Pharmacognosy lab-manual

LABORATORY MANUAL
OF
PHARMACOGNOSY

Experiment-1
Aim-To study the macroscopic characters of datura
References- 1.Kokate C.K., Purohit A.P., Gokhle S. B.,
Pharmacognosy, Edition 41, Reprint 2008, Nirali Parkashan,
Page no. 501-502
2. Shah N. B., Nayak B. S., Experimental Pharmacognosy,
First Edition April,2008, S. Vikas Page no. 109-111
THEORY
Synonym- Datura herb
Bio. Source- It consists of the dried leaves and flowering
tops of Datura metel Linn. And other species of Datura
belonging to Family Solanaceae
MACROSCOPIC CHARACTERS:
Leaf
Color-Lower surface is slightly pale in color Upper surface is
darker than lower surface.
Size- the entire leaf has a length of 6-13 cm. and width of
8-13 cm.
Shape- ovate and petiolate
Lamina- Glabrous and usually has 3 to 4 coarse teeth
Apex-acute
Base- unequal
Texture- smooth and thin
Midrib- Prominent
Seeds
Color-Brown
Shape-Triangular
Flowers- Reddish purple on outside and whitish on innerside
Shape- Funnel Shaped
Fruits
Color- Green
Shape-Oval with spines
Size- Fruit has length of 4.5 cm and width of 3.5 cm
Odour- Disagreeable and characteristic
Taste- unpleasant bitter

Chemical constituents-
Datura leaf mainly contains tropane alkaloids (upto 0.5%) of which hyoscine is main
alkaloid. L-hyoscyamine and atropine are present in less quantity.
Hyoscine is an ester of tropic acid ad scopine.
Chemical tests:
1. Vitali-morin test-It is specific test for tropane alkaloids. Powdered sample is moistened
with nitric acid followed by evaporation to dryness and addition of methanolic
potassium hydroxide solution to the residue, a violet color is produced.
2. On addition of silver nitrate solution to the solution of hyosine hydrobromide, Yellowish
white ppt. formed which is insoluble in nitric acid but soluble in dilute ammonia
Uses-
1. Datura herb and its main alkaloid hyoscine are parasympatholytic with
anticholinergic and central nervous system depressant effects.
2. It is used in cerebral excitement.
3. Along with morphine, it is used as preoperative medication.
4. It is used in treatment of ashma and cough
5. 5. Hyoscine hydrobromide is used in motion sickness, gastric or duodenal ulcers
Cautions:-
Do not taste the crude drug because it might be harmful to the human body in
crude form.
Experiment-2
Aim-To study the macroscopic characters of withania

Reference- 1.Kokate C.K., Purohit A.P., Gokhle S. B., Pharmacognosy, Reprint 2008,
Nirali Parkashan, Page no. 13.79-13.81.
2. Shah N. B., Nayak B. S., Experimental Pharmacognosy, First Edition April,2008, S.
Vikas Page no. 187-189
Theory
Withania
Synonym- Asgandh, Winter cherry, withania root
Bio. Source:- It consists of dried roots and stem bases of Withania somnifera Dunal,
belonging to family Solanaceae
Color- outer surface is Buff to grey-yellow with longitudinal wrinkles

Odour- Fresh roots have smell similar to urine of horse
Taste- Bitter and acrid
Shape- Straight, unbranched, conical and bears a crown.
Fracture- smooth and powdery
Main constituents are alkaloids and steroidal lactones. Among the various
alkaloids, withanine is the main constituent. other alkaloids are somnine,
somniferinine, withananine, pseudo-ithanine, tropine, pseudo-
tropine, cuscohygrine, anferine andanhydrine. Two acyl steryl glucosides, sitoindoside
VII and sitoindoside VIII, have been isolated from root. The leaves contain steroidal
lactones, which are commonly called withanolides. The withanolides have C28
steroidal nucleus with C9 side chain and a six-membered lactone ring.
Uses-
1. Sedative and Hypnotic
2. adaptogenic
3. chronic fatigue

4. dehydration
5. bone weakness
6. neurodegenerative disorders
Cautions:-
crude form.
Experiment-3
Aim-To study the macroscopic characters of Vinca
Edition 39th
, Nirali Parkashan, Page no. 484-487.
Theory:
VINCA
Synonym- Periwrinkle

Bio. Source- It consists of dried whole plant of Catharanthus roseus Don belonging
to
family Apocyanaceae
Leaves
Color- Green
Shape- Simple,petiolate, ovate or oblong, unicostate, reticulate, entire, brittle and
oppositely arranged
Apex- Acute and mucronate Lamina- glabrous somewhat shiny Margin- Entire with
acute base
Flowers
Color- Violet, pink, white or carmine red in color
Shape- Flowers are bractate, pedicillate, complete, hermaphrodite, normally 2-3
cm in cymarose axillary clusters.
Fruits are follicles with several black seeds Odour- characteristics
Taste-Bitter

It mainly contains indole alkaloids (0.1-1.5%) of which vincristine and vinblastine are
medicinally important. Vinblastine contains indole alkaloid part called catharanthine
and dihydroindole alkaloid part called vindoline.Other alkaloids present are
ajmalicine, serpentine and tetrahydroalstonine.
Uses-
1. Vincristine and vinblastine are used in the treatment of cancer.
2. Vincristine sulphate is an antineoplastic agent which may act by arresting mitosis at
the metaphase.
3. It si used in the treatment of Hodgkin disease and choriocarcinoma.
4. It also possess hypotensive and antidiabetic effects
Cautions:- Do not taste the crude drug because it might be harmful to the human
body in crude form.
Experiment-4
Aim-To study the macroscopic characters of Rauwolfia
Edition 39th

Theory
Synonym- Rauwolfia root, serpentine root, chootachand, sarpagandha
Bio. Source- it consists of dried roots and rhizomes of Rauwolfia serpentine Benth,
belonging to Family Apocyanaceae
MACROSCOPIC CHARACTERS-
Color- outer surface- dull greyish brown with faint longitudinal ridges.
Internal surface- pale yellow
Odour-Odourless
Taste- bitter
Size- About 8-15 cm long, 0.5-1.0 cm thick
Shape- Roots are subcylindrical, slightly tapering, somewhat tortuous, rarely
branched
Fracture- Short and irregular
Rauwolfia contains atleast 30 indole alkaloids (0.7-2.4%).madicinally important
indole alkaloids are reserpine, rescinnamine, ajmalicine and serpentine.

Use-
1. Antihypertensive
2. Tranquillising agent and used in certain neuro-psychiatric disorders
Cautions:-
crude
form.

Experiment-5
Aim-To study the macroscopic characters of
Nuxvomica.

Edition 39th
Theory
NUXVOMICA
Synonyms- Kuchla, karaskara, crow fig, semen strychni
Bio. Source- It consists of dried ripe seeds of Strychnos nuxvomica Linn. Belonging to
family loganiaceae
Color-outer surface is grey to greenish grey
Odour-None
Taste- Intensly bitter
Size- 10-30 mm in diameter and 406 mm in thickness
Shape- Disc shaped somewhat flat or irregularly bent and concavo-convex.
Margin- rounded or acute

Surface- silky due to presence o dense, closely oppressed radiating, outwardly
directed trichomes
Hilum is raised in the centre of flat surface.
Nuxvomica seed contains 1.5-5% of bitter indole alkaloids. Chief constituents are
Strychnine and brucine while vomicine, α- Colutrine, pseudostrychnine and
strychnicine are also present. Seeds contain 3% of fat.
Nuxvomica also contain a glycoside i.e. loganin, chlorogenic acid and fixed oil
Uses-
1. Bitter stomachic and tonic
2. CNS stimulant
3. Respiratory stimulant.
4. It is used in CNS depressants poisoning such as Barbiturate poisoning
Cautions:-
crude form.

Experiment-6
Aim-To study the transverse section and powder microscopy of Rauwolfia
References- 1.Kokate C.K., Practical Pharmacognosy, Edition 11th
, Vallabh Parkashan,
Page no. 76-78.
Requirements- sample (rauwolfia root entire & powder), microscope, Glass slide,
Coverslip, glycerine (50%), phloroglucinol, HCl
Theory
Synonym- Rauwolfia root, serpentina root, choota chand, sarpagandha
Bio. Source- It consists of dried roots and rhizomes of Rauwolfia serpentina Benth,
MICROSCOPIC CHARACTERS-
Transverse section of root
1 CORK- stratified with 1-7 laters of small cells, 1-3 layers of larger cells, suberised,
larger cells lignified.
2. PHELLODERM- 10-12 rows of cellulosic parenchyma

3. Phloem- sieve tubes and parenchyma alternating with medullary rays
4. Medullary rays2-4 cells wide
5. Xylem- tatrach with small vessels, parenchyma, fibers,large celled medullary ras
6. Parenchyma of xylem and phloem, both lignified containing prisms and
conglomerate crystals of calcium oxalate and starch granules
7. Granular section staining yellow with iodine in phloem parenchyma
Transverse section of rhizome shows-
In addition to the above structure, cortex, pericycle with unlignified pericyclic fibres,
a small pith and perimedullary phloem in the periphery of pith, stone cells absent
POWDER MICROSCOPY
1 The powder is pale brownish-yellow in color with bitter taste.
2. Microscopic examination shows abundant starch grains, simple and compound
(2-4 components), spherical, with a distinct hilum in the form of split.
3. Cork-stratified iso diametric cells sometimes lignified. Polygonal in surface view.
4. parenchyma- pitted, lignified
5. vessels- lignified, fairly narrow with bordered pits
6. Fibers are lignified, slightly pitted, found with or without attached vessels

Cautions:-
crude
form.
Experiment-7
Aim-To study the transverse section of Datura and Vinca
References - 1.Kokate C.K., Purohit A.P., Gokhle S. B., Pharmacognosy, Edition 41,
Reprint 2008, Nirali Parkashan, Page no. 501-502
Requirements- sample (Datura leaf and catharanthus leaf), microscope, Glass slide,
Coverslip, glycerine (50%)
THEORY
Bio. Source- It consists of the dried leaves and flowering tops of Datura metel
Linn. And other species of Datura belonging to Family Solanaceae
MICROSCOPIC CHARACTERS:
Study of transverse section-
1. Datura leaf is dorsiventral i.e. having distinct upper and lower surfaces.
2. Stomata are of anisocytic type on both surfaces.
3. Epidermal cells are polygonal with thin cuticle, anticlinical walls, slightly straight on
the upper surface, wavy on the lower surface.

4. A single layer of elongated palisade cells is present below upper epidermis.
5. Next to palisade layer is a single row of rectangular cells containing rosette calcium
oxalate crystals and remainder of the mesophyll consists of spongy parenchyma.
6. Leaf bears both covering and glandular trichomes, covering trichomes, uniseriate,
multicellular and conical, usually less than 35µ in diameter at the base, glandular
trichomes with unicellular stalk and uni- and multi-cellular head.
7. Midrib region shows collenchymatous cells on both surfaces.
8. Xylem bundles are arranged in an arc.
9. The space between collenchyma and vascular bundle is occupied by 6-8 rows of
cortical parenchymatous cells some of which are filled with calcium oxalate
THEORY:
VINCA
Synonym- Periwrinkle
Bio. Source- It consists of dried whole plant of Catharanthus roseus Don belonging to
family Apocyanaceae
1. Vinca leaf is dorsiventral.
2. Stomata are of anomocytic type on both surfaces.
3. Epidermal cells are polygonal with thin cuticle; anticlinal walls are straight on the
upper surface and wavy on the lower surface.

4. A single layer of elongated palisade cells is present below upper epidermis.
5. Mesophyll consists of 5-7 layers of spongy parenchymatous cells with intercellular
spaces.
6. the midrib region shows collenchyma on both surfaces blow epidermis
7. A zone of vascular bundles occupies the central region.
Experiment-8
Aim-To study the transverse section and powder microscopy of Withania
References- 1.Kokate C.K., Purohit A.P., Gokhle S. B., Pharmacognosy, Reprint 2008,
Nirali Parkashan, Page no. 13.79-13.81.
Shah N. B., Nayak B. S., Experimental Pharmacognosy, First Edition April,2008, S. Vikas
Page no. 187-189
THEORY
Synonym- Asgandh, Winter cherry, withania root
Bio. Source: - It consists of dried roots and stem bases of Withania somnifera Dunal,
belonging to family Solanaceae
Requirements- sample (Withania root entire & powder), microscope, Glass slide,

Study of transverse section (T.S.)-
1. T.S. of root shows thin cork, composed of several layers of uniformly arranged, thin-
walled cells
2. Cork surrounds a broad cortex, composed of thin-walled, polygonal, irregular
parenchymatous cells containing starch grains.
3. Below cortex is a large wood, which consists of vessels, tracheids and fibres
transversed longitudinally by medullary rays.
4. Medullary rays are single celled and straight.
5. Longitudinal section shows vessels and tracheids with pitted thickening
Powder microscopy-
1. The powder is greyish in colour with characteristic odour and bitter and acrid taste.
2. Microscopic examination shows abundant vessels and tracheids with pitted
thickening.
3. Starch grains, nearly all simple, spherical-ovoid with distinct central hilum are found
scattered or parenchymatous cells of cortex.
4. Cork cells are thin walled and polygonal in surface view.

5. Fibres are narrow, elongated with tapered ends.
Experiment-9
Aim-To study the transverse section and powder microscopy of Nuxvomica
References- 1. Kokate C.K., Purohit A.P., Gokhle S. B., Pharmacognosy, Reprint 2007,
Edition 39th
3. Kokate C.K., Practical Pharmacognosy, Reprint 2007, Edition 4th
, Vallabh
Parkashan, Page no. 66-67.
Requirements- sample (Nux-vomica seed & powder), microscope, Glass slide,

Theory
NUXVOMICA
family loganiaceae
1. Epidermis- large, thick walled lignified cells with slit like pits.
2. Epidermal trichomes- thick walled, bent, twisted, and lignified with about 10
longitudinal ribs on the limb.
3. Inner layer of epidermis with collapsed cells.
4. Endosperm-thick walled cellulosic parenchyma of isodiametric cells.
5. embryo and endosperm both contain aleurone grains and fixed oil globules
6. Plasmodesma- communication of the protoplast of endosperm cells through the
cell walls by means of very fine protoplasmic threads.
Powder Microscopy-
1. The powder is yellowish-grey to greenish-grey in color.

2. Microscopical examination shows epidermis of testa with attached trichomes.
3. Trichomes are narrow lignified rods running longitudinally.
4. Endosperm cells from central region are large and very thick walled while those from
outer part, small and relatively thin walled.
5. A few fragments of endosperm show faint plasmodesma
Experiment-10
Aim-To perform the chemical test of Nuxvomica and datura
References- 1. Kokate C.K., Purohit A.P., Gokhle S. B., Pharmacognosy, Reprint 2007,
Edition 39th
, Nirali Parkashan, Page no. 476-479.&501-502
, Vallabh
Requirements- Sample (Nux-vomica powder), Test tubes, test tube stands and
holders, watch glass, UV lamp, Bunsen burner , Conc. H2SO4, Conc. HNO3,
Ammonium Vanadate, methanolic Potassium KOH, Acetone, K2Cr2O7,

Theory
NUXVOMICA
family loganiaceae
THEORY
Bio. Source- It consists of the dried leaves and flowering tops of Datura metel Linn.
And other species of Datura belonging to Family Solanaceae
Chemical tests:
The thin section of nuxvomica seed is defatted and performs the following tests-
1. A section of endosperm is strained violet by a drop of ammonium vanadate in
sulphuric acid due to presence of strychnine.
2. A section of endosperm is strained crimson or orange-red by a drop of nitric acid due
to presence of brucine.
3. A thick section of drug is treated with iodine solution and concentrated sulphuric
acid. Endosperm cell walls stain blue (due to hemicellulose)
THEORY

Bio. Source- It consists of the dried leaves and flowering tops of Datura metel Linn.
And other species of Datura belonging to Family Solanaceae
Chemical tests:
1. Vitali-morin test-It is specific test for tropane alkaloids. Powdered sample is moistened
with nitric acid followed by evaporation to dryness and addition of methanolic
potassium hydroxide solution to the residue, a violet color is produced.
2. On addition of silver nitrate solution to the solution of hyoscine hydrobromide,
Yellowish white ppt. formed which is insoluble in nitric acid but soluble in dilute
ammonia.
Experiment-11
Aim-To perform the chemical test of Rauwolfia and Cinchona
References- 1.Kokate C.K., Practical Pharmacognosy, Edition 11th
, Vallabh Parkashan,
Page no. 76-78.

Vikas Page no. 97 &160-161
, Vallabh
Requirements- sample (rauwolfia powder), Test tubes, test tube stands and holders,
watch glass, UV lamp, Bunsen burner Vanillin, acetic acid, Bromine water, ammonia,
Conc. H2SO4
Theory
RAUWOLFIA
Synonym- Rauwolfia root, serpentina root, chotta chand, sarpagandha
Bio. Source- it consists of dried roots and rhizomes of Rauwolfia serpentina Benth,
Chemical tests:-
1. Powdered drug is treated with sulphuric acid and p-dimethylaminobenzaldehyde, a
bluish-violet to red color is developed. The test is specific for indole alkaloids.
2. 1% tincture of drug shows fluorescence in UV light.

CINCHONA
Synonym- Peruvian bark, Jesuit’s bark
Bio. Source- It consists of dried stem or root bark of the plant Cinchona succirubra, C.
ledgeriana, C. calisaya, C. officinalis and other species of Cinchona belonging to
family Rubiaceae
Chemical tests:-
1. Heat the cinchona powder with glacial acetic acid. Reddish brown fumes on the
cooler part of the test tube are seen.
2. Coarse powdered bark is treated with lime and extracted with alcohol. The alcoholic
extract is evaporated and residue is taken up with dilute sulphuric Acid, the liquid
gives a blue fluorescence.
3. Thalleioquin test- To 10ml of the extract, add 0.25 ml of bromine water and 2 ml of
dilute ammonia solution are added and heated if necessary, a bright green
colouration is produced.
Experiment-12
Aim-To perform TLC of given sample.

References-Shah N. B., Nayak B. S., Experimental Pharmacognosy, First Edition
April,2008, S. Vikas Page no. 255-257
Requirements- TLC Plate & Chamber, Capillary, oven, spraying bottle, Silica gel G,
Sample
Theory
Thin Layer chromatography
Principle- TLC has been included under both adsorbtion and partition. Separation
may result due to adsoption or partition or by both phenomenon depending upon
the nature of adsorbents used on plates and solvent system used for development.
Thin layer chromatography (TLC) is a chromatography technique used to separate
mixtures. Thin layer chromatography is performed on a sheet of glass, plastic, or
aluminum foil, which is coated with a thin layer of adsorbent material, usually silica
gel, aluminium oxide, or cellulose (blotter paper). This layer of adsorbent is known as
the stationary phase.
After the sample has been applied on the plate, a solvent or solvent mixture (known
as themobile phase) is drawn up the plate via capillary action. Because different
analytes ascend the TLC plate at different rates, separation is achieved.
Thin layer chromatography can be used to:



Monitor the progress of a reaction
Identify compounds present in a given
substance Determine the purity of a substance

Procedure:-1. Prepare the TLC plate and keep it for the saturation. Make the extract
of the powdered drug and the spot it on the saturated TLC plate. Make the solvent
system and let it saturate it for the half an hour. The run the solvent system till 3/4th
of
the plate. Dry the plate on the hot plate and then spray it by the spraying agent. Let
it get air dried and the mark the spot and trace the spot on the tracing paper.
Results: - Report the Rf value.
Rf Value =
Distance travelled by solute
Distance travelled by solvent

Cautions:-
Do not misuse the chemicals and keep the plate properly in the solvent chamber.

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