PHARMACOGNOSY PHYTOCHEMISTRY II LAB MANUAL B.Pharmacy 5th Semester 2023 (2022-2023) Previous Year's Question Papers/Notes Download - HK Technical PGIMS

To be an educational Institute of par excellence and produce competent pharmacy professionals
to serve the community through research and the ever-increasing needs of Industry.
1. Imparting quality education and innovative research for various career opportunities.
2. Creating conducive academic environment to produce competent pharmacy professionals.
3. Indoctrination of students adorned with high human values and make them aware of their
responsibility as health care professionals.
PEO 1: To produce graduates with sound theoretical knowledge and technical skills required
for their career opportunities in various domains.
PEO 2: To incite the students towards research and to address the challenges with their innovative
contributions for the benefit of the mankind.
PEO 3: To instill the essence of professionalism, ethical commitment to become a health
care professional with sound integrity and adherence to the core human values in the service
of the society.

1. Pharmacy Knowledge: Possess knowledge and comprehension of the core and basic knowledge associated
with the profession of pharmacy, including biomedical sciences; pharmaceutical sciences; behavioral, social,
and administrative pharmacy sciences; and manufacturing practices.
2. Planning Abilities: Demonstrate effective planning abilities including time management, resource
management, delegation skills and organizational skills. Develop and implement plans and organize work to
meet deadlines.
3. Problem analysis: Utilize the principles of scientific enquiry, thinking analytically, clearly and critically,
while solving problems and making decisions during daily practice. Find, analyze, evaluate and apply
information systematically and shall make defensible decisions.
4. Modern tool usage: Learn, select, and apply appropriate methods and procedures, resources, and modern
pharmacy-related computing tools with an understanding of the limitations.
5. Leadership skills: Understand and consider the human reaction to change, motivation issues, leadership and
team-building when planning changes required for fulfillment of practice, professional and societal
responsibilities. Assume participatory roles as responsible citizens or leadership roles when appropriate to
facilitate improvement in health and well-being.
6. Professional Identity: Understand, analyze and communicate the value of their professional roles in society
(e.g. health care professionals, promoters of health, educators, managers, employers, employees).
7. Pharmaceutical Ethics: Honour personal values and apply ethical principles in professional and social
contexts. Demonstrate behavior that recognizes cultural and personal variability in values, communication
and lifestyles. Use ethical frameworks; apply ethical principles while making decisions and take
responsibility for the outcomes associated with the decisions.
8. Communication: Communicate effectively with the pharmacy community and with society at large, such
as, being able to comprehend and write effective reports, make effective presentations and documentation,
and give and receive clear instructions.
9. The Pharmacist and society: Apply reasoning informed by the contextual knowledge to assess societal,
health, safety and legal issues and the consequent responsibilities relevant to the professional pharmacy
practice.
10. Environment and sustainability: Understand the impact of the professional pharmacy solutions in societal
and environmental contexts, and demonstrate the knowledge of, and need for sustainable development.
11. Life-long learning: Recognize the need for and have the preparation and ability to engage in independent
and life-long learning in the broadest context of technological change. Self-assess and use feedback
effectively from others to identify learning needs and to satisfy these needs on an ongoing basis.

About MLRIP

Program
Educational
Objectives

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

Dept. Of Pharmacognosy

MLR Institute of Pharmacy Page | 3

EXPERIMENT - 1
INTRODUCTION TO MICROSCOPE

A microscope can be defined as an optical instrument, comprising of a lens or a combination of
lenses which enables to view magnified images of a minute object.

SIMPLE MICROSCOPE (DESSECTING MICROSCOPE)
It helps to reveal the morphological characteristics of the object.
COMPOUND MICROSCOPE
The compound microscope essentially consists of three major systems.
I. Support system:
It comprises of base, stage and body tube.
II. Illumination system:
It throws light on the object for proper viewing. It comprises of light of source or mirror, iris
diaphragm and condenser. The light source may be a plain or concave mirror or electrically
illuminated by a tungsten filament lamp or a halogen lamp. Mirror and electric light source are
generally interchangeable.
III. Magnification system:
This includes a set of lenses aligned in such a manner so that a magnified real image can be
viewed. The objective is a set of lenses placed near the object. It partially magnifies the object,
which can be observed through the EYEPIECE in a more magnified form
DIFFERENT ILLUMINATION SYSTEMS USED IN MICROSCOPES
(A)Plain Mirror
Use plain mirror when the fixed source of light is used.
(B) Concave Mirror
When skylight is used, concave mirror helps to converge the beam onto the condenser.
Sub stage lamp interchangeable with mirror
Where there is no electricity or battery, mirror can be used.
MICROSCOPE

SIMPLE One set of lens Lower
magnification.

COMPOUND Two sets of lenses: Eyepiece and
Objective Higher magnification
Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

(C) Build-in sub stage lamp (Tungsten – Filament or Halogen Lamp) with intensity
adjustment.
LIGHT ADJUSTMENT IN A MICROSCOPE
While viewing an object, sometimes, the object has to be brightly illuminated; on the occasions,
less light is needed.
(A)The light rays on the object can be altered in 2 ways by means of
CONDENSER:
a. Condenser can be moved upwards with the knob so as to make the object brighter.
b. Condenser can be moved downwards to make the objects less bright.
(B) Light illuminating the object may be adjusted with IRIS DIAPHRAGM. The diaphragm
may be opened or closed to increase or diminish the light falling on the condenser hence
making the object more or less bright.
FACTS AND FIGURES ABOUT MICROSCOPE
(A)Magnifying power (M.P)
M.P – Magnification of objective * Magnification of eye piece e.g., if you are observing an
object on a slide using a 10x objective and 5x eyepiece then MP= 10*5=50 times.
Thus, the object viewed is magnified 50 times.
(B) Resolving power of objective (R.P)
Resolving power of objective is defined as the ability to separate distinctly two small elements
of an object which is situated a short distance apart R.P can be measured by Numerical Aperture
(N.A) of an objective Greater the N.A greater is the resolving power.
(C)Working distance:
The distance between the object and the objective is shown as working distance
The higher the power of objective, lesser is the working distance.
(D)Focusing:
Focus an object while viewing through the eye piece by adjusting working distance. This is done,
with the help of coarse adjustment and fine adjustment knob. Coarse adjustment knob is rotated
to bring the object in field of view and the fine adjustment knob is rotated to get a sharp image.
(E) Field of view:
The area of the object which can view through the eye piece is the field of view. The field of
view narrows as magnification increases.
(F) Objectives: Different objectives used in microscopy.
✓ 10X – Low power objective; with the help of this one identifies the part of the observed
in high power. This does not reveal many details.
✓ 40X – This is a high power objective to reveal finer details of the object. This is spring
loaded, which means that a spring is fitted between the front and back lenses of the
objective. This is I protect the front lens, as the working distance is low in high
Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

magnification and the lens may touch the slide while focusing. The spring does not allow
pressure on the front lens when it touches the slide.
✓ 100X – Oil impression lens – This also is a spring loaded objective, requiring very low
working distance and will give an image only when the object is immersed in CEDAR
WOOD OIL. This oil is used as it has high refractivity and allows very high resolving
power.

RECENT ADVANCES IN MICROSCOPES
Since the invention of microscope, it has undergone many changes in size, sharp, material and
uses.
The commonly used student microscope with one eyepiece is the Monocular microscope, with a
vertical body tube and draw tube.
To avoid fatigue to neck and back, this microscope was build with an angular or inclined draw
tube.
To inclined draw tube was made fully rotatable through 360 degrees, so as to enable more than one
person to view the object without changing seats but by only rotating the draw tube head.
Trinocular Microscope is available with a binocular head with a vertical draw tube attached, which
helps in photo micrographic work.
Projection microscopes are available which project the images of the object on a 6”-8” wide screen,
build at the top of the microscope.
Stereo microscopes are available with zoom facilities with greater flatness and contrast and long
working distances ensuring more distinct images.
Advanced photo micrographic equipment is available for taking photographs of the slides observed
under microscopes.
Phase contrast microscope:
In is type of microscopy controlled illuminations of the specimen and special phase contrast
objectives are used. By phase contrast, contrast can be added to normally invisible objects and hence
more details can be observed.
Phase – contrast microscopy technique is largely utilized for studying living objects like cells
and tissues and specifically the cytological details of the organelles. This technique has been discovered
by Fritz Zernike, the Noble prize winner in 1953. Magnification up to 2000 is possible.
Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

Fluorescence microscope:
Some chemical substances absorb light waves of one wavelength and emit visible waves of greater
wavelength. So material under observation appears of one color by ordinary light and of an entirely
different color by ultraviolet light.
The materials are known as fluorescent and the phenomenon is known fluorescence.
By this method, cancer can be detected in early stages, while bacteria of various types and the even
antigen antibody complexes be studied rapidly and also with accuracy.
The technique is used for cinchona, Gambier etc.
Ultra-violet microscope:
When ultra-violet light having short wave length of 180o
-400o
mμ is used as source of radiation,

instead of visible light of 400o
-700o
mμ more magnification can be obtained since it has greater
resolution. (Resolution is the ability of the microscope to differentiate between adjacent objects as
separate which decides the magnifying capacity of microscope.)
The absorption of ultraviolet radiation by certain substances enables them to locate or separate under
the microscope. In ultra-violet microscopy the image is made visible by using photographic emulsion.
Electron microscope:
For the maximum magnification to the tune of 2, 00,000 – 4, 00,000 times, electron microscopy is
used, now-a-days. In electron microscopy, beam of electrons is used instead of light waves to produce
the magnified image.
Electron microscope is installed in dust-free, vibration – free area without magnetic-fields and
in air-conditioned room. It is very useful for understanding the ultra-structure of viruses and different
types of animal and plant cells. It is being using since 1940.

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

INTRODUCTION

PREPARATION OF HISTOLOGICAL SLIDES

SECTION CUTTING TECHNIQUE
Section Of A Stem, Root, Stolon
Different sections can be obtained from a stem, root or stolon, depending on the plane of cutting each
section revealing details from a different angle.
Transverse Section (T.S.):
Transverse section is obtained by cutting along the radial plane of a cylindrical portion of the
stem/root/stolon and perpendicular to the long axis.
This section when prepared and observed under a microscope reveals the radial arrangement of tissues
and shows concentric layers and vascular bundles.
In order to reveal the tissue arrangement longitudinally along the planes parallel to the long axis both
radially and tangentially, we have to cut the longitudinal sections.
LONGITUDINAL SECTION (L.S):

SIGNIFICANCE OF TS, TLS AND RLS
Observation of section of a stem/root/stolon in TS, TLS and RLS reveals the structure and
morphology of a particular cell from all angles. At the same time arrangement of cells in a tissue
is revealed from all angles as is evident from the figure showing a stereogram of Quassia wood.
LONGITUDINAL SECTION

TANGENTIAL
LONGITUDINAL SECTION

A section cut along the
axis parallel to a tangent

RADIAL LONGITUDINAL
SECTION

A section cut along the
long axis & the cutting
plane passing through
the long axis and radius

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

SECTION CUTTING TECHNIQUE
Section cutting is an art which every pharmacognocist most acquire. Thinner the sections, more
clearly the tissues can be observed.

MATERIALS REQUIRED

First of all keep the following materials ready on your laboratory table.
1 Napkin 10 A dropper
2 Watch glass 11 Filter paper / blotting paper
3 Test tubes 12 Stains
4 Painting brush (thin) 13 Drug sample
5 Bunsen burner 14 Forceps
6 A sharp razor blade 15 Test tube holder
7 Micro – slides 16 Test tube stand
8 Cover slips 17 Needle
9 A beaker full of water 18 Camel hair brushes (two)

SELECTION OF DRUG SAMPLE FOR SECTION CUTTING
Selection of appropriate size, part and shape of a crude drug sample is very important in
obtaining a good section.
In case of a stem/root/stolon, select a portion of the drug having a diameter of 3 to 5 mm and a
length of 25mm. A sample shorter in length will be difficult to hold and a sample thicker in
diameter may give rise to thick and wedge shaped sections.
In case of a leaf as it does not have a thick midrib and lamina, hence it is more convenient to
obtain fine sections.

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

PREPATATION OF SAMPLE FOR SECTIONING

1. Put the selected sample in a test tube and add sufficient chloral - hydrate solution or water
so that the sample remains submerged. Boil the sample in water over a Bunsen flame for
a few minutes. This will soften the hard drug sample and will help in obtaining fine
sections. In case of a leaf, this step may not be necessary.
2. For a stem/root/stolon drug, cut a cylindrical portion which is almost straight and cut off
both edges so as to make the edge surface smooth. This sample is ready for section
cutting.
3. Hold the sample vertical between the first, second finger and the thumb and move the
blade back and forth from one end to the other, obtaining fine slices.
4. Take sufficient number of sections, as all sections will not be very fine and uniform.
5. Transfer the sections to a watch glass containing water with the help of a brush. Reject
thick and oblique one.
6. Similarly, cut sections of the leaf in the block of pitch which shall give sections of the
leaf when separated from the pith. Transfer the sections to a watch glass with a brush.
7. Note: Before taking the section, ensure that the blade is having enough amount of water
on its edge, if a dry blade is used, it shall entrap air bubbles in the section, which are
difficult to remove.
8. In case of a leaf drug cut a part of the leaf passing through midrib. The surface area of the
surface to be cut has to be increased. This is done by embedding the sample in a block of
pith. This pith is obtained from red pumpkin (Bhopla) or raw papaya or potato. A cubical
portion of the pith is cut off and used as shown in the figure.
Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

III. STAINING AND MOUNTING OF SECTIONS

Staining is a process in which chemical dyes are used to impart to various tissues in a section of
drug sample, which enables to distinguish the arrangement of various tissues in the sample.
A STAIN is a chemical dye (colorant) which combines chemically or physically with a cell content
to impart color to it. e.g., safranin combines with the lignin present in cell wall and vessels and
imparts a red color to the lignified tissues.
Iodine solution combines with starch grains to give a blue color. Sudan Red III dissolves in the
fixed oil present in the oilseeds to impart red color.
STAINING PROCESS
1. Take a clean watch glass and add the staining solution to it.
2. With the help of a brush, transfer the section taken from water to stain solution and keep
for 2-3 minutes.
3. Pick up the section after 2-3 minutes and transfer it to watch glass containing plain water,
so that excess stain is washed away. This section is ready for mounting on a side.
MOUNTING PROCESS:
1. Take a clean glass micro slide.
2. On this slide transfer the section to be mounted, with the help of brush.
3. Add one or two drops of water on the section with a dropper. See that the section is
submerged in the water.
4. Take a clean cover slip with the help of a forceps and needle. Place the cover slip on the
section gently.
5. If any air bubbles are seen, slightly lift the cover slip and add a drop of water and replace
the cover slip till the air bubble is removed.
6. With the help of a blotting paper, wipe off excess water present outside the cover slip. The
slide is ready for observation.
7. To avoid evaporation of water and drying of section, glycerine water can be used instead
of water. In order to prepare a permanent mount, a special process is adopted.
Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

B) PREPARATION OF COMMONLY USED REAGENTS AND SOLUTIONS IN
MICROSCOPIC WORK
REAGEGNTS PREPARATION AND USES
Acetic acid (1-5 %) Dilute aqueous solution (1-5 %). It does not affect calcium oxalate

crystals, however, dissolves globoids of aleurone grains.

Ammonium -
vanadate
(Sulphuric acid)

Dissolve 1.62g of anhydrous ammonium vanadate in 125ml of
concentrated sulphuric acid, cool and add into 125ml of ice-cold water.
After dilution to 10 fold, used to detect alkaloids, phenolic compounds
and steroids.

Aniline blue Staining reagent for cellulosic tissue. 1% aqueous solution stains callus.
Whereas the alcoholic solution is as a counter stain with safranin.
Aniline sulphate It is saturated aqueous solution of aniline sulphate acidified with
concentrated sulphuric acid. It stains lignified cell walls to yellow in
colour.

Bleaching solution Prepare a solution by dissolving 75g of crystalline sodium carbonate in
125ml of distilled water; add it to a mixture of 50g of chlorinated lime
in 375ml of distilled water. Shake occasionally and filter.

Canada balsam Heat Canada balsam on water - batch to remove all the volatile matter.
Dissolve the residue in xylene or benzene to form a thin viscous fluid.
The reagent is used to prepare permanent mounts.

Chloral hydrate Dissolve 50g in 20 ml of distilled water. It is used as clearing agent. For
the removal of chlorophyll, boil material with a little amount of this
reagent over flame for a short while.

Chlor-zine-iodide
(Schulze's
solution)

Dissolve 30g zinc chloride, 5g potassium iodide and 1g iodine in 14ml
distilled water. It is stains cellulose-blue or violet; lignin-yellow, cutin
and suberin-yellow or brown, starch-blue and protein-brown.
Chromic acid It is the saturated aqueous solution of chromic acid or mixture of 10%
chromic acid in 10% nitric acid. Strong solution dissolves cellulose and
lignified walls.

Cuoxam Mix 0.5g of copper carbonate in a mortar with 10ml of distilled water
and gradually add to it 10ml of strong solution of ammonia with
constant stirring. It dissolves cellulose walls.

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

EXPERIMENT-3

SYSTEMATIC PHARMACOGNOSTIC STUDY OF CINCHONA

AIM: Study of macro, powder and microscopic study of Cinchona.
APPARATUS: Microscope, glass slide, cover slip, camel brush.
CHEMICAL: Glycerine.
BIOLOGICAL SOURCE: It is a dried bark of cinchona species viz. cinchona calisaya,
C.Legeriana, C.Officinalis, C.Succirubra or hybrids of last two species with either of the first
two species belongs to the family of Rubiaceae.

MORPHOLOGICAL CHARACTERS
SHAPE : Flat pieces or quills

Fracture short in the cork and cortex, fibrous in phloem.
COLOUR : Outer surface greyrish green and rough due to presence of
Cracks and wrinkles and often bears epiphytes such as lichens.

Inner surface is reddish brown.
TASTE : Intensely bitter.
ODOUR : Odourless.

DESCRIPTION:
A transverse section shows.
1) Crack is with an occasional presence of lichens.
2) Parenchymatous cortex contains starch grains and microcrystals of calcium oxalate.
3) The cortex consists of several secreatory channels and phloem fibres with Y- Shaped pits,
either isolated or in radial rows of 2 to 4.
4) It consists of sieve tubes with companion cells and phloem parenchyma.
5) Medullary rays are 1 – 3 seriate.
6) Stone cells are absent.
Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

PROCEDURE:
Take a clean glass slide and place little quantity of given alkaloidal drug powder in the centre of
the slide. Few drops of glycerine is added to it and carefully spread by using camel brush. Put the
cover slip with care to avoid enclosing of air bubbles. Then observe the slide under microscope.

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

POWDER CHARACTERISTICS:
1) Cork cells.
2) Lignified fibres with Y – cells Shaped pits.
3) Parenchyma.
4) Micro sphenoidal calcium oxalate crystals.
5) Starch grains.

CHEMICALS CONSTITUENT: It belongs to quinolone alkaloids. The important alkaloids are
quinine, quinidine, cinchonine and cinchonidine.

USES:
1) It is used as antimalarial drug.
2) Preparation of cinchona is employed as bitter stomachic and antipyretics.
REPORT:
The given organized crude drug was identified to be CINCHONA.
Note:
Antipyretic
• a drug used to prevent or reduce fever.
Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

EXPERIMENT-5

SYSTEMATIC PHARMACOGNOSTIC STUDY OF CINNAMON

Aim: To Study macroscopic and microscopic characters of Cinnamon.
Requirements:
Crude drug, Microscope, glass slide, watch glass, brush, needle, cover slip, blade.
Staining Reagents:
Phloroglucinol, conc. HCl (1:1), Ruthenium red, Iodine, Acetic acid Dil. HCl,
Botanical Source: It consists of dried inner bark of Cinnamomum zeylanicum belongs to the
family: Lauraceae
Macroscopical Characters:
Colour : Pale brown in colour, inner surface darker than the outer surface
Odour : Fragrant
Taste : Warm, sweet and aromatic.
Extra Features : Bark is free of cork, single or double quills or single closely packed compound

quill.
Microscopical Characters:
A transverse section shows Pericycle (stone cell layers). It contains light coloured wavy,
longitudinal lines on the outside of the bark. Pericyclic Fibres (lignified) are present in groups of
6 to 15 occur at intervals. 3 – 4 layers of pitted sclerides are present. It contains thickened
lignified walls, isodiametric and slightly elongated tangentially (U – shaped thickening) also
contains Starch grains.
Secondary Phloem:
• Secondary phloem is made up of Parenchymatous cells. Among this Few cells contain
acicular calcium oxalate crystals and starch grains (diameter up to 10 μ).
• It contains Medullary rays. Medullary rays are usually biseriate, narrow at inner side and
wider in the scleride band side. It also contains starch, acicular raphides.
• It contains single, isolated and circular Phloem fibres.
• It contains Mucilage cells. Which can be identified after staining with Ruthenium red (shows
pink / red colour).
• It also contains big and isolated oil cells.
• Cork and Cortex are absent.

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

Microchemical Tests

REAGENTS OBSERVATION CHARACTERISTICS
Phloroglucinol +conc.HCl

(1:1). Pink Lignified cells : Pericyclic fibres, stone cells,

cork cells
Ruthenium red Pink Mucilage cells
Iodine Blue Starch
Dil. Hcl Soluble Calcium oxalate crystals.
Acetic acid Insoluble Calcium oxalate crystals.

Powder characteristics
1) Slender fibres
2) Stone cells with horse-shoe shaped thickening
3) Starch grains
4) Parenchyma
5) Acicular crystals of calcium oxalate

Important constituent

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

Volatile oil; cinnamic aldehyde.
Identification tests
Alcoholic extract of the drug treated with a drop of ferric chloride solution forms green colour.
Chloroform extract of the drug treated with 10% azueous solution of phenyihydrazine shows red
shaped crystals of hydrozone of cinnamaldehyde.
Chemical Constituents:
It contains 0.5-1% of Volatile oil. The volatile oil contains 55-65% of cinnamic aldehyde and 5-
10% of eugenol.
It also contains terpenes, mucilage, starch and calcium oxalate crystals.
Uses:
It is used as Carminative, mild astringent and flavouring agent.
It is also used as an aromatic and stomachic.
It is used in spices.

Report: Transverse section of Clove was prepared and Morphological characters and
microscopic characters of Cinnamon Bark were studied.

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

EXPERIMENT-6

SYSTEMATIC PHARMACOGNOSTIC STUDY OF SENNA LEAF

AIM: To Study macro, powder and microscopic characters of Senna.
APPARATUS AND CHEMICALS REQUIRED: Glass slide, microscope, camel brush,
coverslip, glycerin, chloral hydrate, phloroglucinol etc.
SYNONYMS:- Senna, Cassia Senna, Cassia angustifolia.
BIOLOGICAL SOURCE:-It is a dried of fresh leaflet of Cassia angustifolia belonging to
family leguminosae.
PROCEDURE:
Take the T.S along with the midrib and remove the chlorophy11 by warming with excess
amount of chloral hydrate solution. Then transfer slowly into the plane slide by using
camel brush and observe the characteristics initially at low power and finally at high
power.
Micro chemical tests are performed by adding one drop of phloroglucinol and one
drop of HCL.

MORPMOLOGICAL CHARACTERS

SIZE : 2.5 – 6cm length and 1 – 2cm in diameter leaflet is lanceolate
with entire margin, acute apex, and asymmetrical base.

COLOUR : Pale green to dark green
TASTE : Slightly bitter
ODOUR : Faint Characteristic
MICROSCOPIC CHARACTERS
The following sectional characters are observed as follows.
I. LAMINA:
A. UPPER EPIDERMUS : Single layered, Polygonal cells covered with cuticle and
unicellular thick walled, short covering trichomes and paracytic stomata are observed.
B. PALISADE CELLS :Single layered, compact, enlongated, narrow, columnar cells
are observed.
Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

NOTE: In lower epidermis the palisade cells are smaller having inter cellular spaces.
Both trichomes and paracytic stomata are observed same as in upper epidermis.
C. SPONGY PARENCHYMA :These cells are thin, narrow, loosly arranged in
between upper and lower epidermis. In this region vascular bundles i.e. xylem and
phloem are observed. Xylem is towards ventral surface where as phloem is towards
dorsal surface.
II. MID RIB: In midrib region a prominent vascular bundles are observed which is the
diagnostic characteristic of senna.

NOTE: Calcium oxalate crystals appeared in both spongy parenchyma and in midrib
region as individual clustered crystals and a sheath.

POWDER CHARECTERISTICS:
Epidermal cells:polygonal,straight walled with paracytic stomata(rubiaceous)
Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

Covering trichomes:unicellular, thick warty walls,acute apex,bulbous base,narrow
lumen,conical shape:length:70-260 μ :width:12-18-25 μ
Xylem vessels:angular thickening,lignified
Calcium oxalate crystals:isolated or in parenchymatous cells.very abundant, occur as
prisms and also as cluster crystals.

CHEMICAL CONSTITUENTS:
Anthracene glycosides, sennoside (A, B, C & D)

IDENTIFICATION TEST
BONTRAGER’S TEST:
Take a few places of leaflet and boil with dil. H2SO4 and filter it while it is in hot
condition. Take the filtrate and extract with an equal amount of benzene or chloroform or
Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

petroleum ether. Separate the organic layer and add ammonia solution. The aqueous layer
acquires pink or red colour by standing aside.
USE:
It is used as laxative.

Note:
Cassia acutifolia:leaves with acute apex
Cassia angustifolia:plant with narrow leaves
Laxative:
• A drug facilitates evacuation of the bowels.
REPORT:
Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

SYSTEMATIC PHARMACOGNOSTIC STUDY OF CLOVE

Aim: To Study macroscopic and microscopic characters of Clove.
Requirements:
Microscope, glass slide, watches glass, brush, needle, cover slip, blade,
Staining Reagents:
Phloroglucinol, conc. HCl (1:1), Strong KOH, Dil. HCl, Sudan red -III
Synonym: Lavang (Hindi), Clove flower, Clove bud.
Biological Source:
It consists of dried flower buds of Eugenia caryophyllus belongs to the family: Myrtaceae.
The clove contains not less than 15% v/w of clove oil.
Macroscopical Characters:-
Colour : Dark brown /crimson red.
Odour : Aromatic.
Taste : Spicy pungent followed by numbness.
Microscopic characters:
Epidermis contains Single layered, straight walled cells with anamocytic (Ranuncululaceous)
stomata. The outer layer of epidermis is covered with very thick cuticle.
Cortex contains three zones.
1. Upper zone contains radially arranged parenchymatous cells with two to three layers of
big, ellipsoidal, schizolysigenous oil glands. Parenchymatous cells contain tannins hence
show dark colour when stained with ferric chloride.
2. Middle zone containing a ring of bicollateral vascular bundles where xylem is composed
of three to five lignified spiral vessels. About 15 vascular bundles are present in ring.
Isolated pericyclic fibres may be present around the vascular bundle. Fibres are prominent
& slow distinguished pink colour on staining.
3. Lower zone (or) Inner zone of parenchyma is composed of air spaces (arenchyma),
separated by lamellae, one cell thick which supports the central columella.

Collumella: It contains parenchymatous cells. It is rich in calcium oxalate clusters and a ring of
vascular bundles towards the periphery.

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

It contains 25-30 small vascular bundles at periphery. If T.S. is passing through ovary, instead
of columella, two bean shaped lobes with ovules will be seen in the centre.
Starch, trichomes and stone cells are absent.
Microchemical Tests:

REAGENTS OBSERVATION CHARACTERISTICS
Phloroglucinol + conc.HCl (1:1). Pink Vascular bundles & fibres.
Sudan red III Red Cuticle glands.
Strong KOH solution. Needle shaped potassium

eugenate crystals. Eugenol of volatile glands.

Dil. HCl (60%w/w) sulphuric acid. Crystal’s soluble, needles of

calcium sulphate on standing. Calcium oxalate crystals.

Chemical Constituents: It mainly contains 15-30% of Volatile oils contents. The chief
constituents of volatile oil are Eugenol, isoeugenol, methyl & dimethyl furfural, α & β
caryophylline. It also contains Hydrolysable tannins.
Uses:
1. It is used as carminative, an aromatic, stimulant and flavouring agent,
2. It is used as dental analgesic and antiseptic
3. It is used in microscopic work and also used for isolation of eugenol.
Report:
Transverse section of Clove was prepared and Morphological characters and microscopic
characters of clove were studied.

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

SYSTEMATIC PHARMACOGNOSTIC STUDY OF EPHEDRA

AIM:Study of macro, powder and microscopic study of Ephedra.
APPARATUS AND CHEMICALS REQUIRED: Glass slide, microscope, camel brush,
coverslip, glycerin, chloral hydrate, phloroglucinol etc.
BIOLOGICAL SOURCE: It is consists of dried young stem of Ephedra gerardiana (Wall)
stap F and E. nefrodensis (Tineo) stap F or E. sinica and E. equisetina belonging to the family
Ephedraceae.
MORPMOLOGICAL CHARACTERS
SHAPE : Cylindrical woody stems

Scaly leaves are subulate usually in whorls of two from
each node.
COLOUR : Grey to Greenish
Brachlets are cylindrical and green in colour leaf bases are
dark brown in colour and joined to form a sheath.

TASTE : Strong astringent.
ODOUR : Heavy, aromatic, pine.
DESCRIPTION:
A transverse section shows.
1. EPIDERMIS:
Unicellular Epidermis is made of quadrangular cells along with thick walled cuticle
vertical rows of sunken stomata and papillae on the ridges.
2. CORTEX:
Corte is chlorenchy matous outer one consists of radially elongated cells and inner one
consists of spongy parenchyma. Hypodermal fibres are present below the ridges. Meso
cortical fibres are present in groups, non – lignified to slightly lignified.
3. VASCULAR BUNDLES:
These are open, collateral and about 6 – 10 secondary xylem forms a complete ring in old
stems.
Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

4. PITH:
Pith is made up to parenchymatous large rounded cells with dark brown mucilaginous
substance.

PROCEDURE:
Take a clean glass slide and place a pinch of given alkaloidal drug powder in the centre
of the slide. Few drops of glycerine is added to it and care fully spread by using a brush. Put the
cover slip with care to avoid enclosing of air bubbles. Then observe the slide under microscope.

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

POWDER CHARACTERISTICS:
1. Lignified and non – lignified fibres.
2. Tracheids with bordered pits.
3. Epidermis with ridged outer walls.
4. Dark brown pigmented cells.

CHEMICAL CONSTITUENTS:
Itcontains alkylamine alkaloids i.e. Ephedrine, n – methyl ephedrine, pseudo – ephedrine etc.

USES:
1. It is used as a bronchodilator in asthma.
2. It is also used in the treatment of allergic conditions like hay fever.
3. It is also used to correct the low blood pressure conditions.
REPORT:
The given organized crude drug was identified as EPHEDRA.
Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

EXPERIMENT-7

SYSTEMATIC PHARMACOGNOSTIC STUDY OF FENNEL FRUIT
Aim: To Study Morphological and Microscopical Characteristics of Fennel Fruit.
Requirements:
Crude drug, Microscope, glass slide, watch glass, brush, needle, cover slip, blade, Filter paper.
Staining Reagents:
Phloroglucinol, conc. HCl (1:1), Alcoholic picric acid, Sudan red -III
Synonym: Bari sauf (Hindi), Fructus foeniculi
Biological Source:
It consists of dried ripe fruits of cultivated species Foeniculum vulgare Belongs to the family:
Umbelliferae
It contains not less than 1.4 % of volatile oil.
Macroscopical Characters:
Color: Greenish (or) yellowish brown
Odour: Sweet aromatic and characteristic
Taste: Sweet mucilaginous, Agreeable aromatic and characteristic.
Extra Features:
Fennel exhibits cremocarp. It consists of two equal portions called mericarp connected by a
central stalk called as carpophore.
Microscopic Characters:
Pericarp: It contains Epicarp, Mesocarp, Vittae and Endocarp
Epicarp contains a layer of quadrangular to polygonal cells with smooth cuticle.
Mesocarp contains Reticulate, lignified parenchyma surrounding the vascular bundles.
Vascular Bundles are five in number and bicollateral, present below each ridge (primary ridge)
Vittae contain Schizogenous oil cells. It contains 6 vittae, 4 are present on dorsal side and 2 are
present on commissural surface / ventral surface. About 250μ in maximum width, the walls are
brown.

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

Endocarp: It Consists of narrow elongated cells having a parquetry arrangement (group of
parallel cells arranged in different directions).
SEED:
Testa: It contains Single layered cells and it is yellowish brown in color.
Endosperm: It contains Thick walled polygonal, cellulosic parenchyma containing oil globules
(fixed oil), Aleurone grains and rosette crystals of calcium oxalate.
Raphe is a single ridge of vascular strands appears in the middle of commissural surface.
Carpophore contains very thick walled sclerenchyma in 2 strands.
Micro Chemical Test:
S. NO. REAGENTS OBSERVATION CHARECTERISTICS
1. Phloroglucinol + conc. HCl

(1:1) Pink Lignified reticulate parenchyma
of mesocarp & vascular bundles
2. Alcoholic picric acid Yellow Aleurone grains
3 Sudan red -III Red Oil globules in the cells of
endosperm and cuticle

Chemical Constituents:
The mainly contains 4-6% of Volatile oil.
The volatile oil contains 50-60% of Anethol, 12-18% of Fixed oil and14-22% of Proteins.
Uses:
➢ Used as Carminative and flavoring agent.
➢ Used as an aromatic.
➢ Used as respiratory stimulant.
➢ Used as stimulant
Report:
Transverse section of Clove was prepared and Morphological characters and microscopic
characters of Fennel Fruit were studied.

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

SYSTEMATIC PHARMACOGNOSTIC STUDY OF CORIANDER FRUIT
AIM: Study of macro, powder and microscopic study of Fennel Fruit.
REQUIREMENTS: Microscope, glass slide, coverslip, camel brush.Phloroglycerol + conc.Hcl,
alcoholic picric acid, sudan red III.
SYNONYM: Dhania (hindi), coriander fruit.
BIOLOGICAL SOURCE: Dried ripe fruits of Coriandrum sativum Linn.belonging to the family
Umbelliferae.It contains not less than 0.3% of the volatile oil.
MORPHOLOGICAL CHARACTERS:-
COLOUR: Brownish yellow
ODOUR: Aromatic,
TASTE: Spicy and characteristic.
EXTRA FEATURES: Cremocarps, caelospermous fruit.
MICROSCOPICAL CHARACTERS:
PERICARP:
EPICARP: Single layer, thickened, polygonal tabular cells, few cells contain, calcium oxalate
crystals, covered by smooth cuticle.
MESOCORP: Divided into 3 layers.
1. OUTER LAYER: Poorly arranged tangentially elongated non-lignified parenchyma, lacunae
at dorsal side.
2. MIDDLE LAYER: Fusiform, lignified, sclerenchymata cell in sinous cells.Sclernchymatous
cells are of 2 types: 1.Tangentially elongated sclerenchyma.
2. Longitudinally elongated sclerenchyma.Fine vascular bundles at dorsal side, present above the
longitudinally elongated sclerenchyma.Two vittae at ventral side and No vittae at dorsal side.
3. INNERLAYER: Large irregular, hexagonal lignified parenchyma.
ENDOCARP: Inner pericarp shows typical parquetary arrangement of the cells.
SEED:
TESTA: Single layered and yellowish in colour.

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

ENDOSPERM: Thick walled, polygonal cellulosic parenchyma containing fixed oil aleurone
grains and microrosetta of calciumoxalate

STAINING/ DIAGNOSIS/ MICROCHEMICAL TESTS
S.NO REAGENTS OBSERVATION CHARACTERS
1. Pholoroglucinol+conc.Hcl(1:1). Pink. Lignified sclerenchyma,
vascular bundles.

2. Alcoholic picric acid. Yellow. Aleurone grains present in the

cells, Endosperm.
3. Sudan red III. Red. Oil globules, Cuticle.

MICROSCOPICAL CHARACTERISTICS OF THE POWDERED DRUG
SCLERENCHYMATOUSLAYER – Groups of fusiform, fibres running wavy, crossing each
other, lignified.

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

ENDOCARP: Parquetary arrangement of thin walled lignified cells with polygonal cells (or)
mesocarp.
ENDOSPERM: Polygonal parenchyma with aleurone grain & oil globules. Microrosette crystals
of calciumoxalate in the cells.
VITTAE: Few yellowish brown fragments of vittae thin walled small parenchymatous cells in
group.Trichomes & lignified reticulate parenchyma are absent.

CHEMICAL CONSTITUENTS: Volatile oils (0.2 – 1%), coriandrol(E)-linol (0.01-60 to 70&),
Terpenes (20%),Fixed oil(13- 20%), Protiens (17%).
USES: It is used as a Carminative, aromatic, stimulant, spice, flavouring agent.
SUBSTITUENTS: Bombay coriander-Less volatile oil, Bigger in size, Ellipsoidal in shape.
REPORT:-

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

ISOLATION OF CAFFIENE FROM TEA POWDER

AIM:
To isolate caffeine from tea powder and report the percentage yield.
REQUIREMENTS:
APPARATUS:
Beakers, glass rod, china dish, Electronic heater and Mantle and Tripod stand, Test tubes, weighing
Balance
MATERIALS AND REAGENTS:
Tea powder, lead acetate, distilled water, chloroform, sodium hydroxide solution, Dil.H2SO4,
Ammonia solution, Potassium chlorate

PRINCIPLE:
Caffeine is a purine alkaloid and chemically 1, 3, 7 Trimethyl Xanthine. Caffeine is a white or
colourless powder or white crystalline, bitter in taste. It is sublimed without decomposition.
It is the component of tea leaves (5%), coffee (1-2%) and kola nuts (1-2%)
Casein is present upto 4% in Tea leaves (Thea sinensis) belongs to the family Theaceae.
Caffeine is soluble in hot water. Therefore caffeine can be extracted by boiling tea dust with water.
Hot extract (decoction) is obtained by filtration.
The extract is then treated with lead acetate solution to precipitate tannins as lead tannate, which
is filtered off. Subsequently the excess lead is removed by treating with Dil.H2SO4. By this, lead
is precipitated as lead sulphate and separated by filtration.
The filtrate is then made neutral with NaOH solution. The liberated purine (Caffeine) can be
suitably extracted with chloroform. Finally, the chloroform extract is concentrated and evaporated
to get crystals of caffeine.
and responsible for stimulating the action of these beverages on the nerves and heart and for that
reason it is employed as medicine.
PROCEDURE:
Take 30gm of tea powder in a beaker and boil for 30min with 150ml water. Filter in hot condition
and collect the filtrate. Press the marc and combine both the extracts.
Add sufficient quantity of lead acetate solution to precipitate the tannins completely.
Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

STRUCTURE: CAFFEINE

IUPAC NAME:

1, 3,7-trimethyl xanthine (or) 1, 3, 7-trimethyl- 2, 6- dioxo purine.

Calculation:
Theoretical yield of casein:
30gm of Tea powder ---------- gms of Caffiene
10gm of Tea powder gives ----------- gms
Practical yield of Caffiene ------------ x

%yield of Caffiene =

practical yield
theoritical yieldX 100

.
.
. %yield of caffiene = X
0.5
X 100 _______%

To ensure this, collect few drops of filtrate in a test tube. Filtrate should be pale yellow colour for
further confirmation, add few drops of lead acetate solution. Complete precipitation of tannins is
confirmed if there is no precipitate in test tube.
Filter to remove precipitate (lead tannate) and collect the filtrate.

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

Add sufficient amount of Dil.H2SO4 to the filtrate to precipitate excess lead as lead sulphate,
filter and collect the filtrate.
Make the filtrate neutral by the addition of NaOH solution.Concentrate the solution to 50ml and
transfer to separating funnel. Finally extract with repeated portions of chloroform 10 ml each
and combine the chloroform extracts.
Note: though chloroform is an organic solvent its specific gravity is more than 1 therefore
collect the bottom layer.
Concentrate the chloroform extract and cool to get the product.
Perform murexide test for its identification and Report the yield of caffeine.
Murexide test for identification:
Murexide test is useful for the identification of purine alkaloids like caffeine.
Take 10mg of caffeine (isolated product) in a porcelain dish. Add 2-4 drops of Nitric acid.
Evaporate to dryness and then add 2 drops of ammonium hydroxide to the residue. Development
of purple colour confirms caffeine.
Uses:
It acts as a CNS stimulant.
It is useful in the treatment of migraine along with ergotamine.
Report:
Caffiene was isolated from Tea powder and the percentage yield was calculated. The
percentage yield of caffeine was found to be __________________%
Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

INTRODUCTION TO TLC

Adsorbents: Silica gel, aluminium oxide, cellulose, derivatives of cellulose, calcium hydroxide,
magnesium phosphate, polyamide, and precoated TLC plates are available in market.
Saturation of chromatographic chamber:
The chromatographic tank was filled with the solvent system till the level of few centimeters and
it was kept closed for few hours for saturation.
Preparation of TLC plates:
The TLC plates can be prepared by using any one of the following techniques.
Pouring technique:
The slurry is prepared and poured on to a glass which is maintained on a leveled surface. The
slurry is spread uniformly on the surface of the glass plate. After setting, the plates are dried in an
oven.
Dipping technique:
Two plates are dipped into the slurry and are separated after removing from slurry and later dried.
The disadvantage is that the layer thickness cannot be maintained uniformly all over the plate.
Spraying technique:
The suspension of an adsorbent or slurry is sprayed on a glass plate using a sprayer. The
disadvantage is that the layer thickness cannot be maintained uniformly all over the plate.
Spreading:
This is the best technique where a TLC spreader is used. The glass plate of specific dimensions
are stacked on a glass plate (20x20/10/5) are stacked on a base plate. The slurry after preparation
is poured inside the reservoir of TLC spreader. The plates are allowed for setting. This is done to
avoid cracks on the surface of adsorbent. after setting; the plates are activated by keeping in an
oven at 100 0C to 120 0C for 1hr.
Spotting of the sample:
It is done on the specifically prepared TLC plates with the help of micropipette or capillary tube.
A horizontal line is drawn with the pencil 0.5 inches from the bottom. This line is called as base
line or origin line. On this line, the samples are spotted and the TLC plate is labeled as A,B for
each alkaloidal or glycosidal sample. These spots are dried in the atmospheric air or a stream of
hot air or in the special drying cabinets.

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

DEVELOPMENT OF THE TLC PLATES:
Ascending technique is used for the development of TLC plate in which solvent flow is against
the gravity and travels up the TLC plates. A few milliliters of solvent system (mobile phase) is
taken in the TLC development chamber. The spotted TLC plate is placed vertically in the
development chamber and quickly covered with a lid. The solvent level in the chambers should be
below the level of the spots on the plate. The solvent runs up the plate by capillary action. This
slow movement of the solvent, carrying the compounds with continued till the solvent runs upto
about 4/5 of the coated plate. Later the plate is removed, solvent front is marked and the TLC plate
into that air oven.
PREPARATION OF PLATES:
Mix 30gm of adsorbents with 60ml deionised water in a stopper glass bottle or flask and shake it
thoroughly for 90 seconds and spread it on the glass plate (slurry sufficient to coat 10 (10×20)
cm). Keep them in horizontal position till the adsorbent layer is firmly set. Precoated plates are
available with different adsorbents, coated on glass, aluminium sheers or plastic. These are also
available with the fluorescent indicator that allows the detection of compound which quenches
the fluorescence when the plate is observed in UV light of 254nm.

VISUALIZATION OR DETECTION TECHNIQUE:
The dried TLC plates are removed. If all the compounds in the sample examined are colored, a
visual inspection is sufficient to locate the spots. If the compounds in a sample are colorless, the
position of each compound can be visualized by iodine vapor or by using ninhydrin solution or in
UV chamber in short wavelength.
QUALITATIVE ANALYSIS OF TLC:
The TLC data of a compound are expressed in terms of Retardation factor or Resolution factor
(Rf) values.
Rf = Distance travelled by the component
Distance travelled by the solvent front.
Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

Activation of plates:
Air dried plates are activated by heating in an oven at 100 to 110°C for 30- 60 mins.
Application of spots:
1 to 10 ml samples are spotted at 2 to 3 cm intervals across the line 15 to 20mm from the base of
the plate. Then spots are allowed to dry.
Development of the plate:
The plate is placed GC tank with the solvent placed in the bottom of the tank to the depth of
10mm. A lid is sealed to the top of the tank. The solvent is allowed to run to 10cm. Note the
temperature of the lab (sufficient for qualitative separation). The plate is removed; solvent front
marked immediately and then allowed to dry.
Detection of spots:
Compound separated on the plate is visualized by general or specific methods. Fluorescent
compounds are examined in both long (565nm) and short (263 nm) wavelength in UV light.
Different reagents in the form of sprays are used for detecting different chemical constituents.
Documentation:
A copy of TLC plate is made as a tracing paper placed on the back of the plate holding it against
light. Note the base line, number of spots on th sheet and solvent front and calculated Rf value for
each. Rf value is always 1
To avoid this fraction multiply with 100.
Preparative TLC: It is carried out by using thick layer (1-2mm) of adsorbent. The area of
separated constituents are scrapped off, the development plate. The adsorbent is then eluted with
solvent like ether, which is centrifuged to remove adsorbent.

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

EXPERIMENT-9

DETECTION OF EUGENOL IN CLOVE OIL BY TLC

Aim:
To determine content of eugenol in clove oil by TLC
Requirement:
Clove oil, Eugenol Standard, Silica gel-G, TLC plates, TLC Chambers, Glass plates, Oven.
Solvent system:
(1:9) i.e. 10Volumes of ethyl acetate and 90 Volumes of Toluene R
Spraying agent:
Anisaldehyde Solution.
Principle:
The main principle involved in TLC is principle of separation. The separation depends on the
relative affinity of compounds towards stationary and mobile phase. The compounds under the
influence of mobile phase (driven by capillary action) will travel over the surface of stationary
phase. During this movement the compounds with higher affinity to stationary phase travel slowly
while the others travel faster. Thus separation of components in the mixture is achieved. Once
separation occurs individual components are visualized as spots at respective level of travel on the
plate. Their nature of character is identified by means of suitable detection techniques. This process
involves a suitable adsorbent (the stationary phase), solvents or solvent mixtures (the mobile phase
or eluent), and the sample molecules. For thin layer chromatography the adsorbent is coated as a
thin layer onto a suitable support (e.g. glass plate, polyester or aluminium sheet). On this layer the
substance mixture is separated by elution with a suitable solvent.

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

Procedure:
1. Prepare slurry of silica gel G by mixing one part of the adsorbent with 2.5 parts of water in
a glass mortar. Coat the slurry uniformly (thickness0.3mm) with the help of an applicator
on clean dry glass plate. Allow the plate to dry at room temperature and activate in an oven
at 1200
C for 30 minutes.

2. Prepare the solvent system and pour it to a depth of 3cm in the chamber lined from inside
with a filter paper to maintain the equilibrium of the mobile Phase.
3. Prepare the standard reference solution by dissolving 50μ litres of Eugenol in alcohol and
dilute to 25ml with the same solvent.
4. Prepare the test solution by dissolving 50μ litres of the substance to be examined (Clove
oil) in alcohol and dilute to 25ml with the same solvent.
5. Apply to the plate 5μ litre of each solution i.e. Test Solution and Standard reference
solution at a distance of 2-5 cm from each other with the help of micropipette or capillary
tube. Allow the spots then dry at room temperature.
6. Place the glass plate gently inside the chamber develop the Chromatogram by ascending
technique till the solvent front had moved by about 15cm using a mixture of 10Volumes
of ethyl acetate and 90 Volumes of Toluene. Then take out the plate, Mark the Solvent
front and dry at room temp.
7. Spray the plate with anisaldehyde solution then heat at 100-1050C for 10 minutes. The
Principal spot in the chromatogram obtained with the test solution is similar in position,
colour and size to the principal spot in the chromatogram obtained with the reference
solution.
8. Calculate the Rf value of test solution and standard solution.
Rf = Distance travelled by the component
Distance travelled by the solvent front.
Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

Identify the Eugenol in clove oil by comparing their location and Rf values with that of
Standard reference solution.
Report:
Eugenol in clove oil is identified by comparing their location and Rf values with that of Standard
reference solution.

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

EXPERIMENT-2

IDENTIFICATION OF CRUDE DRUGS BY ORGANOLEPTIC METHOD
AIM:
To identify crude drugs by using organoleptic method

1. Cinnamon
Synonym: Cinnamon bark, Kalmi-Dalchini, Ceylon cinnamon.
Biological Source:
Cinnamon consists of the dried inner bark of the shoots of coppiced trees of Cinnamomum
zeylanicum, Cinnamomum verum belongs to the family Lauraceae.
Chemical Constituents:
Cinnamon bark contains about 0.5 to 1.0% of volatile oil, 1.2% of tannins (Phlobatannins),
mucilage, calcium oxalate, starch and sweet substance known as mannitol. Volatile oil is
the active constituent of the drug. Cinnamon oil contains 60-70% of cinnamaldehyde, 5-
10% eugenol, benzaldehyde, cuminaldehyde and other terpenes like phellandrene, pinene,
cymene, caryophyllene etc.
Uses:
1. Bark is used as carminative, stomachic and mild astringent.
2. It is also used as flavouring agent and stimulant.
3. It is used as an aromatic and antiseptic.
4. Commercially is used as spice and condiment.
2. Clove
Synonym: Caryophyllum, Clove flower, Clove buds.
Biological Source:
Clove consists of dried flower buds of Eugenia caryophyllus, belongs to the family
Myrtaceae.
Chemical Constituents:
Clove contains about 15 to 20% of volatile oil, 10 to 13% of tannin (gallotannic acid),
resin, chromone and eugenin. The volatile oil of the drug contains 70-90% eugenol,
eugenol acetate, caryophyllenes.
Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

Uses:
1. It is used as Dental analgesic, carminative and stimulant.
2. It is used as flavouring agent, aromatic and antiseptic.
3. The oil is used as perfumery and also in the manufacture of vanillin.
3. Fennel
Synonyms: Fennel fruits, Fructus Foeniculi
Biological Source:
Fennel consists of dried ripe fruits of the plant konown as Foeniculum vulgare, family
Umbelliferae, obtained by cultivation.
Chemical Constituents: Fennel contains of 3 to 7% of volatile oil, about 20% each of
proteins and fixed oils. The chief active constituent of the volatile oil is a ketone,
fenchone 20%, and a phenolic ether anethole 50%. The other constituents are
phellandrene, limonene, methyl chavicol, anisic aldehyde etc.
Uses:
1. It is used as carminative, aromatic and stimulant.
2. It is also used as expectorant.
3. Pharmaceutically it is used as flavouring agent.
4. Myrobalan
Synonym: Chebulic myrobalan, Harde, Haritaki
Biological Source:
It consists of dried, ripe and fully matured fruits of Terminalia chebula belongs to the
family Combretaceae.
Chemical Constituents:
Myrobalan fruits are an important source of tannin. The tannins of Myrobalan are of

pyrogallol type (hydrolysable tannins), which on hydrolysis yield chebulic acid and d-
galloyl glucose. Chebulagic, Chebulinic, ellagic and gallic acids are the other contents of

myrobalan. Myrobalan also contains glucose and sorbitol (about 3.5%).
Uses:
1. Myrobalan is mainly used as astringent, laxative, stomachic and tonic,
antihelementic.
2. Fruit pulp is used to cure bleeding.
Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

3. It is an ingredient of Ayurvedic preparation ‘Triphala’, used for the treatment of
ailments.
4. Commercially, it is used in dyeing and tanning industry.
5. Myrobalan is used in the treatment of piles and external ulcers.
5. Black catechu:
Synonym: Kattha, Cutch, Khadir-catechu, Catechu
Biological Source:
It consists of dried aqueous extract prepared from the heart-wood of Acacia catechu and
Acacia chundra, family Leguminosae.
Chemical Constituents:
Black catechu contains about 10% of acacatechin. It is distereoisomer of 5, 7, 3’,4’
tetrahydroxy falavan-3-ols. Acacatechin is also knowns as acacia catechin. Acacatechin
undergoes oxidation to catechutannic acid in presence of water and the latter constitutes
about 30% of the drug. The other contents of black catechu are catechu red, quercetin,
gum and quercitrin.
Uses:
1. It is used as an astringent externally for boils, skin eruptions and ulcers.
2. It is also used in cough and diarrhea.
3. It has cooling and digestive properties.
Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

Viva Questions:
Explain the principle involved in hydrodistillation?
What are essential oils.
2. Give the synonym of Eucalyptus leaves.
3. Write the chemical Constituents of Eucalyptus leaves.
4. what is the use of clavengers apparatus.
5. Write the the uses of Eucalyptus oil .
6. Give the organoleptic characters of eucalyptus oil.
7. Write the Biological source of Eucalyptus .
8. What is the family of Eucalyptus.
9.what is the yield of eucalyptus oil after extraction.
10.what is the main chemical constituent of eucalyptus leaf.
Clove oil is ............... oil.
2.What is TLC and What is the principle involved in TLC.
3.What is the Solvent System used for detection of eugenol in clove oil by TLC.
4.What is the Spray reagent used to identify the spots in tlc of clove oil.
5.what is Rf value.
6.How Rf Value is calculated ?
7.Write the Family of clove.
8.write the biological source of clove.
9Give the Biological source of Clove.
10.Write the Uses of Clove.
1.What is the the Solvent System used in TLC of Eucalyptus oil.
2.What is the Spray reagent used in TLC of Eucalyptus oil.
3.What is the odour of Eucalyptus oil.
4.What is the Thickness of Silica gel on the TLC glass plate.
5.Write the Chemical Constituents of Eucalyptus oil.
6.Write the different uses of Eucalyptus oil.
7.Why the activation of TLC plate is Required .
8.Why the Saturation of TLC Chamber is required.
9.Which Chemical constituent is responsible for its camphoraceous odour of the oil..
10. What is Silicagel.
.What are Curcuminoids.
2.Curcumin is extracted from which particular drug.
3.Write the Biological Source of Turmeric.
4.Write the Chemical constituents of Turmeric.
5.write the family of Turmeric.

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

6.Write the Uses of Turmeric.
7.What is the Solvent Used for Extraction of Curcuminoids.
8.What is the method of Extraction for curcuminoids.
9.What is the Solvent System Used For TLC of Curcuminoids.
10.What is the difference between Curcuminoids and curcumin.
What are Tanins.
2.What are the Different Types of Tanins.
3.Give the Examples of Tanins.
4.What are pseudo tannins.
5.Write the Synonym of Black Catechu.
6.What is the Family of Myrobalan.
7.Write the Chemical constituent of Arjuna bark.
8.What is the General chemical test for Tanins.
9.What is Match stick test.
10.What is the Difference between Black catechu and Pale catechu.
What are Resins.
2.What are the Different Types of Resins.
3.Give the Examples of Resins.
4.What are the Chemical test of Resins.
5.What are oleo gum resins..
6.What is the Family of Benzoin.
7.Write the Chemical constituent of Asafoetida.
8.What is the Common name for Benzoin.
9.What is the chemical constituent of tolu balsam.
10.What is the use of Asafoetida.
. Write the Synonym of Fennel.
2. Write the Biological source of Fennel.
3. Write the family of Fennel
4. Write the Morphological characters of Fennel
5. What are the different microscopic characters of fennel.
6. What are the different stains /micro chemical test for fennel.
7. How many vittae are present in TS of fennel
8. How many Vascular bundles are seen in TS of Fennel.
9. What is the main chemical constituent of Fennel.
10. Write the Different Uses of Fennel
Write the Synonym of Clove.
2. Write the Biological source of Clove
3. Write the Family of Clove

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

4. Write the Morphological characters of Clove
5. What are the different microscopic characters of Clove.
6. What are the different stains /micro chemical test for Clove
7. What is the specific microscopic character of Clove.
8. What is the adulterant for Clove.
9. What is the main chemical constituent of Clove.
10. Write the Different Uses of Clove.
Write the Synonym of Cinnamon.
2. Write the Biological source of Cinnamon
3. Write the Family of Cinnamon
4. Write the Morphological characters of Cinnamon
5. What are the different microscopic characters of Cinnamon
6. What are the different stains /micro chemical test for Cinnamon
7. What is the specific microscopic character of Cinnamon
8. What is the adulterant for Cinnamon
9. What is the main chemical constituent of Cinnamon.
10. Write the Different Uses of Cinnamon.
. Write the Synonym of Ginger.
2. Write the Biological source of Ginger
3. Write the Family of Ginger
4. Write the Morphological characters of Ginger
5. What are the different microscopic characters of Ginger
6. What are the different stains /micro chemical test for Ginger
7. What is the specific microscopic character of Ginger
8. What is the adulterant for Ginger
9. What is the main chemical constituent of Ginger
10. Write the Different Uses of Ginger.
Write the Synonym of Eucalyptus.
2. Write the Biological source of Eucalyptus
3. Write the Family of Eucalyptus
4. Write the Morphological characters of Eucalyptus
5. What are the different microscopic characters of Eucalyptus
6. What are the different stains /micro chemical test for Eucalyptus
7. What is the specific microscopic character of Eucalyptus
8. What is the adulterant for Eucalyptus
9. What is the main chemical constituent of Eucalyptus
10. Write the Different Uses of Eucalyptus

Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

. Write the Synonym of Black Catechu.
2. Write the Biological source of Black Catechu
3. Write the Family of Black Catechu
4. What is the colour of Black Catechu
5. What is the odour of Black Catechu
6. What is the Taste of Black Catechu
7. What is the shape of Black Catechu
8. What is the adulterant for Black Catechu
9. Black Catechu is an extract obtained from ................... Wood of..................
10. Write the Different Uses of Black Catechu
1. Write the Synonym of Pale Catechu.
2. Write the Biological source of Pale Catechu
8. What is the adulterant for Pale Catechu
9. What is the main chemical constituent of Pale Catechu
10. Write the Different Uses of Pale Catechu
Write the Synonym of Myrobalan.
Write the Biological source of Myrobalan
What is the main chemical constituent of Myrobalan
Write the Different Uses of Myrobalan
2. Write the Biological source of Arjuna bark
What is the main chemical constituent of Arjuna bark
10. Write the Different Uses of Arjuna bark
Write the Synonym of Balsam of Tolu.
2. Write the Biological source of Balsam of Tolu.
7. What are Balsams
9. What is the main chemical constituent of Balsam of Tolu.
10. Write the Different Uses of Balsam of Tolu.
Viva questions
• What is macroscopy? What are the macroscopic characteristics of senna
• Define microscopy. what are the microscopic characteristics of senna
• Name some species of senna?
• What is the difference between senna and datura with respect to palisade parenchyma?
• What type of leaf is senna? How you differentiate palisade tissue and spongy tissue?
Downloaded from HK Technical PGIMS (pgims.hktechnical.com)

• What type of stomata is present in senna?
• What are trichomes? What type of trichomes are obsereved in senna?
• What are the powder characteristics of senna?
• what are the chemical constituents in senna
• .what type of alkaloids are present in senna?
• What are the general tests for alkaloids?
• What are the uses of senna?