Herbal Pharm Prac Manual 2 (1)

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


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.


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.


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%


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.


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.


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%


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%


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.


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.


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)


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.)


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.


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.


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.


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.


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.


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.


Volatile oil 0.8-4% (chiefly fenchore and anethole)

Fixed oil, aleurone grains, calcium oxalate


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.


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.


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.


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.


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


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.


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.

Documents

Herbal Pharm Prac Manual 2 (1)