1

Pharmacognosy – 1

(PHG 222)

A Laboratory Manual

2

Course contents

Lab.

No.

Date

Sunday

Lab. Titels

1 92 Jan. Introduction , instruction

Botanical Garden, Herbarium, Basic Principles For

Plant Taxonomy and microscope

2 5 Feb. Plant Morphology & Inclusions

3 29 Feb. Microscopic Characters of Powdered Leaves,

Flowers, Seeds, Fruits and Barks Containing Volatile

Oils

4 22 Feb.

Microscopic Characters of Powdered Leaves,

Flowers, Seeds, Fruits and Barks Containing Volatile

Oils

5 92 Feb Physical characters and Chemical tests of

Carbohydrates

6 5 Mar

Unorganized Drugs

Gums and Resins

7 29 Mar Unorganized Drugs

Latice, Juices & Aqueous Extracts

8 22 Mar.

Chromatography

9 21- 29 Mar.

MIDTERM HOLIDAY

10 9 Apr Chromatography

11 2 Apr. Different Extraction Techniques

Preparation of Some Extracts

I-Classical Techniques (volatile oils extraction)

12 22 Apr.

Different Extraction Techniques

Preparation of Some Extracts

II-Modern Techniques(other compounds

extraction)

13 92 Apr Application of extraction method and

chromatography

14 23 Apr.

Application of extraction method and

chromatography

15 7 May.

Final exam

3

Lab (13)

Introduction

Instructions for Students

Students shall read the points given below for understanding theoretical

concepts and practical applications.

1. Students should wear white lab coat, mask, and gloves before

entering into the laboratory.

2. Students should keep their belongings in locker which are not

required during practical like bag, extra files, etc.

3. Students should always carry Laboratory Manual, rough notebook

and practical requirements without exception.

4. Listen carefully to the lecture given by teacher about importance of

subject, skills to be developed, information about equipment,

instruments, procedure, method of continuous assessment, tentative

plan of working laboratory, and total amount of work to be done.

5. Students should perform the practical only at the place which

allocated to her. (No change can be done without permission of

subject teacher)

6. Students shall undergo study visit of laboratory for types of

equipment, instruments, material to be used, before performing

experiment.

7. Read write up of each experiment to be performed, a day in advance.

8. Organize the work in the group and make a record of all

observations.

9. Understand the purpose of experiment and its practical applications.

10. Write the answer of the questions allotted by teacher during

practical hours if possible or afterwards, but immediately.

11. Students should not hesitate to ask any difficulty faced during

conduct of practical.

12. Students shall study all the questions given in the laboratory manual

and practice to write the answers to these questions.

13. Students shall develop maintenance skill as expected by the

industries.

14. Students should develop the habits of pocket discussion, group

discussion related to the experiments so that exchanges of

knowledge, skills could take place.

4

15. Students shall attempt to develop related hands-on -skills and gain

confidence.

16. Students shall focus on development of skills rather than theoretical

or codified knowledge.

17. Students shall visit nearby workshops, workstation, industries,

technical exhibitions, trade fair etc. even not included in the lab

manual. In short, students should have exposure to the area of work

right in the student's hood.

18. Students shall insist for the completion of recommended

laboratory work, industrial visits, answers to the given questions,

etc.

19. Students shall develop habits of evolving more ideas, innovations

skills etc. than included in the scope of the manual.

20. Students shall develop technical magazines, proceedings of the

seminars, refers websites related to the scope of the subjects and

update their knowledge and skills.

21. Students should develop the habit of not to depend totally on the

teachers but to develop self- learning techniques.

22. Students should develop the habit to react with the teacher without

hesitation with respect to the academics involved.

23. Students should develop the habit to submit the practical exercise

continuously and progressively on the scheduled dates and should

get the assessment done.

24. Students should be well prepared while submitting the write up of

the experiments. This will develop the continuity of the studies and

he will not be over loaded at the end of the term.

25. Students should clean the platform before leaving the laboratory.

Marks distribution

15

Practical part:

Attendance, Continues evaluation and

report

10 Practical exams

30 Total

5

Part I: Botanical Garden

Definition of Botanical garden A place/ venue (controlled or semi controlled environment) to cultivate/grow different

plant species for different purposes (public, conservation, recreation, global

conservations, genetic conservation…etc)

Objectives

Create a controlled environment to cultivate plants.

To provide samples of medicinal, aromatic and poisonous plants for academic and

researches purposes.

To conserve the endangered medicinal plant species.

To create a storage of the required plants for academic and research purposes.

Components of the HBG

Green houses.

High land area

Low land area

Irrigation system

Storage

Main farms.

Management of the HBG

Setting up of the green houses and preparation of the areas.

Irrigation management.

Different irrigation methods (drip irrigation, surface irrigation, sprinkling

irrigation).

Hydroponic system which is method of growing plants using mineral nutrient

solutions, in water, without soil.

Fertilization (phosphor and calcium, carbon dioxide and germination).

How to define the medicinal plant

All the plant or part of the plant or product/extract out of the plant used for medication

either traditional or based on scientific evidences.

Part II: Herbarium Definition of Herbarium

A herbarium is a collection of dried, pressed plants mounted on herbarium sheets

bearing detailed data, labelled and stored in a herbarium cabinet in a climate controlled

room, preferably fumigated and one without windows that open. A herbarium can be

thought of as a dried plant library, the pages of the books are the sheets of plants. Like

a library, the “books” or dried plant specimens are arranged in a systematic order by

plant family, eg: solanaceae. Usually the plant families are arranged either

alphabetically or phylogenetically (by their evolutionary relationships).

Herbarium cabinet

6

The least expensive models are metal with rubberized seals around the doors to keep

the humidity constant and help prevent insect infestation. Insects and mold can

devastate a herbarium. The cabinet has deep shelves to hold the herbarium sheets. When

not in use, keep the herbarium cabinet doors closed.

Pressing herbarium specimens from fresh plant material

A field notebook should be used to record important morphologic characters, especially

those that might be lost when the plant is dried, such as flower colour. Each specimen

should get its own specimen number. Record the date, location, directions to site etc.

Fresh plant material can be collected in plastic bags, large containers or pressed right

on the spot. Arrange the fresh plant material in the plant press, so that the top and

underside of leaves will be available for examination. Arrange floral parts and fruits for

easy inspection. Each specimen is placed in folded sheet of newsprint or any paper large

enough to cover most of the specimen. Each newsprint wrapped specimen is then

sandwitched between special water absorbent papers. Foam sheets are also used to

flatten large, bulky material such as fruits, nuts, thick stems and leaves etc. The foam

sheet can be placed against the newsprint wrapped specimen. This is all placed in a

plant press and compressed. Typical plant specimens will be dry in a few days.

Plant dryer

Fresh plant materials placed in a plant press must be dried so that it can be glued onto

a sheet of paper. A slow gentle drying is the best as this prevents the colour of the plants

from fading and delicate plant parts are less likely to shrivel.

Mounting specimens

Once the dried plant specimen is dried, it is ready for mounting. There are two methods

for mounting specimens. One method involves the use of glass plate which is coated

with glue. After coating the glass, place the plant specimen on the glass sheet and gently

press into the glue surface. This will coat the underside of the specimen, which can then

be carefully removed and placed on a herbarium sheet. The other method is probably

more time consuming but best for smaller numbers of specimens. In this method glue

is placed at selected areas on the underside of the specimen by hand or by using a small

brush dipped in glue. Once the glued specimen has dried onto the herbarium sheet,

straps may be used for fixing it.

Specimens

Straps help ensure that the specimen does not pop off the herbarium sheet when the

sheet is flexed. The plant specimen is labelled and arranged on the sheet.

Plant labels The label helps to identify the plant. It specifies the botanical name first and may also

include common names. The botanical name should also include the author of the name.

Important aspects of the plant specimen including the height of plants, flower colour,

plant/floral odour, habitat details, soils, geologic features, chromosome number, etc.

7

can be noted. Other important data include the date of the collection, location (city,

county, state, and country), collection number, collector and any authority that may

have verified the identification of the specimen.

Typical plant labels are in the 4-5 inch (width) x 3-5 inch (length) range. The label is

traditionally placed in the lower right-hand corner of the herbarium sheet.

Why create a herbarium? Plant identification is increasingly becoming a lost art and science. Because there are

so few trained taxonomists, it is a good idea to have in hand those plants that you might

encounter with some frequency. Once you have a plant specimen correctly identified,

you can use this for comparison to any future plant collections. In addition plant

specimens become an important learning tool. Herbarium specimens allow you to show

someone distinctive morphological characters important for correct identification.

The following are four main reasons to make a herbarium collection.

Identification of plant specimens.

Basis for research and teaching.

Preservation of specimens.

Part III: Basic Principles For Plant Taxonomy

Taxonomy is the science of systematically naming and organizing organisms into

similar groups. Plant taxonomy is an old science that uses the gross morphology (flower

form, leaf shape, fruit form, etc.) of plants to separate them into similar groups. Quite

often the characteristics that distinguish the plants become a part of their name. For

example, Quercus alba is a white oak, named because the underside of the leaf is white.

The development of more sophisticated microscopes and laboratory chemical analyses

has made this new science possible.

Common Taxonomic Divisions

Plants belong to the Kingdom of Plantae. Other Kingdoms include Fungi, Protista

(one-celled organisms including yeasts, bacteria, and protozoans), and Animalia

(animals). The scientific system of classification divides all living things into groups

called taxa (singular, taxon).

The plant kingdom is divided into two taxa, broyophytes (including mosses and

liverworts) and vascular plants (plants with a vascular system of xylem and phloem).

Vascular plants (sometimes called higher plants) are divided into two subgroups:

seedless and seeded. The seeded plants divides into two taxa, Gymnospermae

(Gymnosperms) and Angiospermae (Angiosperms).

These taxa divide into Divisions (or Phylum). Division names end in ‘phyta’. Examples

of phyla include Ginkgophyta (Ginkgo), Pinophyta (conifers), and Magnoliophyta

(flowering plants). Gymnosperms (meaning naked seed) do not produce flowers, but

rather produce seeds on the end of modified bracts, such as pine cone. Many have scale

or needle-like leaves. Arborvitae, fir, ginkgo, pine, and spruce are examples of

Gymnosperms. Angiosperms (Magnoliophyta or broadleaf flowering plants) produce

seeds through flowering. Most have broad leaves. Angiosperms are divided into two

taxa, monocotyledon (monocots) and dicotyledon (dicots).

8

Families

Families of higher plants are separated from one another by characteristics inherent in

their reproductive structures (flowers, fruit, and seed). Many family members share

common characteristics in plant appearances, seed location and appearance and growth

habit. However, some families have a lot of diversity in appearance.

Family names end in ‘aceae’. Examples of common families include the following:

Caprifoliaceae – Honeysuckle family, including Snowberry and Viburnum

Fabaceae – Pea family including Japanese Pagoda, Locust and Siberian Peashrubs

Oleaceae – Olive family including Ash, Forsythia, Lilac and Privet

Rosaceae – Rose family including Apples, Peach, Plum

Genus and Species

The taxonomic divisions beyond the family level are the genus and specific names,

together called the species. Plants are named using a binomial system. The genus name

comes first and is analogous to a person’s last name (like Smith). The specific names

follows as a more specific identifier. It would be analogous to a person’s first name

(like John).

Genera (plural of genus) are groupings whose members have more characteristics in

common with each other than they do with other genera within the same family.

Similarity of flowers and fruits is the most widely used feature, although roots, stems,

buds, and leaves are also used. Common names of plants typically apply to genera. For

example Acer is the genus of maples, Fraxinus is the ash, and Juniperus is the junipers.

Specific name generally refers to interbreeding sub-groups of genus or groupings of

individual plants that adhere to essential identification characteristics but display

sufficient variation so as not to be categorized as replicas of one another. The specific

name is always used in conjunction with the genus. When genus and specific names are

written, they should always be italicized to denote they are Latin words. The genus

name is always capitalized, but the specific name is not. For example, Japanese Maple

would be written Acer palmatum Thunberg or Acer palmatum T. The Irish potato would

be written Solanum tuberosum Linnaeus or Solanum tuberosum L.

Common names

On the other hand, common names are often local in use and many times do not clearly

identify the specific plant.

THE MICROSCOPE

To study the microscope and its parts.

To study microscope types.

To learn how the optical microscope works and its applications.

To get an idea about the electron microscope and its uses.

Requirements:

Compound microscope, slides, cover slips.

9

Theoretical introduction:

A microscope (from the Greek: mikrós, "small" and, skopeîn, "to look" or "see") is an

instrument designed to make fine details visible. The science of investigating small

objects using such an instrument is called microscopy. Microscopic means invisible to

the eye unless aided by a microscope. It is an invaluable tool in today's research and

education. It is used in a wide range of scientific fields, where major discoveries in

biology, medicine and materials research are based on advances in microscopy.

The microscope must accomplish three tasks: produce a magnified image of the

specimen (magnification), separate the details in the image (resolution), and render the

details visible to the eye, camera or other imaging devices.

Optical microscopy:

The optical microscope, often referred to as the "light microscope", is a type of

microscope which uses visible light and a system of lenses to magnify images of small

samples. Optical microscopes are the oldest and simplest of the microscopes.

Since so many microscope users rely upon direct observation, it is important to

understand the relationship between the microscope and the eye. Our eyes are capable

of distinguishing color in the visible portion of the spectrum. The eye also is able to

sense differences in brightness or intensity ranging from black to white and all the gray

shades in between. Thus, for an image to be seen by the eye, the image must be

presented to the eye in colors of the visible spectrum and/or varying degrees of light

intensity. The eye receptors, used for sensing color and intensity of image, are located

on the retina at the back of the inside of the eye. The front of the eye (see Figure below),

including the iris, the curved cornea, and the lens are respectively the mechanisms for

admitting light and focusing it on the retina. For an image to be seen clearly, it must

spread on the retina at a sufficient visual angle.

Electron Microscopy

(Electron beams)

MICROSCOPY

Optical Microscopy

(electromagnetic waves)

10

Because of the limited ability of the eye's lens to change its shape, objects brought very

close to the eye cannot have their images brought to focus on the retina. The accepted

conventional viewing distance is 10 inches or 25 centimeters. The "simple

microscopes" could spread the image on the retina by magnification through increasing

the visual angle on the retina.

Simple microscope:

A simple microscope is a microscope that uses only one lens for magnification, and is

the original light microscope. Light microscopes are able to view specimens in color,

an important advantage when compared with electron microscopes, especially for

forensic analysis, where blood traces may be important.

Early microscopes were called simple because they only had one lens. These early

microscopes had limitations to the amount of magnification no matter how they were

constructed. The creation of the compound microscope by the Janssens helped to

advance the field of microbiology. The Janssens added a second lens to magnify the

image of the primary (or first) lens.

Compound Light Microscope:

11

The term light refers to the method by which light transmits the image to your eye.

Compound deals with the microscope having more than one lens. Simple light

microscopes of the past could magnify an object to 266X as in the case of

Leeuwenhoek's microscope. Modern compound light microscopes, under optimal

conditions, can magnify an object from 1000X to 2000X (times) the specimen's original

diameter.

Parts of a compound microscope

1) Eyepiece: It is the top part of the microscope; it is the lens you look through to see

your specimen.

2) Arm: It is the large metal band attaching the base to the lens and eyepiece. When

you carry a microscope, use one hand to hold the arm, and place the other hand under

the base.

3) Coarse Adjustment Knob: Of the two knobs on the side of a microscope, it is the

largest. It is used to focus on the specimen; it may move either the stage or the upper

part of the microscope (in a relative up and down motion). Always focus with the

coarse knob first.

4) Fine Adjustment Knob: It is the smaller round knob on the side of the microscope

used to fine-tune the focus of your specimen after using the coarse adjustment knob.

12

5) Objective Lenses: Most microscopes have two, three or more lenses, typically made

of glass to collect light from the sample, that magnify at different powers. Always start

with the lowest power and work your way up to the strongest when examining a

specimen. The shortest lens is usually the lowest power.

On a typical compound optical microscope, there are three objective lenses: a scanning

lens (4X), low power lens (10X) and high power lens (ranging from 20 to 100X). Higher

magnification lenses must be physically closer to the specimen itself, which poses the

risk of jamming the objective into the specimen. Be very cautious when focusing. Some

microscopes have a fourth objective lens, called an oil immersion lens. To use this lens,

a drop of immersion oil is placed on top of the cover slip, and the lens is very carefully

lowered until the front objective element is immersed in the oil film. Such immersion

lenses are designed so that the refractive indexes of the oil and of the cover slip are

closely matched so that the light is transmitted from the specimen to the outer face of

the objective lens with minimal refraction. An oil immersion lens usually has a

magnification of 50 to 100X.

The actual power or magnification of an optical microscope is the product of the powers

of the ocular (eyepiece), usually about 10X and the objective lens being used. The

magnified image seen by looking through a lens is known as a virtual image, whereas

an image viewed directly is known as a real image.

6) Stage: It is where the sample or specimen is placed for examination. The stage

usually has arms to hold slides (rectangular glass plates with typical dimensions of

25 mm by 75 mm, on which the specimen is mounted).

7) Iris Diaphragm: It is what allows you to control the amount of light on the specimen

that comes through the stage.

8) Light Source: It can be a bulb or a mirror, and is usually found near the base of the

microscope shining up through the stage.

9) Aperture: It is the hole in the stage that allows light through for better viewing of

the specimen.

Applications:

Optical microscopy is used extensively in microelectronics, nanophysics,

biotechnology, pharmaceutical research and microbiology.

It is used for medical diagnosis, the field being termed histopathology when dealing

with tissues, or in smear tests on free cells or tissue fragments.

Microscopy is also becoming an important tool for forensic scientists who are

constantly examining hairs, fibers, clothing, blood stains, bullets, and other items

associated with crimes.

13

The smallest objects that are considered to be living are the bacteria. The smallest

bacteria can be observed and cell shape recognized at a 100X magnification.

Optical microscopy is best suited to viewing stained or naturally pigmented

specimens such as stained prepared slides of tissue sections or living photosynthetic

organisms.

Stereo microscope:

The stereo or dissecting microscope is designed differently from the compound

microscope, and serves a different purpose. It uses two separate optical paths with two

objectives and two eyepieces to provide slightly different viewing angles to the left and

right eyes. In this way it produces a three-dimensional visualization of the sample being

examined.

The stereo microscope is often used to study the surfaces of solid specimens or to carry

out close work such as sorting, dissection, microsurgery, watch-making and small

circuit board manufacture or inspection.

The stereo microscope or dissecting microscope usually has a binocular eyepiece tube,

a long working distance, and a range of magnifications typically from 5X to 35 or 40X.

Digital microscope:

A digital microscope is a variation of a traditional optical microscope that uses optics

and a charge-coupled device (CCD) camera to output a digital image to a monitor. A

digital microscope differs from an optical microscope in that there is no provision to

observe the sample directly through an eyepiece.

A primary difference between an optical microscope and a digital microscope is the

magnification. With an optical microscope the magnification is found by multiplying

the lens magnification by the eyepiece magnification. Since the digital microscope does

not have an eyepiece, the magnification cannot be found using this method. Instead the

magnification for a digital microscope is found by how many times larger the sample

is reproduced on the monitor. Thus the magnification number of an optical microscope

is usually 60% larger than the magnification number of a digital microscope.

Since the digital microscope has the image projected directly on to the CCD camera, it

is possible to have higher quality recorded images than with an optical microscope.

With the optical microscope, the lenses are made for the optics of the eye. Attaching a

CCD camera to an optical microscope will result in a image that has compromises made

for the eyepiece.

14

Electron microscope

An electron microscope is a type of microscope that produces an electronically-

magnified image of a specimen for detailed observation. The electron microscope (EM)

uses a particle beam of electrons to illuminate the specimen and create a magnified

image of it. The microscope has a greater resolving power than a light-powered optical

microscope, because it uses electrons that have wavelengths about 100,000 times

shorter than visible light (photons), and can achieve magnifications of up to

1,000,000X, whereas light microscopes are limited to 2000X magnification.

The electron microscope uses electrostatic and electromagnetic "lenses" to control the

electron beam and focus it to form an image. These lenses are analogous to, but different

from the glass lenses of an optical microscope form a magnified image by focusing

light on or through the specimen.

Electron microscopes are used to observe a wide range of biological and inorganic

specimens including microorganisms, cells, large molecules, biopsy samples, metals,

and crystals.

Image of glandular hairs under electron microscope

Care of the microscope

Everything on a good quality microscope is unbelievably expensive, so be careful.

Hold a microscope firmly by the stand, only. Never grab it by the eyepiece holder,

for example.

Since bulbs are expensive, and have a limited life, turn the illuminator off when you

are done.

Always make sure the stage and lenses are clean before putting away the

microscope.

Never use a paper towel, your shirt, or any material other than good quality lens

tissue or a cotton swab (must be 100% natural cotton) to clean an optical surface.

Be gentle! You may use an appropriate lens cleaner or distilled water to help remove

dried material. Organic solvents may separate or damage the lens elements or

coatings.

Cover the instrument with a dust jacket when not in use.

15

Focus smoothly; don't try to speed through the focusing process or force anything.

For example if you encounter increased resistance when focusing then you've

probably reached a limit and you are going in the wrong direction.

Preparation of the Specimen:

Add the plant material in a clean slide.

Add few drops of chloral hydrate.

Heat using Bunsen flame for few minutes (never let the slide dry).

Put the cover slip on the slide in an angle 45° and lower slowly to drive out any air

bubbles.

Before examination be sure that the area not covered by the cover slip is clean and

dry.

Notes on using microscopes:

1. Place your microscope on a secure table, free from vibration, to begin. Try to have

the microscope at least one foot away from any edge to avoid an accidental fall.

2. Turn on the lamp and set the intensity for comfortable viewing.

3. Place a specimen slide on the stage.

4. You should now begin to learn an important skill that will significantly increase

your enjoyment of the instrument. You must learn to view through the eyepiece(s)

with both eyes open! Whether you have a monocular microscope (one eyepiece,) or

a binocular microscope (two eyepieces,) start from the beginning to use both eyes.

5. Start from the beginning by low power (4 X).

6. If you wish to move to a higher power objective, it should take very little movement

of the fine adjustment knob to bring the image into focus. Similarly, a particle in

the image which is centered in the field of view should remain in the center as

objectives are changed.

7. Initially, slowly focus back (turn the fine focus knob to raise the optical tube) while

looking through the eye piece. Once the specimen comes into focus, you can make

fine adjustments up or down with the fine focus knob without fear of damaging the

slide or the microscope.

8. If the specimen does not come into view (does not focus), raise the tube a little with

the coarse focus knob and attempt to focus again with the fine focus knob. Once the

object is in focus, switching objective lenses (to a higher power) should be possible

without any further coarse adjustments.

16

Procedure for Using an Oil Immersion Objective:

1. Focus the specimen with the 10X objective. Then switch to the 40X “dry” objective

and center a desired feature in the field of view.

2. Lower the stage and gently place a drop of immersion oil on top of the cover slip.

3. Rotate the oil immersion objective (usually the 100X) into the light path.

4. While looking at the microscope from the front or side (not through the observation

eyepieces), slowly raise the stage until the front of the oil immersion objective makes

contact with the oil drop. You will see a sudden flash of light.

5. Now, using the fine adjustment only, continue to raise the stage until the specimen

comes into focus.

17

Lab (2)

Plant Morphology & Inclusions

A plant has two organ systems:

1-Leaves

Consists of: - Blade (= lamina): Flat expanded area.

-Petiole (= stalk): Stalk that connects leaf blade to stem, and transports

materials

-Stipule: Is an outgrowth of the lower zone of a young leaf, part of the

leaf base

- The shoot system:

Is above ground and

includes the organs such as

leaves, buds, stems, flowers,

and fruits.

2- The root system:

Includes those parts of the

plant below ground, such as the

roots, tubers, and rhizomes.

18

Shapes

Apices Bases

Margins

19

2-Stem

Types of stem

A) Over ground: Normal types in majority of plants

B) Underground stem

1. Bulb, in which the shoot consists of very short vertical stem (bearing roots below) and fleshy storage leaves (e.g. Onions).

2. Corm, in which the shoot consists mostly of vertical storage stem(e.g Colcicum)

3. Rhizome, in which the stem is horizontal and underground (e.g. Zingiber officinale, Ginger).

4. Tuber, which consists of a thick, underground storage stem, usually not upright, (e.g., Solanum tuberosum, Potato)

20

3-Flowers

The typical flower consists of four sets of flower parts arranged on a short swollen

structure called receptacle. The four parts from outside are:

Sepals (collectively called calyx) are the outermost organs below the petals

Petals (collectively called corolla) are the showy part of most flowers. In some flowers,

the petals are green and are called sepaloid.

Stamens (collectively called androecium) are the male sexual organ of the flower. A

stamen consists of an anther which contains the pollen, supported by a thin filament.

Pistils, which are often called carpels, (collectively called gynoecium) are the female

sexual organ of the flower which are usually vase-like in appearance

.

21

A. Plant cell inclusions by microscopy

Background

The use of herbs as medicine is the oldest form of healthcare known

to humanity and has been used in all cultures throughout history. Early

humans recognized their dependence on nature for a healthy life and since

that time humanity has depended on the diversity of plant resources for

medicine to cure myriads of ailments. Primitive people learned by trial and

error to distinguish useful plants with beneficial effects from those that

were toxic or inactive, and also which combinations or processing methods

had to be used to gain consistent and optimal results.

Nowadays, microscopical examination is one of the most important

tools in herbal drug identification. Some structures are very important in

this process and sometimes called “Key elements”. The most important

elements that should be observed while studying the powder microscopy

of herbs are:

Trichomes

Fibers

Sclerides

Pollen grains

Calcium oxalate crystals

(1) Trichomes (Hairs)

Outgrowths of the epidermis called trichomes vary greatly in size

and complexity. Trichomes are present on the outer part of plants

(dermal tissue) of leaves and stems. They may reduce water loss

and/or reduce herbivory. Trichomes may also secrete a variety of

compounds.

Types of trichomes

a. Covering trichomes (non-glandular): they have a protective

functions. It may be unicellular or multicellular (branched and

unbranched).

22

b. Glandular trichomes: These secrete essential oils or oleo-resins.

According to their stalk, it may be uniseriate (unicellular or

multicellular head), biseriate, pluriseriate, and branched.

Epidermal trichomes; A, papillae of lower epidermis of Coca leaf. B-G,

unicellular hairs. H, group of unicellular hairs from Harnamelis leaf. I, T-

shaped hair of Artemisia absinthium.

23

Epidermal trichomes; A-H, uniseriate covering hairs. I, multicellular

branched hair. J, biseriate hair.

Glandular hairs. A and B, Atropa belladonna. C, Datura strarnonium. D,

Digitalis purpurea. E, multicellular labiate glandular hair. F, Hyoscyamus

24

niger. G and H, Primula vulgaris. I, Digitalis lutea. J, Cannabis sativa. K,

Arternisia maritirna.

(2) Fibers and Sclerides

The ground tissue of a plant body is composed of parenchyma,

collenchyma, and sclerenchyma. Parenchyma cells are the most common

type of plant cells and have functional nuclei and are capable of dividing,

commonly also store food and water. Collenchyma cells provide much of

the support in young stems and leaves. Sclerenchyma cells strengthen plant

tissues and may be non-living at maturity. There are two types of

sclerenchyma: fibers and sclereids.

Fibers are long, slender cells that are usually grouped together in

strands. Linen, for example, is woven from strands of sclerenchyma fibers

that occur in the phloem of flax.

Cinchona barks fibers:

Cascara barks, Senna leaves

fibers with crystal sheath

Sclereids are variable in shape but often branched. They may occur

singly or in groups; they are not elongated, but may have various forms,

including that of a star, isodiametric, elongated or branched forms. It

consist of cell wall deposit with lignin. The thickening may be uniform or

irregular and sometime stratification and pitting may occur. These are

25

present in the hard outer coats of seeds and fruits, bark and pericyclic

regions of woody stems. The gritty texture of a pear is caused by groups of

sclereids that occur throughout the soft flesh of the fruit. Both of these

tough, thick-walled cell types serve to strengthen the tissues in which they

occur.

Cinnamon bark Cascara bark

Horse-shoe shape

One wall much thinner

Irregular sclereids

Irregular, solid, many in groups

(3) Pollen grains

A microspore in flowering plants, which germinates to form the

male gametophyte, a structure made up of the pollen grain plus a pollen

tube. It is produced in anthers. It varies considerably in size, shape, external

characters. It can be useful diagnostically for drugs containing floral parts.

The outer surface can be smooth, pitted or spiny. Each pollen grain has 3

germinal pores and 3 germinal furrows. Pollen grains of clove are

triangular in shape.

26

(5) Calcium oxalate crystals:

These are considered as excretory products of plant metabolism.

These provide valuable information for identification of crude drugs in

entire and powdered forms. Prisms of calcium oxalate may occur singly or

in small groups.

Types Examples

Prisms Senna

Rosettes

(cluster)

Rhubarb

Bundles of

acicular

crystals

Squill

27

B. Different Types of Starch Powder

Background

Starch (Amylum) is a complex polysaccharide with very high molecular

weight and consists of large number of monosaccharide units linked

together through glycosidic linkage. Starch is a natural plant product which

is a mixture of amylose (25%) and amylopectin (75%).

Amylose:

- Linear molecule consists of 250 – 300 glucose units of α – D –

glucose units linked together through α – 1, 4 glycosidic linkage.

- More water soluble than amylopectin.

- Amylose + Iodine (I2) Blue color

Amylopectin:

- Branched molecule consists of more than 1000 units of α – D –

glucose units linked together through α – 1, 4 and α – 1, 6 glycosidic

linkage.

- Less water soluble than amylose.

- Amylopectin + Iodine (I2) Violet color

Sources of starch

Starches are obtained from maize (Zea mays L.) (Graminae), wheat

(Triticum aestivum L. (Graminae), rice (Oryza sativa L.) (Graminae), and

potato (Solanum tuberosum L.) (Solanaceae).

Properties of Starch

- White mass powder, odourless with starchy taste.

- Insoluble in water (form colloidal solution).

- Starch + Iodine (I2) Deep blue color.

- Starch + H2O gel (with heat).

- Corn and wheat starches have neutral pH, rice starch has slightly

alkaline pH, and potato starch has slightly acidic pH.

Measurement of starch are necessary for the quantitative

identification of closely allied substances. In most cases, the original drugs

are adulterants or substitutes. By comparing the sizes of starch grains in

different species, the adulterants and/or substitutes in crude drugs can be

distinguished using optical microscopy

28

Microscopical characteristics:

- Maize starch

Rings (striations) are usually absent

Spheroidal and polygonal

Polyhedral

Usually stellate hila can be found

X – Y hilum

- Wheat starch

Contain large granules

Lenticular

Smaller ones are globular

Hilum is centric

Faint striations

29

- Rice starch

Very small, polyhedral and polygonal

Aggregated from 2 – 150 component

Sharp angles

Rings and hila can not be detected (striations are absent)

Very rare we can detect the presence of centric hila.

- Potato starch

Central and eccentric hilum (dot shape)

Horse shoe-shaped hila are eccentrically situated, small and

unapparent

Clear striations (rings are clearly evident)

Mussel-shaped

30

Procedure:

- Observe the physical characters (colour, odour, texture and taste) of

maize, wheat, rice and potato starches.

- Observe the solubility of starch in water before and after heating.

- Determine the pH of starch: mix 0.2 g of starch with 5 ml water, dip

in a piece of pH paper and read the pH.

Fill the results for these physical and identification testes in the following

table:

Corn starch Wheat

starch

Rice starch Potato

starch

Colour

Texture

Odour

Taste

Solubility in

cold water

Solubility in

hot water

- Microscopical examination:

Prepare amount of starch in water (half spatula of starch on a

clean slide and add 2- 3 drops of water).

Smear the paste on the slide using cover slip to make a thin

smear.

Observe the starch granules and identify the structure of different

starch grains by microscope using 10X and 40X.

31

Draw the microscopically features of the following:

1. Corn starch

2. Wheat starch

3. Rice starch

4. Potato starch

5-Maize starch

32

Lab (3&4)

Microscopic Characters of Powdered Leaves, Flowers,

Seeds, Fruits and Barks Containing Volatile Oils

Background

Powder analysis includes the examination of cell form and arrangement of different

cells. This is done by destroying the morphology (external characters) of the plants so

that the microscopical cell characters can be evaluated easily. The powder analysis of

different drugs play a major role in the identification of drugs. Powder analysis is done

to ensure the identity and quality of herbal drugs.

Theoretical introduction:

Name Biological

Name Family

Parts

Used Active Constituents

Peppermint

Mentha

piperita

Labiatae leaves

V.O: l.5%, containing menthol,

menthone and menthyl acetate as

the major components.Flavonoids;

(menthoside&rutin).miscellaneous;

(rosmarinic acid, azulenes, choline,

carotenes )

Buchu

Barosma

betulina

Rutaceae

leaves

V.O: diosphenol, pulegone, 8-

mercapto-p-menthan-3-one,

(responsible for odour). Flavonoids

(rutin, diosmin, hesperidin,

quercitin) tannin and mucilage.

Clove

Eugenia

caryophyllata

Myrtaceae

Flowers

V.O, 60 to 90 % eugenol, which is

the source of its anesthetic and

antiseptic properties.

Chamomile

Anthemis

nobilis

Compositae Flowers

V.O (1-2%) containing alpha-

bisabolol, alpha-bisabolol oxides A

& B, and matricin (usually

converted to chamazulene).

Other constituents (bioflavonoids

apigenin, luteolin, and quercetin).

Fennel Foeniculum

vulgare Umbelliferae Fruits

V.O: terpenoid anethol, in addition

to fenchone and anisaldehyde.

Coriander Coriandrum

sativum

Umbelliferae Fruits

V.O: D-(+)-linalool (coriandrol),

including among others borneol, p-

cymene, camphor, geraniol. The

unusual smell is caused by the

trans-tridec-2-enale content.

33

Fatty oil (petroselic acid, oleic

acid, linolenic acid).coumarins:

(umbelliferone, scopoletine.

Cinnamon Cinnamomum

zeylanicum Lauraceae Bark

V.O: (0.5 – 1 %)cinnamicaldehyde,

ugenol, tannin and mucilage.

Cassia Cinnamomum

cassia

Lauraceae

Bark

V.O (1-2 %) cinnamic aldehyde,

cinnamyl acetate, phenylpropyl

acetate, tannin and mucilage.

Procedure

1. Observe physical characters of plants (color, odor, and taste).

2. Make sure that your slide and cover are clean.Prepare the microscope and adjust the

light. Put a certain amount of powdered plants of our interest on the slide.

3. Add few drops (1-2) of chloral hydrate solution to the powdered plant on slide.

4. Heat the slide gently over a low flame Bunsen burner.

5. Put the cover slip on the slide in an angle 45°.

6. Make sure that the solution is covering the place under the cover and no

heterogeneous element under it.

7. The prepared slide put over the stage of microscope.

8. Find the most important key elements of the interested plant starting from the

smallest objective lens (4 X).Use the course adjustment to a view.

9. Clear the view with fine adjustment.

10. Change the objective (10 X) and clear the view with the fine adjustment. [ Note:

don't use the course adjustment when you change from the smallest objective lens]

11. Search the elements by moving the stage right and lift.

12. When you find the element change the microscope to the objective (40 X) to draw

the element.

Identification marks of Leaves

1) Mentha

The fresh or dried leaves and stems are a dark purplish-green (Black Peppermint) or

paler green with purple patches (White Peppermint). The odour and taste are strongly

aromatic and characteristic.

34

2) BUCHU

A greenish-yellow powder with a characteristic aromatic odour and taste.

35

Identification marks of Flowers

1) Clove

A dark brown powder with a characteristic, spicy odour and an aromatic, pungent and

slightly astringent taste.

2) Chamomile

A light brown to buff powder with a greenish tinge; it has a very strong,

characteristic and aromatic odour and a slightly bitter and aromatic taste.

36

Draw the powdered characteristics of the following:

a) Mentha

b) Clove

c) Chamomile

37

Microscopic Examination of Seeds & Fruits

1) Fennel

A yellowish-brown to greenish-brown powder with a pleasant, aromatic odour and

taste somewhat reminiscent of Anise.

2) Coriander

A medium brown powder with a characteristic, aromatic odour and a spicy taste.

38

Microscopic Examination of Barks

1) Cinnamon A reddish-brown powder with a characteristic, pleasant and aromatic odour and taste.

2) Cassia

A reddish-brown powder with a characteristic, pleasant odour similar to that of

Cinnamon and a characteristic, slightly mucilaginous taste.

39

Draw the powdered characteristics of the following:

a) Fennel

b) Coriander

c) Cinnamon

d) Cassia

40

Lab. (5)

Physical characters and Chemical tests of Carbohydrates

(Monosaccharides and Disaccharides)

Background

Carbohydrates [CX(H2O)Y] are usually defined as polyhydroxy aldehydes and ketones

or substances that hydrolyze to yield polyhydroxy aldehydes and ketones.

Physical characters:

Condition: Sugars are white, crystalline in shape and with sharp melting points.

Taste: Sugars have a sweet taste.

Solubility: Sugars are soluble in cold water and hot alcohol.

Chemical tests: 1. Molisch’s test:

2. Fehling’s reduction test (All sugars but not sucrose):

3. Benedict's test:

4. Barfoed’s test (for monosccharides only):

5. Resorcinol test (for keto-hexoses):

6. Furfural test (Differentiate between Pentoses and Hexoses):

41

Tests name Image observation

1. Molisch’s test:

Any carbohydrate + Alcoholic α-

naphthol then add conc. H2SO4 on

the wall of the test tube: Violet

ring between the two layers.

2. Fehling’s reduction test (All

sugars but not sucrose):

Sugar solutions + Fehling’s A

(CuSO4) + Fehling’s B (NaOH,

NaK tartarate rochell salt), heat

on water bath: Red Precipitate.

3. Benedict's test:

1 ml of Sugar solutions + 5 ml of

Benedict's (alkaline solution

containing cupric citrate

complex) were added. The

mixture was heated; red

precipitate

4. Barfoed’s test (for

monosccharides only):

Sugar solution + Barfoed’s

reagent (Cu Acetate/Acetic acid),

heat for 3 minutes on boiling

water bath: Red ppt with

monosaccharides only.

5. Resorcinol test (for keto-

hexoses):

Sugar solution + few crystals of

Resorcinol + Equall volume of

conc. HCl and warm on water

bath: Rose Red Colour.

6. Furfural test (Differentiate

between Pentoses and

Hexoses):

Pentose + Coc. Acid and heat,

expose the vapours to Aniline

acetate paper: Red colour.

Date :

42

Lab (6)

Unorganized Drugs

(Physical Characters & Chemical Tests)

Gums and Resins

Backgrounds:

They are crude drugs of plant or animal origin and having no cellular structure. They

are either mixture of chemical substances or they are decomposition products.

They are classified into several groups:

1. Gums and mucilage's (Gum Arabic & Gum Tragacanth).

2. Resins and resin combinations (Myrrh, Guar Gum, Benzoin, Balsam).

3. Dried Latices (Opium) 4. Dried Juices (Aloe) 5. Aqueous Extracts. (Agar)

1. Gums and mucilage's

A-Gum Acacia (Gum Arabic)

Source:

The dried gummy exudates from the trunk and branches of Acacia Senegal or of some

other African species of Acacia F. Leguminosae

Physical Characters:

Color( pale yellow or white),Odor( Odorless ),Taste( Mucilaginous ),Solubility

(Soluble in Water and Insoluble in ethanol & in ether).

Chemical constituents:

Arabin (a complex mixture of calcium, magnesium and potassium salts of Arabic acid,

Arabic acid (branched polysaccharide) & enzymes (oxidases, peroxidases and

pectinases).

Chemical tests:

1- Oxidase Enzyme: In a Porcelain dish add few drops of water to coarse powdered

gum and Triturate. Add one drop of H2O2, few drops of Bezidin or Tr.Guaiacum: a deep

blue to greenish blue color is produced.

2- Solution in water + Borax solution: a stiff mass is formed.

3- Solution in water + Lead Sub acetate: a white ppt is formed. 4- Solution in water + Lead Acetate: negative reaction.

5- Fehling’s A & B: a slight reduction is observed.

43

Uses: 1- Emulsifying and suspending agent. 2- Demulcent and emollient. 3- Adhesive

and binder in tablet manufacture

B-Gum Tragacanth (Gum Dragon)

Source:

The dried gummy exudates obtained by incision from Astragalus gummifer and other

Asiatic species of Astragalus F. Leguminosae

Physical Characters:

It is a viscous, odorless, tasteless, mixture of polysaccharides. Solubility: slightly

soluble in water, swelling at first into a homogeneous adhesive mass. It is insoluble in

ethanol.

Chemical constituents:

It’s a mixture of polysaccharides named as; Bassorin (a complex of polyhydroxylated

acids) and Tragacanthin (demethoxylated bassorin)

Chemical tests:

1. Deep yellow precipitate is formed, on boiling the solution of tragacanth with few

drops of 10 % aqueous ferric chloride solution.

2. Stringy precipitate is formed, on dissolving the tragacanth and precipitated copper

oxide in concentrated ammonium hydroxide (conc. NH3OH).

3. Canary yellow colour is developed, on warming tragacanth with sodium hydroxide

(NaOH) solution.

4. It gives green colour with strong iodine solution

Uses:

As gum arabic, but due to its resistance to acid hydrolysis it is preferred for use in highly

acidic conditions

Results and comment:

44

2-Resins and resin combinations

Resins, in general, are amorphous solid or semisolid substances that are invariably

water insoluble but mostly soluble in alcohol or other organic solvents. However,

physically they are found to be hard, translucent or transparent and fusible i.e., upon

heating they first get softened and ultimately melt. But chemically, they are complex

mixtures of allied substances

I) Guar Gum

Source:

Guar gum, also called guaran, is a galactomannan. It is primarily the ground endosperm

of guar beans. The guar seeds are dehusked, milled and screened to obtain the guar

gum. It is typically produced as a free-flowing, off-white powder of endosperm of the

seeds of Cyamopsis tetragonolobus Linn and other species of Cyamopsis, Family:

Leguminosae

Physical Characters:

Colour (Almost colourless or pale yellowish white powder). Odour and Taste:

Characteristic. Shape and Size (When examined in lactophenol mount under

microscope, it shows irregular particles of angular shape and size). Solubility (In water

it swells rapidly forming a translucent suspension. When the gum is stirred with 50

parts of water, a thick jelly is formed which with further addition of 150 parts of water,

yields a thick transparent suspension. It is insoluble in alcohol.

Chemical constituents:

Chemically, guar gum is a polysaccharide composed of the sugars galactose and

mannose. The backbone is a linear chain of β 1,4-linked mannose residues to which

galactose residues are 1,6-linked at every second mannose, forming short side-

branches

Uses: most frequently used gums in gluten-free recipes and gluten-free products. In canned

soup, it is used as a thickener and stabilizer. thickening agent, emulsifying agent, binding

agent, plasticizer

Chemical tests:

1. A 0.5% w/v solution of gum is neutral to litmus.

45

2. To 0.5% w/v solution of gum add 20% w/v of lead acetate, a flocculant precipitate

is produced (distinction from acacia and sterculia gum).

II) Balsams

Aromatic resinous substances. If the resin contain balsamic acid, cinnamic &/ or

benzoic acid it is called balsam. Ex. Benzoin, balsam of Tolu & Balsam Peru.

a. Balsam Tolu & Balsam Peru

Both types are quite similar to each other in their names the following table sumarizese

the major difference between them regarding their

Balsam Tolu Balsam Peru

Synonyms Tolu balsam, Resin Tolu. Peruvian Balsam, China Balsam,

Indian Balsam.

Botanical

Source

It is obtained by incision of stem of

Myroxylon balsamum, Family

Leguminosae.

It is obtained by incision of stem

of Myroxylon pereirae Family

Leguminosae.

Physical Characters

Texture Semi-solid or plastic solid Soft, viscous liquid or semi-solid

Colour Yellowish-brown or brown Yellow, on keeping it becomes

dark brown or nearly black

Solubility Insoluble in water and petroleum

ether; soluble in alcohol.

Insoluble in water and petroleum

ether; soluble in alcohol.

Odour and

Taste

Aromatic odour and taste is vanilla

like and slightly pungent.

Aromatic odour and taste is

vanilla like and slightly pungent.

Chemical

constituents

Toluresinotannol cinnamate, benzyl

benzoate, cinnamic acid, benzoic

acid, vanillin

Cinnemein, benzyl benzoate,

cinnamic acid, peruviol

Uses Expectorant, stimulant and

antiseptic.

miticide, to aid in healing of

indolent wounds, in skin

ulcer therapy, scapis.

Chemical tests for Balsam Tolu and Balsam Peru

1. Alcoholic solution of Balsam Tolu (1g) gives green colour with ferric

chloride.

2. Alcoholic solution of Balsam Tolu is acidic to litmus paper.

46

3. To filtered solution of Balsam Tolu (1g) in water (5 ml) aqueous potassium

permanganate solution is added and heated for 5-10 minutes. Odour of

bezaldehyde is produced due to oxidation of cinnamic acid.

b. Benzoin (Gawi)

Benzoin resin is a balsamic resin obtained from the bark of several species of trees in

the genus Styrax. There are two types of Benzoin; Siam benzoin and Sumatra benzoin

Apart from the distinction already made between Siam benzoin and Sumatra benzoin,

there are two English terms used to describe the resinous product from styrax trees:

benzoin (or gum benzoin, although use of the word gum is strictly incorrect since

benzoin is not a water-soluble polysaccharide) and gum benjamin.

Sumatra Siam

Physical Characters

Color White masses embedded in

Reddish matrix

Color White masses embedded

in Reddish matrix

Odor Balsamic odor Vanilla like

Taste Slightly acrid balsamic

Solubility Solubility Totally soluble in

Alcohol Totally soluble in

Alcohol

Solubility Totally soluble in

Alcohol Totally soluble in

Alcohol

Chemical Constituents

Balsamic acids Benzoic & Cinnamic

(20%)

Balsamic acids Benzoic (22%)

Esters 75 % 60- 70 % Mainly Coniferyl

Benzoate

Resin Alcohols & Esters Alcohols & Phenols

(Siaresinotannol) & Esters

Triterpene acids Triterpene

acids

Triterpene acids Triterpene

acids

Test for identity

Warm 0.5 g powder with

KMNO4

Odor of benzaldehyde Balsamic acids Benzoic (22%)

Extract with Ether, Filter,

to 1 ml of Filtrate add 2-

3 drops of H2SO4

Deep Reddish-brown Deep Purplish-red color with

Alcoholic solution +

Alcoholic sloution of

FeCl3

Green colour due to

Siaresinitannol

Negative

Sublimation Test : Crystals of Benzoic

(Plates) & Cinnamic acid

(Rods).

Crystals of Benzoic (Plates) &

Cinnamic acid (Rods).

47

III) Myrrh

Myrrh from the Arabic مر (mur), is the aromatic resin of a number of small, thorny tree

species of the genus Commiphora, which is an essential oil termed an oleoresin. Myrrh

resin is a natural gum. It has been used throughout history as a perfume, incense and

medicine

Source: It is an oleo gum-resin obtained from the stem of Commiphora molmol,

Family: Burseraceae. When a tree wound penetrates through the bark and into the

sapwood, the tree bleeds a resin. When people harvest myrrh, they wound the trees

repeatedly to bleed them of the gum. Myrrh gum is waxy, and coagulates quickly. After

the harvest, the gum becomes hard and glossy. The gum is yellowish, and may be either

clear or opaque. It darkens deeply as it ages, and white streaks emerge.

Physical characters

Occurrence: It occurs in solid, irregular or rounded tears. Colour and

fluorescence: Translucent, almost transparent. It is reddish-brown with a rough, dull

and dusty surface. Hardness and fracture: Hard, brittle break with granular fractures.

Fracture: Breaks with granular fracture. Odour and taste: Agreeable aromatic odour,

bitter, acrid, but not unpleasant taste.

Chemical constituents

Volatile oil, resin, gum, bitter principle. It contains 40% of resins, α-, β-,γ- commiphoric

acids, resenes etc.

Chemical Tests:

1. It is triturated with water to give a yellowish emulsion.

2. The ethereal extract of Myrrh becomes red when treated with bromine vapours and

change purple when treated with nitric acid solution.

Uses: carminative, antiseptic, uterine stimulant, in mouth washes and perfumes

Result and comments:

48

Lab (8)

Unorganized Drugs

(Physical Characters & Chemical Tests)

Latice, Juices & Aqueous Extracts

3. Dried Latices

Opium

Source:

The air dried latex obtained by incision of the unripe capsule of Papaver somniferum

F. Papaveraceae.

Chemical Constituents:

Opium contains a large number of alkaloids, the most important of them: morphine,

codeine, papaverine and contains 3-5% meconic acidwhich exists free or in

combination with alkaloids.

Chemical tests:

1. Extract with alcohol containing a few drops of dil. HCl, evaporate to dryness.

2. Residue, boil with water + MgO, filter while hot, concentrate the filtrate which

contains Mg meconate.

3. Acidify with HCl + few drops of FeCl3 → brown red to purplish red colour.

Uses: opium is CNS depressant. It acts as analgesic and hypnotic.

Result and comments:

49

4. Dried Juices (Aloe)

Aloe

Source: Origin: the solid residue obtained by evaporating the liquid juice, which drains

from the leaves, cut transversely near their bases, of Aloe ferox and its hybrids known

in commerce as Cape Aloes, or of A. perryi known in commerce as Socotrine or

Zinzibar Aloes or of A. vera known in commerce as Curacao Aloes F. Liliaceae.

Physical Characters

Texture: Solid, waxy mass. Colour: Dark brown.Hardness and fracture: Hard,

uneven porous fracture. Solubility: partially soluble in water, chloroform and ether,

completely soluble in ethanol, alkali and glacial acetic acid. Effect of heat: At high

temperature produce black mass. Odour and Taste: Characteristic unpleasant odour,

very bitter and nauseous taste.

Chemical constituents Aloe

Number of anthraquinone glycosides, the major one is barbaloin. and number of free

anthraquinones

Uses: purgative and improve digestion, cosmetics

Chemical tests:

Boil 0.5 g with 50 ml water until nearly dissolved, cool and add 0.5 g kieselguhr and

filter, apply the following tests to the filtrate.

1. Modified Borntragers test:

5 ml filtrate + 10 ml FeCl3 + 5 ml dil HCl, heat for about 10 min, filter, cool, extract

with organic solvent (Benzene). Separate the organic layer. Shake with dil

Ammonia: Rose Red color in the ammonia layer.

2. Cupraloin test for Isobarbaloin:

To 10 ml of 0.1% solution of aloes in distilled water, add 1 drop of 5% solution of

copper acetate, 0.5 ml of saturated solution of sodium chloride, 1 ml of alcohol and

warm: Pale wine red colour.

50

Results and comments

51

5. Aqueous Extracts.

AGAR

Source: Origin: it consists of polysaccharides obtained by extracting various species of

Rhodophyceae, mainly those belonging to the genus Gelidium, with boiling water,

filtering whilst hot and evaporating to dryness Family: Gelidiaceae

Physical characters: Solid long thick strips up to 60 cm long, thickness 0.5 to 1 cm

and width 2.5 cm.

Colour and Fluorescence: Greyish white, translucent with yellowish tint.Hardness

and fracture: Tough, difficult to break.Solubility: insoluble in cold water, slowly

swells and is soluble in boiling.Odour and taste: Characteristic slight odour of marine

algae, mucilaginous taste.

Chemical constituents

Constituents: it is the calcium salt of strongly ionized, acidic polysaccharides. It can

be resolved into two major fractions, agarose and agaropectin

Chemical tests

1. Iodine test: Powdered agar + few drops of dil. I2 crimson red colour

2. BaCl2 test: Powdered agar + dil. HCl prepared in distilled water and heat on boiling

water bath for 30 min., cool, add BaCl2 white precipitate of BaSO4. (gum

tragacanth and gelatin).

3. Powder + rhuthenium red → the particles are stained deep red.

Uses: Suspending agent, emulsifier, gelating agent

Result and comments:

52

Lab. (9)

Chromatography

Chromatography (from Greek chroma = color and graphein = to write)

Chromatography is a physical method of separation in which the components to be

separated are distributed between two phases

One of which is stationary (stationary phase) while the other (the mobile phase) moves

through it in a definite direction.

The chromatographic process occurs due to differences in the distribution constant of

the individual sample components.

Techniques by chromatographic bed shape are planar chromatography & Column

chromatography

I-Plane Chromatography

Planar chromatography is a separation technique in which the stationary phase is

present as or on a plane. The plane can be a paper, serving as such or impregnated by a

substance as the stationary bed (paper chromatography) or a layer of solid particles

spread on a support such as a glass plate (thin layer chromatography). Different

compounds in the sample mixture travel different distances according to how strongly

they interact with the stationary phase as compared to the mobile phase. The specific

Retention factor (Rf) of each chemical can be used to aid in the identification of an

unknown substance.

Plane chromatography could be classified into thin layer chromatography (TLC) and

paper chromatography (PC).

S.N

Thin-Layer Chromatography

Paper Chromatography

1 A technique to separate mixture of chemicals

based on differential partitioning between the

mobile and stationary phases

Chromatography is used to separate

mixtures of substances into their

components. All forms of

chromatography work on the same

Stationary phase

2 ►Solid such as silica gel or its derivatives,

alumina, cellulose, sephadex----etc + Binder +

Fluorescent material

► Filter paper

Mobile phase

53

3 ►It is a suitable liquid organic solvent or mixture

of solvents

Solvents like: petroleum ether, Cyclohexane,

Ether, Acetone, Ethyl acetate, Chloroform,

Methanol, Ethanol, Water or buffer solutions

►It is a suitable liquid organic

solvent or mixture of solvents or

buffer solutions

4

Procedure

5 Step: 1 (Chamber)

It contain suitable mobile phase, Pour the suitable solvent in TLC chamber & Closed the

chamber opening and leave for some time enough for the atmosphere to be saturated

with solvent vapors.

TLC Plate

Mark the place of sample application (1cm apart) on the line by pencil, Apply small

amount of a sample solution on the specified position using capillary tube & Dry the

place of spot to get rid of any solvents (All sample must be dissolved in volatile solvents

or solvents)

6 Step-2 (Development )

Put TLC plate of paper inside the TLC chamber. It is important that the solvent level is

below the start line & Cover the chamber to keep atmosphere saturated with solvent

vapors.

►When the solvent front gets close to the top of the plate, the plate is removed from the

jar and the position of the solvent is marked with another line.

►Allow the plate to dry using air gun, oven ►Allow the plate to dry using air

gun,

Note: The beaker is often lined with some filter paper soaked in solvent to help saturating

the atmosphere in the beaker with vapors.

Glass Cover Pencil line

(Start line)

Jar

Solvent

TLC

Plate/

Paper

Sample

spot

mark

54

7 Step 3-Measuring Rf values

►These measurements are the distance travelled by the solvent, and the distance

travelled by individual spots.

The distance travelled relative to the solvent is called the Rf value. For each compound

it can be worked out using the formula:

For example, if a component travelled 1.7 cm from the base line while the solvent had

travelled 5.0 cm, then the Rf value for the component is:

►These measurements are the distance traveled by the solvent, and the distance traveled

by individual spots.

Step 4. Visualization

1. UV light

Both TLC & PC can have coulers under UV according to the nature of the comopounds

on them.

2. Spray reagent

Many spraying reagent are used to give different coulers. some of them needs heat

reaction in oven (TLC only) and some needs gentle worm (Both TLC & PC)

The Superiority Of TlC Over Paper Chromatography

S.N TLC chromatography PAPER chromatography

1 Sulfuric acid spray No use

2 Less time require (3h) More time require (18h)

3 Choice of adsorbent allow Not Possibile

Start line

Solvent front

55

TLC & Paper chromatography of amino acids

Drugs: Standard (Amino acid) Alanine, arginine, asparagine, aspartic acid, cysteine,

glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,

phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine,

Chemicals: spraying reagent (0.2% w/v Ninhydrin in acetone), n-butanol and glacial

acetic

Procedure:

1. Take filter paper(Whitman No 1) cut in size 5×10 cm, draw a line with pencil

1.5 cm from the lower end of the paper.

2. Using pencil mark position for sample application 1cm from the edge and 1 cm

from each other.

3. Prepare solvent system; n-butanol- acetic acid- water (4: 1: 5 v/v/v) (organic

phase)) is poured in chamber to a depth of 1 cm and allow to saturate (for PC).

4. Prepare solvent system; ethyl acetate: acetic acid: methanol: water (60:15:15:10

v/v/v/v) For TLC

5. The chamber is lined with filter paper to maintain equilibrium of mobile phase.

6. Prepare 2 ml standard solution of two amino acids (1mg/ml) in a test tube and

mixture of two amino acids (1ml each) in other test tube.

7. The solutions are loaded on the marks with the help of capillary tube and keep

in room temperature for 5 minutes.

8. Put the Paper in the chamber and wait until solvent move close to the paper end.

9. Remove the paper mark the solvent front and keep for some time at room

temperature and then dry using air-gun.

10. Spray the plate with Ninhydrin reagent and dry by air-gun.

11. Identify the location of amino acids which takes radish violet colour.

12. Calculate the Rf value for both PC & TLC. Compare both results (TLC & PC)

Result & comments:

56

Perform Radial development using paper chromatography

Requirement: Filter paper, Petri dishes, Gentian violet, Sudan III, Picric acid, Toluene,

Methyl chloride, acetic acid

Procedure:

1. Prepare mobile phase composed of Toluene: Methyl chloride: Acetic acid,

(10:10:4) in Petri dish

2. Cut a wedge in a filter paper using scissor

3. Apply sample to the centre of the filter paper using capillary tube.

4. Put the filter paper in the Petri dish containing the mobile phase with the wedge

immersed in the mobile phase

5. Allow solvents to reach near the edge of filter paper

6. Remove filter paper from the Petri dish, dry, record your observation, prepare a

report along with your chromatogram for grading

Result and comments:

57

Lab. (10)

Chromatography

II- Columnar Chromatography (CC)

Column chromatography in chemistry is a method used to purify individual chemical

compounds from mixtures of compounds. It is often used for preparative applications

on scales from micrograms up to kilograms. The main advantage of column

chromatography is the relatively low cost and disposability of the stationary phase used

in the process.

Open Column Chromatography

(Traditional column chromatography

Traditional column chromatography is characterized by addition of mobile phase under

atmospheric pressure and the stationary phase is packed in a glass column.

Packing & operating the column

1- Packing: The selection of the method of packing depends mainly on the density of

the solid. Techniques used are the wet and dry methods. In all cases avoid inclusion of

air bubbles.

a. Wet Packing: First prepare slurry of adsorbent with the solvent, shake well

to derive out air bubbles and then pour inside the column. The solvent allow to down 1

cm above the top

Definition Term

Mobile liquid phase with no affinity to the stationary phase (i.e. inert towards it)

& no effect on solutes. Solvent

Any liquid with more affinity to the stationary phase than the solvent but less

than solutes and just capable to move them through the column. Developer

Any liquid that passes out of the column. Effluent

Any liquid that has lesser affinity to the stationary phase than solutes but is

capable to move them out of the column. Eluent

Fraction of eluent containing a required specific substance. Eluate

(Or retardation volume): Volume of mobile phase that passes out of the column,

before elution of a specific substance.

Retention

) R

(Vvolume

58

b. Dry Packing: Pack column with adsorbent. Pass solvent until equilibrium

reaches.

The most common are Alumina & Silica gel in which the interactions with

solute molecules is due to OH groups present on their surface.

2- Sample Application: Apply evenly & in a concentrated solution to the top of the

column which is protected from disturbance (e.g. add glass wool or filter paper

3-Elution techniques

We can use any of the following solvent as eluent; Petroleum ether, cyclohexane, Ether,

acetone, Ethyl acetate, Chloroform, Methanol, Ethanol, Water.

Procedure Technique

Addition of solvent mixture of fixed composition during the whole

process.

Isocratic

elution

Continuous or linear elution: in which there is continuous change in

the composition of the mobile phase over a period of time (e.g.

polarity, pH or ionic strength).

Gradient

elution

Step wise or fractional elution: in which the change is not continuous

i.e. a sudden change in the composition of the mobile phase is

followed by a period where the mobile phase is held constant.

Selection of the mobile phase

The liquid stationary & mobile phases should have a considerable difference between

their solvent strength parameters.

Pure water > Methanol > Ethanol > Propanol > Acetone > Ethyl acetate> Ether >

Chloroform > Dichloromethane >Benzene > Toluene > Carbon tetrachloride >

Cyclohexane > Hexane > Pentane.

4- Detection: On-column detection for colored or fluorescent compounds directly after

developing the chromatogram. Monitoring of eluted fractions (PC or TLC). Using

special detectors connected to the column such as refractive index, UV detectors, etc…

Preparation of column

The bottom portion packed with cotton wool or glass wool Pack column with adsorbent

Procedure:

59

1- Make slurry of activated silica gel (60-120 mesh) with the mobile phase (CHCl3).

2- Insert a little bunch of cotton at the bottom of column.

3-Pour the slurry into the column uniformly

4-Open the tap at the bottom of column and add more mobile phase into the column

and run it for 20 minutes, and closed when 1cm above

5-Dissolve the substance (gentian violet, suddan III )(about 1g) in small part of

mobile phase.

6- Add the solution of substance over the top of column and open tap till the mixture

adsorbed on the silica gel.

7- Add more mobile phase open the tap at the bottom of column and collect the

fractions of volume around 10 - 20 ml in test tubes.

8-Increase polarity by using 2%, 5%, 10% of MeOH.

9- Collect fraction containing the violet zone and other containing the red zone.

10- The fractions with single spot can be merged and evaporated, pure substance is

isolated which is then subjected to different spectra like IR, NMR, Mass etc. for

confirmation of structure of substance. Remaining fractions can be rejected.

Result:

60

Lab. (11)

Different Extraction Techniques

Preparation of Some Extracts

I-Classical Techniques

Principle

Extracts can be defined as preparations of crude drugs which contain all the constituents

which are soluble in the solvent used in making the extract Plant constituents are usually

contained inside the cells. Therefore, the solvent used for extraction must diffuse into

the cell to dissolve the desired compounds whereupon the solution must pass the cell

wall in the opposite direction and mix with the surrounding

Extraction includes Liquid-liquid extraction, and Solid phase extraction. A plant extract

must be obtained from a solid-liquid extraction

Solid-liquid extraction is defined as an operation to separate elements contained

in a solid body by solubilization with a solvent, and it may be followed by purification.

The extract is contained in the solvent. If the solvent is an edible solvent, it is not

necessary to dissociate it from the extract. If the solvent is not an edible solvent,

separation allows obtaining a dry extract.

Methods of Extraction

I-Classical Techniques

Maceration, Percolation, Infusion, Decoction, Digestion,. Distillation

II-Modern Techniques

Microwave-assisted Extraction, Supercritical Fluid Extraction, Counter-Current

Extraction, Ultrasound Extraction (Sonication), Continuos hot extraction technique

(Soxhlet extraction).

61

I-Classical Techniques

1) Volatile oil preparation and Analysis

Background

“Volatile oils “is a term to designate the odoriferous principal obtained mainly from

plant and rarely “volatile” and ”ethereal” are added to indicate that they easily evaporate

on exposure to air at ordinary temperature (volatile, from the latin “volare”=to fly).

They are also called “essential oils “ after the latin “essential” meaning a liquid easily

changed to gas and most probably because they represent the efficient fraction of the

drug in which they occur. “Volatile oils” are generally mixtures of hydrocarbons and

oxygenated compounds derived from them .they differ entirely in both chemical and

physical properties from “fixed oils”.

Therefore, may be defined as: “complex liquid mixtures of odoriferous

principals of varying chemical composition, with easily evaporate when exposed to air

at ordinary temperature and which used for either there specific therapeutic activity or

there aroma “.

Volatile, ethereal oils or essential oils, as their name implies, are volatile at room

temperature and in steam. They differ entirely in both chemical and physical properties

from fixed oils. They are secreted in oil cells, in secretion ducts or cavities or in

glandular hairs. They are frequently associated with other substances such as gums and

resins and they tend to resinify on exposure to air

Chemical composition of volatile oils:

• Volatile oils are generally mixtures of hydrocarbons and oxygenated compounds

derived from these hydrocarbons.

• It is not uncommon for a volatile oil to contain over 200 components, and often

the trace constituents are essential to the odor and flavor. The absence of even

one component may change the aroma.

Methods of extraction or preparation

1. Distillation

2. Enfleurage

3. Expression

4. Maceration

5. Solvent extraction

62

1. Extraction by Distillation

Distillation takes place when the sum of the vapour pressures is equal to the

atmospheric pressure when two immiscible liquids are heated together the boiling-

point of such a "mixture" would be lower than that of the constituent with the lower

boiling-point-in other words, below the boiling-point of water (100°C) when we make

distillation of volatile oils. .

e.g.Clove oil

Principle

The boiling point of most oil constituents range between 150ºC-300 ºC, distillation at

such high temperatures may cause either decomposition or polymerization .The

presence of water during distillation allows the process to be carried at temperature

below 100 ºC. This is explained by Dalton’s law of partial pressure which states that:”

When two immiscible liquids are heated together, they will boil at a temperature below

the boiling point of either one”.

Description of the apparatus

Clavenger apparatus for volatile Clavenger apparatus for

Oil lighter than water volatile oil heavier than water

A distillation apparatus is basically formed of 3 parts:

1- The body of the still (distillation flask): made of copper lined with lead-free tin or

better stainless steel (the use of Fe is avoided as any Fe+++ may catalyze hydrolytic &

oxidative decomposition of constituents).

2-The condensing system (condenser): designed to provide proper cooling to avoid

reflux of the distillate.

3-the receiver (collecting flask): specifically designed to allow separation of the oily

layer from water in the distillate.

Procedure

63

1- Put 10 grams of powdered in 500 mL round bottom flask.

2- Cover the powder with 150 mL of water,

3- Boil the mixture using heating mantle, Bunsen burner or a hot plate. For 2 hours.

Powdered cloves may lead to foaming if too much heat is applied

3- Separate the oil and add to it few grams from anhydrous sodium sulphate to remove

any water residue .Measure the volume using measuring cylinder.

4-Calculate the oil content in ml per 100g of plant material.

5- Run TLC using system (Benzene-ethyl acetate 9:1 v/v) &(50/50 mixture of

hexane/CH2Cl2).

6-Spray (with vanillin sulphuric acid). Calculate the Rf value & Compare to the Rf value

with eugenol. Locate the spots with a UV lamp.

2- Extraction by Expression

e.g. Orange oil

Principle

Mechanical procedures carried at room temperature and based on puncturing and

/or squeezing of the plant material to liberate the oil which is collected.

Application

Used for preparation of heat sensitive oils which are present in large amount in outer

peels of fruits e.g. Citrus fruits (Rutaceae) such as orange, lemon and bergamot.

Preparation and purification of the oil

It should be noted that the peel of Citrus fruits if formed of: An outer coloured zone

rich in waxes and pigments and containing the oil gland, and an inner white zone formed

of pectin and cellulose.

The process involves:

(a) squeezing of the peel under a stream of water yielding an emulsion formed of

essential oil + water +pectin + cellulose + pigment + traces of waxes

64

(b) Removal of water +pectin +cellulose by centrifugation.

(c) Removal of waxes by strong cooling (chilling ) followed by filtration or

decantation

1-Spong Method

Is based on squeezing the removal peels to collect the oil and is used in Sicily for

preparation of orange oil as follows:

(a)Fruits are washed, cut into halved and fleshy parts removal.

(b) Peels are soaked in water, turned inside out and pressed between a convex

projection and a sponge.

© The saturated sponge is periodically squeezed in a vessel and the emulsion

obtained centrifugated and cooled.

N.B

The tissue of the sponge serves for both:

(a)Collection of the oil, and

(b) Filtration of the product from any particles of the inner white zone of the peel.

Gas Chromatography

Theory

GC has a column where different volatile chemicals pass through at different rates

depending on their interaction with the column filing. As the chemicals exit the end of

the column, they are detected and identified electronically. By comparing the GC chart

of the compound extracted in laboratory to a commercial grade product, the purity can

be determined. If a single compound is supposed to be in the product, it should be

represented by a single peak in the chart.

Application of GC and TLC in volatile oil analysis

1. Qualitative analysis

2. Quantitative analysis

3. Identification of mixture of volatile oils

4.

65

Result & Comments:

66

Lab. (12)

Different Extraction Techniques

Preparation of Some Extracts

II-Modern Techniques

Continuos hot extraction technique (Soxhlet extraction)

Extraction of Hisperidin

Hesperidin is a flavanone glycoside found

in citrus fruits. Its aglycone form is called hesperetin. Hesperidin is also referred to by

names like bioflavonoid, citrus bioflavonoid, and hesperin methyl-chalcone.

Flavonoids like hesperidin used to be called “vitamin P.”. It is so abundant in citrus

fruits that it is an inexpensive by-product of citrus production.

1) Collection of Plant Material: The fruits of Citrus sinensis (Orange) were peeled

off and peels were dried under shade.

2) Extraction of Hesperidin: Hesperidin can be extracted by two methods.

(Conventional Method & Modern Method)

a.Conventional Method :The dried orange peels (50gm) were macerated with

60ml of aq. Alkaline solution (10% KOH) pH 8-9 for overnight, After complete

maceration the mixture was filtered by large Buchner funnel and filtrate was evaporated

on water bath to make it syrupy mass. Cool, acidify to PH 4-5 using con HCl and re-

Concentrate on water bath and separate the solid formed. Recrystallize from water.

b.Modern Method: 500 mL ethyl alcohol (40 – 60°C) is filled in a 250 mL

round bottom flask with magnetic stir bar. 50g dried and powdered orange peel are

placed in the extraction sleeve of a Soxhlet extractor and covered with a little glass

wool. A reflux condenser is put on the Soxhlet extraction unit, and then the reaction

mixture is stirred and heated for 1 hours under strong reflux. The alcoholic extract is

evaporated using water bath or rotatory evaporator.

67

Using TLC examine the extract for the presence of flavonoids, using solvent

system Chloroform-methanol (95:5 v\v) and Ethyl acetate-methanol- water (30-5-4

v\v\v\).

Results and Comments:

68

Lab. (13)

Application of extraction method and chromatography

Orange peel extract from previous lab. Will be subjected to column chromatography

for its isolation and identification.

Write down the expected techniques for the isolation and identification of this

compounds

I. Column Chromatography

Baking material (type and quantity)

Elution system

Volume of fraction

69

Lab. (14)

Application of extraction method and chromatography

Following up methods

1-UV

2-Sray reagents

Results and comments

70

Right down what have you got from this Lab Course?