PHARMACOGNOSY LABORATORY EXPERIMENTS

COLLEGE OF PHARMACY AND NURSING

School of Pharmacy

COLLEGE OF PHARMACY AND NURSING

School of Pharmacy

Dr. Mohammad Sohail Akhtar

Table of Content1

Experiment

No.

Title of the Experiment Page

No. Preface 3

Appendix A 4

1 Chemical tests of carbohydrate 5-7

2 Identification of different starch by microscope 8

3 To study the morphological characters of following fruits and

flowering bud containing drugs e.g. Fennel, Black pepper and

Clove.

9-12

4 Thin layer chromatography of Cumin oil 13-14

5 Microscopy of Ginger powder 15-17

6 Microscopy of Senna leaf powder 18-20

Preface

2

The laboratory manual in Practical of Pharmacognosy aims to develop the

student’s ability and to train them to different methods used for qualitative and

quantitative assay of different natural products, how to identify the different

chemical constituent present in the plant with the help of chemical test. How to

identify plant drugs with the help of their morphological characters. How to

identify powder drugs with the help of compound microscope.

Separate plant constituents into their components with the help of chromatographic

methods. Then the students were asked to identify the unknown plant sample with

help of these methods.

The Author

Revised: 2009 - 2015

APPENDIX

3

WORKING WITH SAFETY AND PRECAUTIONS IN THE

PHARMACOGNOSY LABORATORY

1. READ THE LABORATORY INSTRUCTIONS BEFORE COMING TO THE

LABORATORY.

2. SAFETY GLASSES MUST BE WORN AT THE TIMES REQUIRED IN THE

LABORATORY.

3. A LABORATORY COAT MUST BE WORN AT ALL TIMES DURING

LABORATORY EXPERIMENTS.

The laboratory coat must be buttoned up. Loose clothing like scarfs must be tucked

inside the laboratory coat.

4. EATING, DRINKING AND SMOKING ARE NOT ALLOWED IN THE

LABORATORY.

Students are not allowed to stay inside the Laboratory if there are no Laboratory

Activities.

5. IN CASE OF ACCIDENT OR FIRE, FOLLOW INSTRUCTIONS FROM THE

LECTURER OR TECHNICIANS.

6. KEEP YOUR WORKING AREA AND GLASSWARES CLEAN AND DRY

BEFORE LEAVING THE LABORATORY.

7. OBSERVE PROPER HYGINE. WASH YOUR HANDS AFTER DOING THE

EXPERIMENT.

Experiment-1

4

CARBOHYDRATES

Carbohydrates are compounds composed of carbon; hydrogen and oxygen with the last two

elements are in the same proportion as in water. They have the general formula (CH 2O)n

Carbohydrates are classified into:

a. Simple sugars (saccharides)

b. Polysaccharides

Saccharides (simple sugars) are divided into:

i. Monosaccharides as glucose and fructose

ii. Disaccharides either reducing disaccharides such as maltose, lactose or non-reducing

disaccharides such as sucrose

iii. Trisaccharides

Polysaccharides include starch, gums and agar

Physical properties of carbohydrates

Colour

Shape

Taste: Simple sugars are sweet while polysaccharides are tasteless

Odour: All are odourless

Solubility: Simple sugars are soluble in cold water while polysaccharides are insoluble in cold

water

Chemical tests for carbohydrates

5

1. Molisch's test:

Prepare 2 ml of carbohydrate solution in a test tube

Add few drops of α-naphthol 95% ethanol)

Add few drops of concentrated sulphuric acid on the walls of test tube

A violet colour is formed at the junction between two layers

It gives a positive result with all carbohydrates

2. Fehling test:

Prepare 2 ml of carbohydrate solution in a test tube

Add 2 ml of Fehling A solution and 2 ml of Fehling B solution

Fehling I consists of 7 g of hydrated copper (II) sulfate dissolved in 100 ml of dist.

water. Fehling II is made by dissolving 35 g of potassium sodium tartrate and 10 g of

sodium hydroxide in 100 mL of dist. water.

Heat in a boiling water bath for five minutes

Any change in colour is considered as positive result

This test gives positive result with monosaccharides and reducing disaccharides

(glucose, fructose, maltose and lactose)

3. Osazone test:

To 0.1 g of powder carbohydrate add 0.2 g of phenylhydrazine hydrochloride and 0.3 g of

sodium acetate

Add 10 ml of distilled water

Heat on boiling water bath for 30 minutes

Glucose and fructose will give ppt on hot while maltose and lactose will give ppt after

cooling.

4. Resorcinol test (Seliwanoff's test)

6

Prepare 1ml of test solution

Add 2 ml of dilute HCl

Add 1-2 crystals of resorcinol

Heat on boiling water bath.

Deep red colour is obtained with fructose and sucrose

5. N/50 I2 test to differentiate between polysaccharides

Add small amount of powder carbohydrate in porcelain dish

Add 2 drops of N/50 I2

Mix well with the bottom of a test tube

An olive green colour will be formed with gum tragacanth

A crimson red colour will be formed with Agar

A deep blue colour will be formed with starch

No change in colour will occur with gum acacia.

Experiment-2Microscopy of different Starch powders

7

Starch is an example of polysaccharides, which comprise a major class of carbohydrates, a group

of primary metabolites found naturally in different plant organs.

Four kinds of starch:

1. Potato starch

2. Wheat starch

3. Maize starch

4. Rice starch

Hilum: The first point formed in starch granules around which deposition of different layers of

granule occurs. It may be point, cleft or stellate.

Centric hilum: hilum present in the center of granule.

Eccentric hilum: hilum present at one end of granule.

Striations: Lines running in the granule due to difference in composition of different layers of

the granule.

Concentric striations: Lines running around the hilum forming complete rings

Simple granules: Separate from each other

Compound granules: Aggregated with each other

Differentiation between four kinds of starch

RiceMaizeWheatPotato

Polyhederal with sharp angles

Polyhederal with blunt angles

RoundPyramidalShape

SmallestSmallerSmallerLargestSize

InvisibleVisible, cleft or stellate, centric

Invisible or faint, point, centric

Visible, point, eccentric

Hilum

AbsentAbsentVery faintConcentric

PresentConcentric

Striations

Mostly compound

Mostly simpleMostly simpleMostly simpleAggregation

Experiment No.

8

Aim:--To study the morphological characters of following fruits and flowering bud containing

drugs e.g. Fennel, Black pepper and Clove.

FENNEL (Fennel fruit, Foeniculum) BPC, 1968

Fennel consists of the dried fruits of the cultivated plants of Foeniculum vulgare (fam.

Umbelliferae).

Organpleptic characters:

Colour: Greenish or Yellowish brown

Odour: Sweet and Aromatic

Taste : Sweet, mucilaginous, Aromatic

Morphology:

Morphological characters

9

(1) Fennel exhibits CREMOCARP.It consists of two equal portions called as

MERICARP, connected by central stalk called as CARPOPHORE.

(2) 5-10 mm long, 2-4 mm broad, staright and oval in shape.

(3) Five primary ridges on the surface with a bifid stylopod at the apex.

(4) Pedicel a plant stalk that supports a fennel fruit.

BLACK PEPPER

10

Black pepper consists of dried unripe fruit of Piper nigrum family Piperaceae.

Organpleptic characters:

Colour: Dark brown or grayish black

Odour: Aromatic

Taste : Pungent

Morphology:

(1) Size 3.5-6 mm in diameter.

(2) Shape almost globular

(3) Surface strongly reticulated

CLOVE

11

Clove consists of dried flower bud of Eugenia caryophyllus family Myrtaceae.

Organpleptic characters:

Colour: Dark brown or crimsons red.

Odour: Aromatic

Taste : Spicy pungent followed by numbness.

Morphology:

(1) Size about 10-17.5 mm.

(2) Shape is sub-cylindrical slightly flattened.

(3) Corolla is dome shaped

(4) Petal four numbers.

(5) Crown is made up of corolla and petals.

(6) Sepal four in numbers and thick in appearance.

(7) Hypanthium is 10-13 mm long.

Experiment No. 4

12

Thin layer chromatography of Cumin oil

Objectives:

To separate the constituents of Cumin oil using thin layer chromatography

Thin Layer Chromatography (TLC)

A method based on adsorption chromatography, which is at present an important analytical tool

for qualitative and quantitative analysis of a number of natural product. After the development of

chromatogram by ascending technique, the resolved spots are revealed by spraying with suitable

detecting agent or observation under UVlight. The TLC technique is useful in analysis of

alkaloids, glycosides, isoprenoids, lipid components, sugar and derivatives and practically all bio

constituents. The Rf values (distance traveled by component/ solvent front) may be vary

depending upon purity of solvent, presence of impurities, adsorbent used, polarity of the solvent,

substance and adsorbent etc.

II. Equipment / Material Chemicals:

1. Cumin oil

2. TLC Chamber 1. Benzene

3. TLC Plate 2.Chloroform

4. Capillary Tube

5. Sprayer

6. Measuring cylinders

7. UV Chamber

III. Procedure:

13

1. Prepare the solvent system by taking Benzene and Chloroform in the ratio of 1:1 (50 ml

0f each solvent and put it in the chromatographic chamber.

2. Take a TLC plate of 10 x 10 cm dimension. Apply a spot of diluted cumin oil sample

with the help of a capillary tube at least 2-3 cm above the base of the plate.

3. Keep the plate in the chamber such that the lower portion below the spot is dipped in the

solvent. Take care that the spot of the sample is not dipped in the solvent.

4. Allow the solvent to ascend on the chromatographic plate up to a height of 5-6 cm.

5. Take out the plate and mark the level of solvent. Dry the plate at room temperature

6. Keep the plate in oven for heating at 100 C

7. Find the Rf value of each component isolated on TLC plate and record the Rf values of

the spots.

IV. Result

Rf Values:

Spot No. 1 ___________

Spot No. 2 ____________

Spot No. 3 ____________

Spot No. 4.______________

14

Experiment-5

Microscopy of Ginger powder

Drug name: Zingiber officinale

Family: Zingeberaceae

Other name: Ginger

Characterization of Ginger powder:

a. Mount in water:

Abundant starch granules, which are mostly simple, fairly large, oblong to oval in outline with a

small pointed eccentric hilum with faint transverse striations.

b. Mount in chloral hydrate:

i. Groups of fibers: Usually occur in groups and may be found associated with the vessels, they

are frequently large and one wall is frequently dentate. Very faint transverse septa occur at

intervals

ii. The oleoresin cells are seen as bright yellow ovoid to spherical cells occurring singly or in

small groups in the parenchyma

ii. The very abundant parenchyma composed of thin-walled cells, rounded to oval in outline

with small intercellular spaces, filled with starch granules or oleo-resin.

15

16

Microscopical characters

1. Parenchymatous cells showing wrinkled walls.

2. Starch granules.

3.Fragments of parenchyma with adherent oleo-resin and starch granules.

4. A small reticulately thickened vessel with

5. Part of a fibre. an oleo-resin cell.

6. Part of a group of fibres.

7 Fibres with dentate walls

8 Part of a larger reticulately thickened vessel resin and starch granules with

associated pigment cell.

9 Parenchyma and an oleo-resin cell (o.r.).associated pigment cells, fibres and paren.

10. Parenchyma with associated collapsed tissue.chyma.

11. Parenchyma with adherent starch granules and5 Part of a fibre, an oleo-resin cell.

Result:

17

Experiment-6

Microscopy of Senna leaf powder

Senna: A very famous drug whose leaves are medicinally used for the strong purgative effect of

their anthraquinone content

SENNA LEAF

Cassia angustifolia 

Family: Leguminosae

A greyish-green or yellowish-green powder with a faint, characteristic odour and a mucilaginous,

slightly bitter taste.

Microscopy of Senna powder:

Two key elements are highly characteristic and used for identification of Senna powder:

1. Unicellular non-glandular bent trichomes.

Trichomes are found scattered as well as attached to fragments of the epidermises; they are

unicellular, conical, with thick and distinctly warted walls; they are sometimes curved near the

base.

2. Fibers accompanied with crystal sheath

The fibres which occur in groups; they are thick-walled, lignified with few pits and are

surrounded by a calcium oxalate prism sheath.

18

19

Microscopical characters

1. Epidermis in surface view showing paracytic stomata, mesophyll cells containing cluster

crystals.

2. Epidermis in surface view showing paracytic stomata.

3. Covering trichomes.

4. Epidermis in surface view with paracytic stomata.

5. Part of the lamina in sectional.

6. Part of the lamina in sectional view with a striated cuticle and an attached trichome.

7. Xylem elements from one of the larger veins,stomata and underlying palisade cells

8 Part of a pitted vessel.

9. Cluster crystals of calcium oxalate.

10 Part of a group of fibres with calcium oxalate.

11 Groups of fibres with calcium oxalate prism shaped crystals.

Results:

20