate solution + 50% glycerine
iii. Phloroglucinol / HCL +50% glycerine
iv. Sudan IV solution + glycerine
Examine and draw features seen
2. Make mounts of powdered drug in the above 4 reagents, and
also in iodine water + glycerine 50%(aleurone grains stain
yellow-brown and plasma desmata become more easily visible)
3. Clear 50mg of powdered drug, centrifuge and make mounts in:
i. Chloral hydrate solution + 50% glycerine
ii. Phloroglucinol /HCL + 50% glycerine
NOTE: Nux vomica seeds are extremely hard and require soaking in
water for at least 48 hours prior to microscopical examination.
CARDAMOM
Cardamom fruit consists of the dried, nearly ripe fruits of Lettaria
cardamom var mimuscula.
Note: Although the drug appears in pharmacy as a whole, only the
seeds are utilized in the making f pharmaceutical preparations. The
pericarp of the fruit contains little aromatic principle, while seeds freed
from the pericarp and stored in this condition are prone to
considerable loss of volatile oil by evaporation (usually following
rupture of the seed coat during decortication). The fruits are therefore
stored in the entire condition, the seeds being removed immediately
prior to use. In this manner, adulteration of the seeds of other closely
allied and outwardly similar species may also be prevented.
MICROSCOPICAL FEATURES OF THE SEED
1. Aril: a thin membrane enveloping the seed composed of several
layers of flattened cells that are yellow in colour.
2. Test: Consist of:
i. Outer epidermis: Of thick-walled, narrow, elongated cells
prosenchymatous, yellowish-brown in colour.
ii. Two to three layers of thin-walled parenchymatous cells,
elongated at right angles to the long axis of the overlying
epidermal cells.
iii. A single layer (becoming 2 -3 layers in the region of the
raphe) of large, thin-walled rectangular parenchyma
containing volatile oil.
iv. Several layers of small, flattened parenchymatous cells,
with strongly thickened inner and anticlinal walls. Lumen is
bowl-shaped, containing a nodule of silica. Individual cells
are about 35 microns long and 20 microns wide, of a
reddish-brown (mature seeds) or pale brown (immature
seed) colour. A layer of flattened cells is to be seen below
these stegmata.
3. Perisperm: Of thin-walled cells packed with minute starch grains
(1-6 microns in diameter) having no hilum, and frequently also
several small prisms of calcium oxalate (10-20 microns long.
4. Endosperm: Of thin-walled parenchyma containing a granular
proteinaceous ground mass.
5. Embryo: Of small, thin-walled cells containing aleurone grains.
The raphe shows small lignified,spirally-thickened vessels but
fibrous sclerenchyma and large vessels are absent from the
seed. (Seen only in pericarp of the fruit).
Starch is absent from the endosperm and embryo of the seed.
DIAGNOSTIC MICROSCOPICAL FEATURES OF THE SEED
1. Masses of adherent starch granules and calcium prisms
embedded in the starch mass
2. The red-brown sclerenchymatous layer of the testa (stegmata)
3. The elongated cells of the outer epidermis
4. Thin-walled rectangular cells containing volatile oil.
5. Presence of small spiral vessels from the raphe; absence of
elements of the pericarp as described above
CHEMICAL CONSTITUENTS
Volatile oil 3-8% (not less than 4% v/w) in which the chief aromatic
principles appear to be:
i. Borneol, terpinylacetate, limonene, terpineol and cineole
ii. Much starch
iii. Calcium oxalate
LABORATORY
Preliminary tests
i. Note the colour, odour and taste
ii. Mix a small quantity of the powder with a few drops of water and
allow to stand. Note any changes.
MICROSCOPICAL EXAMINATION
A. Using material that has been soaked in water for 4 days, cut
both transverse and longitudinal sections of the seed and mount
in the following reagents:
Glycerine 505
Chloral hydrate solution +50% glycerine – warm to clear on a
covered slide.
Phloroglucinol/HCL+ 50% glycerine
Iodine water + glycerine 50% - stains starch and protein matter.
Make diagrammatic representations of the tissues.
B. Use a little powder to prepare mounts in the same four reagents.
Clear about 50 mg of powder in the usual manner, prepare
mounts in reagents (i), (iii) and (iv) and sketch the features seen.