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
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