Pharmacognosy: The Science of Natural Drugs

Pharmacognosy
Dehnnet A.(Bpharm, MSc in Pharmacognosy)
E-mail: DAGMZT24@gmail.com
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Egyptians (Ebers papyrus, 1550 BC)
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Introduction
Pharmakon Drug
Pharmacognosy
to acquire
Gignosco/gnosis
knowledge of
❖is an applied science which deals with the biogenic, biochemical, and
economic features of natural drugs and their constituents.
❖Pharmacognosy is defined as:
➢Scientific & systematic study of crude drugs along with its
History, method of cultivation, collection, preparation, & storage
of crude drugs.
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Introduction ….
Origin and History of pharmacognosy
❖Despite the variation in philosophical premise
❖The universal role of plants in the treatment of disease was

recognized by different systems of medicine like
❖Egyptians medicine, Unani medicines, Allopathic

medicines.
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Introduction ….
❖ By trial and error a primitive man:
❖ Identify natural products with food, medicinal value, &

poisonous/ unpleasant nature.
❖ This observations were handed from generation to

generation.
❖ The healing power of plant roots, fruits, and juices were discovered
by accident and learned.
❖ Individuals like Hippocrates (460-370 B.C), Aristotle and others

contribution to medicine.
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Introduction ….
❖ The term "pharmacognosy" was coined by the Johann Adam

Schmidt “Lehrbuch der Materia Medica.”
❖ Pharmacognosy is the oldest of all pharmacy sciences.
❖ During the 19th C & the beginning of the 20th C
❖Pharmacognosy refers branch of medicine which deals with

drugs in their crude or unprepared form.
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Introduction ….
1. Ancient Egyptian period
The most complete medical documents includes
• Ebers Papyrus (1550 B.C.): 800 prescriptions, and 700 drugs
• Edwin Smith Papyrus (1600 B.C.): contains surgical instructions and
formulas for cosmetics.
• The Kahun Medical Papyrus (1900 B.C.): deals with women’s health,
including birthing instructions.
• Some of the commonly used herbs included: senna, honey, thyme, aloe,
coriander, elderberry, fennel, garlic, onion, peppermint, papyrus, saffron,
watermelon, and wheat Myrrh, turpentine and acacia gum were also used.
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2. Ancient India
• Knowledge of medicinal plants is very old.
• Medicinal (Ayurvedic) writings are:(Charaka Samhita, Susruta

Samhita, and Astanga Hrdayam Samhita).
• Most of medicines origin from plants & animals
• The collection of plant materials was done only by an

innocent, pure, religious person.
• The fresh plants were considered to be the most effective.
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3. Ancient Greece and Rome
• Hippocrates (460-370 BC)
– Considered as ‘father of medicine’
– Wrote Hippocratic Corpus (oldest writings on medicine).
• Recommended a healthy diet & physical exercise
• Describes on the use of plants, repositioning of joints,

relationship of weather and illness.
– Hippocratic oath.
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4. Ancient Greece and Rome….
Dioscorides (70 A.D.) wrote De Materia Medica
– 600 plants described: Belladona, ergot, hyoscyamus, etc.
– considered as authoritative source of pharmacological info.
• 80% medicinal plants and 20% animals and minerals
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4. Ancient Greece and Rome…
Galen (131–A.D. 200)
• Devised methods of preparations of plant and animal drugs,

galenical preparations.
• He assumed that the only certain constituent not the herbs

themselves are beneficial to human body
– Prepare extracts using menstruum (water)
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5. The ancient Babylonians
• Health care relied on magic and incantations.
• The drugs used were mainly of vegetable origin.
• They were known for Laws of Hamorabi (772 B.C.)

– “Eye for an eye and tooth for tooth”
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6. The old Chinese medicine
• Stems from Shen Nung (about 2700 B.C.) emperor
– investigated the medicinal value of several hundred herbs.
– tested many of them on himself, and
– write the first Pen T-Sao, or Native Herbal, recording 365
drugs.
• Acupuncture was the major TMP in Chinese
• Some of the medicines include; Ginseng, Rhubarb, Ephedra,
Star Anise, Pomegranate, Aconite....
➢ Opium is a very old Chinese drug for diarrhea and
dysentery.
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Scope of pharmacognosy
History
Preservation
Distribution
& Use
Pharmacognosy
Evaluation Cultivation
Collection Identification
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Scope of pharmacognosy…..
• It also deals with natural products without any
pharmacological value: natural fibers, suspending agents,
colorants, disintegrates, stabilizers.
• It also deals with
• Poisonous plants
• Hallucinogenic plants
• Allergens, insecticides
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R/ship with other fields
Pharmacology
Quality
control Botany
Biochemistry Pharmacognosy Phytochemistry
Biology Physiology
Pharmaceutics
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Function of pharmacognosist
• Identification of the source of a drug
• Determination of its morphological character
• Investigation of the potency, purity and quality of the drug
• Planning on proper methods of cultivation of the medicinal plants

yielding these drugs
• Prescriptions of the details of process of collection and preparation.
• Detailed knowledge of the constituents and chemical nature of drugs.
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Crude Drugs
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Introduction
Definition
Drug???
Drug means any substances:
✓Recognized in the pharmacopeia
✓Used for diagnosis, mitigation, treatment, or prevention

of disease in human or animal.
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Introduction…
• Crude drugs are plants or animals, or their parts subjected only to
drying or transverse or longitudinal cutting after collection.
❖ Crude drugs can thus be defined as:
➢Natural products that has not been advanced in value or

improved in condition
➢ By any process or treatment
➢Other than essential for Proper packing & prevention

from deterioration.
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Introduction…
Natural products:
❖ They can be
✓ Entire organism (plant, animal, micro-organism)
✓ Part of an organism (a leaf or flower of a plant, an

isolated gland or other organ of an animal)
✓ An extract or an exudate of an organism
✓ Isolated pure compounds
❖ Can be obtained from plants, animals, microorganism, and

minerals. 22
Introduction…
Role of natural products in modern medicine:

➢ Provide a number of extremely useful drugs that are
difficult to produce commercially by synthetic means
➢ Supply basic compounds that may be modified slightly to

render them more effective or less toxic
➢ Serve as prototypes or models for synthetic drugs

possessing physiologic activities similar to the originals.
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Nomenclature of Crude Drugs
• A plant needs a name that applies to it and only to it. It can have
1. Common name
• Local language names (vernacular names).
• May vary from one country to an other, one state to an other.
Thus, it is unreliable
• May change as people move to an area or as old common
names fall out of favor due to a reason.
• The names are not capitalized unless the word is a proper
noun. Eg. hydrilla, bulrush, Florida.
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Nomenclature of Crude Drugs…
Cyphostemma adenocaule
▪ Tigrigna: Acerkuka (Et.) (Araya, et al., 2015)
» : Hareg Temen (Er.) (Yemane, et al., 2017)
▪ Amharic: Aserkush (Birhanu, et al., 2015).
▪ Uganda: kibombo
▪ Shinasha : Emen (Giday et al., 2007)
▪ Tanzania: Lwengere
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2. The Binomial system
• tells a plant’s closest relatives
Genus • Start with capital initial in italics
• Start with small initial,

Species • However, the species start with capital
initial letter when named after a person.
Eg: Cinchona Ledgeriana ( named after Charles
Ledger)
• Comes after binomial name & tells who first
descovered the plant
Authority • Not written in italics
• Cyphostemma adenocaule (Steud. ex A.
Rich)
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The specific name may indicates
1. Some striking characteristics of the plant:
❖Conium maculatum (maculate = spotted)
➢stem with reddish, spotted patches.
❖Glycyrrhiza glabra (glabrous = smooth).
➢Refers to its smooth fruit
➢Hyoscyamus muticus (muticus = short).
➢The plant being short.
❖Atropa belladonna (bella = beautiful, donna = lady)
➢the juice of the berry placed in the eyes causes
dilatation of the pupils, thus giving a striking
appearance).
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The specific name may indicates……
2- A characteristic colour:
▪ Piper nigrum (= black)
▪ Veratrum viride (= green)
▪ Citrus aurantium (= golden yellow)
▪ Digitalis purpurea (= purple)
▪ Digitalis lutea (= yellow)
3- An aromatic plant or certain aroma:
✓Myritaceae fragrans (having a fragrant, nice aroma)
✓Caryophyllus aromaticus (refers to the aroma)

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The specific name may indicates……
4- A geographical source or history of a drug:
➢ Cannabis indica (growing in India)
➢ Tamarinds indica (India)
5- A Pharmacological activity of an active constituents:
❖Papaver somniferum (sleep inducing)
❖Strychnos nux vomica (causing vomiting)
❖Ipomoea purga (laxative).
6- A general meaning or a special indication
✓ Allium sativum (= cultivated)
✓ Triticum vulgaire (= wild)
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Classification of Crude Drugs
❖ A method of classification should be
• Simple
• Easy to use
• Free from confusion and ambiguities.
❖ Because of their wide distribution, each arrangement of

classification has its own merits and demerits.
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Classification of Crude Drugs….
❖ Drugs are classified in the following different ways:

• Alphabetical classification
• Morphological classification
• Taxonomic classification
• Pharmacological/therapeutic classification
• Chemical/biogenic classification
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A. Alphabetical Classification
➢ Classification of any disconnected items.
➢ Latin and English names are used.

❖ Used by reference books, dictionaries, and
pharmacopoeias like:
• Indian Pharmacopoeia • USP and NF
• British Pharmaceutical Codex.
• British Pharmacopoeia
• European Pharmacopoeia(EP)
• British Herbal Pharmacopoeia
• In EP drugs are arranged according to their Latin names where
in U.S.P. & B.P. they are arranged in English names
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Merits:
• It is easy and quick to use
• There is no repetition of entries and confusion.
• Tracing, addition, and deletion of drug entries is easy.
Demerits: no relationship b/n successive drug entries.
Examples: Acacia, Benzoin, Cinchona, Dill, Ergot, Fennel,

Gentian, Hyoscyamus, ……
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B. Morphological Classification
❖ Arrangement based on the morphological or external characters of

the whole or part of plant/animal .
➢ e. g. leaves, roots, stem, glands etc.
❖ Organized drugs: obtained from the direct parts of the plants &
contain cellular tissues.
➢ e. g. Rhizomes, barks, leaves, fruits, entire plants, hairs and fibers.
❖ Unorganized drugs: prepared by incision, drying or extraction with

a solvent and don’t have any cellular tissues.
➢Aloe juice, opium latex, agar, gelatin, benzoin, honey,

beeswax, lemon grass oil etc.
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Organized Drugs
 Woods– Quassia, Sandalwood, Red Sandalwood.
 Leaves– Digitalis, Eucalyptus, Senna, Spearmint, Squill, Tulsi, Vasaka, Coca, Buchu,
Hamamelis, Hyoscyamus, Belladonna, Tea.
 Barks–Ashoka, Cascara, Cassia, Cinchona, Cinnamon, Kurchi, Quillia, Wild cherry.
 Flowering parts– Clove, Pyrethrum, Saffron, Santonica, Chamomile.
 Fruits– Amla, Anise, Bael, Bahera, Bitter Orange peel, Capsicum, Caraway, Cardamom,
Colocynth, Coriander, Cumin, Dill, Fennel, Lemon peel, Tamarind, Vidang.
 Seeds– Bitter almond, Black Mustard, Cardamom, Colchicum, Ispaghula, Kaladana, Linseed,
Nutmeg, Nux vomica, Physostigma, Psyllium, Strophanthus, White mustard.
 Roots and Rhizomes– Aconite, Ashwagandha, Calamus, Calumba, Colchicum corm, Galanga,
Garlic, Gention, Ginger, Ginseng, Ipecac, Ipomoea, Rauwolfia, Rhubarb, Turmeric, Valerian,
 Plants and Herbs– Ergot, Ephedra, Andrographis, Kalmegh, Yeast, Vinca, Datura, Centella.
 Hair and Fibres– Cotton, Hemp, Jute, Silk, Flax.

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Unorganised Drugs.
– Dried latex– Opium, Papain
– Dried Juice– Aloe, Kino
– Dried extracts– Agar, Alginate, Pale catechu, Pectin
– Waxes - Beeswax, Spermaceti, Carnauba wax
– Gums – Acacia, Guar Gum, Indian Gum, Sterculia, Tragacenth.
– Resins– Asafoetida, Benzoin, Mastic, Coal tar, Tar,
– Volatile oil– Turpentine, Anise, Coriander, Peppermint, Rosemary, Cinnamon,
Lemon, Caraway, Clove, Eucalyptus, Nutmeg,
– Fixed oils and Fats– Arachis, Castor, Coconut, Cotton seed, Linseed, Olive,
Sesame, Almond, Cod-liver, Halibut liver
– Animal Products – Bees wax, Cantharides, Cod-liver oil, Gelatin, Halibut
liver oil, Honey, Shark liver oil, shellac, Spermaceti wax, wool fat, musk,.
– Fossil organism and Minerals– Bentonite, Kaolin, Talc.
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➢ Merits:
➢ Helpful to identify and detect adulteration.
➢ Convenient for practical study of drug with unknown
chemical nature
➢ Demerits:
➢ No co-relation of chemical constituents with the therapeutic
actions.
➢ Repetition of drugs or plants occurs.
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C. Taxonomical Classification
• Botanical classification based on
– natural relationship and evolutionary developments.
• Arranged according to the plants from which they are obtained

Kingdom Phylum Classes Order Family Genera Species.
• It allows for a precise and ordered arrangement and

accommodates any drug without ambiguity.
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Merits
➢Helpful for studying evolutionary developments.
Drawback
➢Does not co-relate chemical constituents with biological
activity of the drugs.
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D. Pharmacological Classification
❖ Grouping of drug according to their pharmacological

action/therapeutic use of most important constituent.
❖More relevant and is the mostly followed method.
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Merits
✓Used for suggesting substitutes of drugs
Demerits
✓Ambiguity and confusion : as drugs having different
action on the body gets classified separately in more
than one group .
✓Cinchona--- antimalarial due to quinine
------antiarrythymic due to quinidine.
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E. Chemical/Biogenetic Classification
❖ The crude drugs are grouped in based on their
➢ Constituents or biosynthetic pathways
Carbohydrates– Carbohydrates are polyhydroxy aldehydes or ketones

containing an unbroken chain of carbon atoms.
✓ Gums Acacia, Tragacanth, Guargum
✓ Mucilages Plantago seed
✓ Others Starch, Honey, Agar, Pectin, Cotton
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Glycosides –upon hydrolysis give rise to one or more sugars (glycone) &
non-sugar (aglycone).
✓ Anthraquinone Glycosides Aloe, Cascara, Rhubarb, Senna
✓ Saponins Glycosides Quillaia, Arjuna, Glycyrrhiza
✓ Isothiocyanate Glycosides Mustard
✓ Cardiac Glycosides Digitalis, Strophantus
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Tannins–non-nitrogenous polyhydroxy benzoic acid derivatives. Examples-
Pale catechu, Black catechu, Ashoka bark, Galls, Amla
Volatile oils– plant derived monoterpene and sesquiterpenes. Examples-

Cinnamon, Fennel, Coriander, Cardamom, Orange peel, Mint, Clove,
Lipids - Fixed oils – Castor, Olive, Almond, Shark liver oil
- Fats – Theobroma, Lanolin
- Waxes – Beeswax, Spermaceti
• Resins– Complex mixture of compounds like resinols, resin acids,

resinotannols, resenes. Examples: Cannabis, Jalap, Capsicum, Turmeric,
Balsam of Tolu and Peru, Myrrh, Ginger
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Alkaloids – Nitrogenous substance of plant origin
– Pyridine and Piperidine – Lobelia, Nicotiana
– Tropane - Coca, Belladonna, Datura, Stramonium, Hyoscyamus, Henbane
– Quinoline – Cinchona
– Isoquinoline – Opium, Ipecac, Calumba
– Indole – Ergot, Rauwolfia
– Amines – Ephedra
– Purina – Tea, coffee
Protein – Gelatin, Ficin, Papain
Vitamins - Yeast
Triterpenes – Rasna, Colocynth

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Merits
➢ It is a popular approach for phytochemical studies
Demerits
➢ Ambiguities arise when particular drugs possess a number

of compounds belonging to different groups of compounds.
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The Evaluation of crude drugs
• To evaluate a drug means to identify it and determine its quality and purity.
• Methods of Evaluation of crude drugs include:
✓ Organoleptic
✓ Microscopic
✓ Biologic
✓ Chemical
✓ Physical
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Evaluation of crude drugs…
A. Organoleptic Evaluation
– Is evaluation by means of sense organs based on:
✓Macroscopic appearance of the drug
✓Sensory character of the drug: gross morphology, colour,

odour, taste, size, shape, and special features, such as:
touch, texture.
▪ The simplest, yet the most human form of analysis.
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B. Microscopic Evaluation
– Evaluation and identification of powdered drugs,
– Detection of adulterants, and
– Identification of plants by characteristic tissue features
• Determine microscopic characters like stomata number,

stomata index, palisade ratio, vein-islet number, vein
termination number, etc.
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Evaluation of crude drugs….
C. Biologic Evaluation:-
• evaluation of pharmacological activity, potency, and toxicity.
• More time consuming and expensive.
• It can be
– in vitro: done in lab; test tubes/ dish
– ex-vivo: done in isolated organs of organism
– in vivo: in the body of experimental animal
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D. Chemical evaluation:- determinations of their active principles.
➢ Qualitative chemical tests: presence of the intended class of Cpd.
➢ Mayer’s test: detection of alkaloids
➢ Benedict's test: for carbohydrates….
➢ Quantitative chemical tests :
➢ Acid value: number of carboxylic acid groups in a chemical cpd,
➢ Saponification value: number of total free and combined acids.
➢ Instrumental analyses: to analyse the chemical groups of cpd.
➢ chromatographic and spectroscopic methods

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E. Physical evaluation
– Application of physical constants in crude drug evaluation.
• Specific gravity. drugs that sink or not sink in water.
• Elasticity
• Uv interaction: Aconite--light blue, Berberine --yellow,

Emetine---orange
– Physical constants : solubility, specific gravity, optical rotation,

refractive index, melting point and water content, viscosity .
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Preparation from plants
• Herbal Tea
• Tinctures
• Herbal Capsules
• Herbal Decoction
• Herbal Infusion
• Herbal Preparation with Syrup
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Adulteration of crude drugs
Adulteration: partial or full substitution of the original drug with
other substance
➢Free from or inferior in therapeutic and chemical properties.

Types of Adulteration
✓ Adulteration with inferior commercial varieties

✓ Adulteration by artificially manufactured substitutes.
Eg: Artificial invert Sugar for Honey
✓ Adulteration by Exhausted drugs

✓ Adulteration by addition of Heavy Metals.
✓ Adulteration by Synthetic Principles. 55
Official and unofficial drugs
Official Drugs
❖ That are approved by F.D.A for uses
❖ listed and described in a book recognized by the
government as the legal authority for standards
"Pharmacopoeia"
E.g: Advil for headache for an example.
Unofficial Drugs
❖ Used "off label" or not as directed by the F.D.A.
❖ drugs are substances that have never been appeared in
Pharmacopoeia
E.g: Using Benadryl for a sleep aid is an example.
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Phytochemical Studies
on Medicinal Plants
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Purpose of the study
❖ After this lecture you will be able to know.

• What is phytochemical investigation?
• Steps in phytochemical investigation
• Extraction methods
• Bioactivity/bioassay-guided fractionation
• Different isolation techniques:-
» Crystallization/precipitation
» Fractional distillation
» Derivatization
» Chromatography
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Case study
• Imagine while you are on vacation in your village you have

found one medicinal plant which is used for the treatment of
rabies virus, snake bite, and various tumours by a traditional
herbal medicine practitioners. So you wandered by the healing
power of this plant as pharmacists (discoverer of drugs) you want
to carryout phytochemical study on this plant so that this plant
could be a potential source of new pharmaceutical drug. How can
you start your investigation?
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Introduction
• Natural products are sources
– For more than 25% of modern medicine
– For lead compounds for another 25% modern medicine
• Only 15% of higher plants have been investigated for

biological activity.
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Phytochemical screening
❖ Phyto = plant
❖Phytochemical:- different chemical constituents of a plant
❖ Phytochemical screening:- study related to isolation,

identification, and characterization of all or individual chemical
constituents of a plant in a pure form
❖It follows the biological investigation of each constituent in

pure form to find their biological /therapeutic activities
❖It is an interdisciplinary work of botany, pharmacognosist,

pharmacologist, chemist, and toxicologist.
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Steps of phytochemical screening
1. Selection
2. Collection
3. Botanical identification
4. Literature review on identified plant
5. Preparation of the plant for extraction
6. Extraction and preliminary phytochemical screening
7. Biological/pharmacological test of the crude extract
8. Bioactivity-guided fraction and isolation of active constituents
9. Structural elucidation of each active constituent isolated.

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Steps of phytochemical screening…
1. Selection of Plant Material
Depends on
• Its medicinal use in traditional medicine

• Trial and error/blind screening
• Chemotaxonomic data on related families,
genus or species.
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2. Collection Should consider the:-
» Time/season
» Distribution Affect the qualitative &
quantitative accumulation
» Part of plant used of the active constituents
» Age of the plant.
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• Collection should be done at optimal state of development of the
plant/its part.
1. Roots and rhizomes are collected at the end of the vegetation
period, i.e. usually in the autumn. In most cases they must be
washed free of adhering soil and sand.
2. Bark is collected in the spring.
3. Leaves and herbs are collected at the flowering stage.
4. Flowers are usually gathered when fully developed.
5. Fruits and seeds are collected when fully ripe.
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3. Botanical identification of the plant
• Identification of the plant can be done by:-
Consulting a taxonomist or a pharmacognosist
Comparing the characteristic features of the sample plant

with the reference authentic plant/published monograph
• The whole plant at flowering stages provide to the taxonomist or

pharmacognosist.
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The voucher letter should contain:
• Site of collection: Angacha river side, North Gondar, Ethiopia
• Local name: Aserkush (Amharic)

• Scientific name: Cyphostemma adenocaule (Steud. ex A. Rich)
Descoings ex. wild & Drummond
• Habitat: a climber found on the river side of Teda woreda.
• Collector(s) Name: Dehnnet Abebe
• Date of collection: 07/11/2018
• Specimen number: DA01/2018
• Identified by: Getnet Chekole (Botanist, UoG)
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4. Literature review on the identified plant
• Searching any work done on your study plant in published
literatures such as scientific journals, books, internet, etc.
• Literature review helps:-
Identification of proper plant to work on
Proper preparation, extraction method, and appropriate

solvent to be used can be obtained.
Identification of different constituents of the plant
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5. Preparation of the study plant for extraction
Involves:-
Removal of foreign extraneous matters,
Drying,
Size reduction/powdering
Drying
– Artificial drying E.g. Oven drying
– Natural drying
✓Shade drying
✓Light drying
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• Drying:-
Prevents enzymatic hydrolysis of active ingredients (<10% moisture

content, enzymes are inactive)
Prevent attack by molds and bacteria
Facilitate size reduction or powdering, etc.
• Grinding: improve extraction by
– Making the sample more homogenous,
– Increasing the surface area of contact b/n solvent & powder,
– Decrease the amount of solvent required for extraction by allowing the

powder to be packed densely, and
– Facilitating the penetration of solvent into the cells. 70

6. Designing appropriate extraction methods
• Involve -
Selection of appropriate solvent
Selection of appropriate extraction methods
• If the plant traditional medicine, use the same extraction

solvent and extraction method.
• Choice of extraction method depends on Nature of
– The plant/part used: dry/fresh, root, leaf, stem, bark, ...
– Active ingredients: thermolabile/stable, polar/nonpolar…
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• Choice of extraction solvent follows the principle of “like
dissolves like”
– Non polar/lipophilic are used for extraction of non polar

constituents.
– Polar/ Hydrophilic solvents are used for extraction of polar

compounds.
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Extraction: separation of components soluble in the solvent
– Transfer of mass of soluble material from a solid to a fluid.
– Both the active and inactive impurities can be extracted.
• Extraction involves:
– Diffusion of the solvent into cells
– Dissolution of the metabolites in the solvent
– Diffusion of the solution out of the cells
• Continued till equilibrium is set up between the solution in

the cells and external env’t
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• Crude extract: - plant constituents left after removal of the
dissolving media by evaporation.
• Residue or mark: - the undissolved constituents of the plant
left in the filter paper.
• Filtrate: - Solvent + dissolved plant constituent
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Methods of extraction
Cold extraction methods Hot extraction methods
Maceration Soxhlet extraction
Percolation Infusion
Expression/cold press Digestion
Infusion Decoction
SCF extraction
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7. Checking extracts/ fractions for pharmacological activity
➢ In-vivo, in-vitro, and ex-vivo evaluation methods
➢ Can be evaluated for:
➢Anti-bacterial
➢Anti-fungal
➢Anti-inflammatory and analgesic
➢Immunomodulatory activity etc…
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8. Bioassay guided fractionation
Testing of non polar to polar fraction for biological activity followed by

the isolation of individual pure components from biologically active
fraction Crude extract
Bioassay
Active crude extract

Fractionation
Fractions
Bioassay
Active fraction
Repeated bioassay
Repeated fractionation
Repeated bioassay
More purified active fraction

Repeated bioassay
A single bioactive compound

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Ideal bioassay should be
Inexpensive
Sensitive to small amount of active material
Selective for specific type of bioactivity
Simple to run and maintain
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9. Chemical analysis of the crude extract/fractions/

isolates/Phytochemical screening
– Identification of classes of compounds presents
(alkaloids, glycosides, tannins, steroids, etc.).
– Conducted by chemical tests (using specific reagent)
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10. Isolation and purification of active ingredients

Classical methods Modern methods
Filtration
various chromatographic
Centrifugation methods
Crystallization
Solvent extraction
Distillation.
Fractional distillation
Sublimation
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General methods in studying constituents of
crude drugs
Extraction
• Extraction can also defined as removal of soluble components
from solid, liquid or semisolid material
• Extraction can be
– Initial extraction
• Performed on small amount of crude drug
• To get preliminary knowledge on the nature of exact
& class of metabolites present.
– Bulk extraction
• Is large scale extraction following initial extraction
Extraction…
• Extraction can be
A. Liquid—liquid
➢ based on relative solubility of constituents in two liquids
that do not mix
➢Extraction from one liquid into another/ partitioning
➢ Performed using Separatory funnel: in case of fractionation
B. Solid—liquid
➢ Separation of soluble components from a solid into a
solvent
Extraction…
• Methods of solvent extraction
– Continuous extraction (percolation and Soxhlet extraction),
• Solvent continuously flows through the plant material
• Saturated solvent replaced with fresh solvent
– Discontinuous extraction (Maceration)
• the solvent is added and removed in batches.
• Extraction stops when equilibrium is reached b/n the
concentration of solute inside the plant material & the solvent
• Needs decanting and replacement with new solvent.
Choice of suitable solvents
• An ideal solvent used for extraction should:
– Have good selective solubility for the target compound
– Have high capacity of extraction (high coefficient of saturation)
– Poor solubility of impurities
– Have a low price.
– Be non reactive with the target/ other compounds in the crude drug
– Be completely volatile
– Be non toxic and non flammable

Choice of suitable solvents…
The speed with which this equilibrium is established depends on:
➢ Temperature: enhance diffusivity of solvent by decreasing its viscosity
➢ Particle size: the lower the particle size the higher the surface are f
contact between the solvent and the crude drug
➢ The movement of the solvent

Choice of suitable solvents
❖ Alcohol used with water during extraction
✓ Induce swelling of the plant particles
✓ Increase the porosity of the cell walls.
– Enhance diffusion of the solvent into the cell, and
– facilitates the diffusion of extracted substances from inside the
cells to the surrounding solvent.
❖ For extraction of barks, roots, woody parts and seeds the ideal
alcohol/water ratio is about 7:3 or 8:2.
❖ For leaves or aerial green parts the ratio 1:1 is usually preferred in order to
avoid extraction of chlorophyll.
Cold extraction methods Hot extraction methods
Maceration Soxhlet extraction
Percolation Infusion
Expression/cold press Digestion
Infusion Decoction
SCF extraction
88
Methods of extraction…
1. Maceration
• Soaking of the crude drugs in a bulk of the solvent for 3-7 days
with frequent shaking.
• The process is intended to soften and break the plant cell walls
to release phytochemicals
– Pressing & Filtration of the solution is needed after 3 days
followed by re-maceration of the marc.
– For thermolabile phytoconstituents
89
• Repeated maceration is better than single maceration as
appreciable amount of active constituent left in the marc.
– When the marc can’t be pressed, triple maceration is

employed.
2. Percolation: involves
i. Size reduction to fine/coarse powder
ii. Imbibition: Powder moistened in menstrum and stand in well
closed container for 4 hours
iii. Packaging: moistened powder packed in the percolator
(cylindrical/conical container with a tap at the bottom).
iv. Maceration: additional solvent is poured on top of the plant
material and allowed to stand for 24 hrs.
v. Percolation: the lower tap opened and liquid allowed to drip
– No need for filtration as there is a filter at the outlet of the
percolator
91
• The percolation process:
– is dependent on the flow of solvent

through the powered drug, and
– yields products of greater

concentration than the maceration
Diagrammatic representation of percolation
92
Methods of extraction….
3. Expression/cold press
– Fragrance extraction where raw materials are pressed, squeezed,
or compressed and the oils are collected.
– The oil is forced from the material under high mechanical

pressure
– Suitable for oils that decompose upon distillation
• Lemon oil, orange oil
– May also extracted by puncturing the oil glands by

rolling fruit on sharp material
93
4. Supercritical fluid(SF) extraction.
– Using supercritical fluid as extraction solvent.
• Critical point: the highest temperature and pressure at which the
substance can exist in vapor–liquid equilibrium.
• Supercritical fluid/ dense gas has
– Is homogenous liquid
– Liquid like density
High
– Reduced surface tension
➢ Penetration power, and
– Low/gas like viscosity
➢ Extraction efficiency
– High compressibility like gas
– Higher diffusivity than liquid
• Co2 -is the widely used fluid for SF extraction above 31.1 0c and
73.8 bar.
– Non toxic, cheap, nonflammable, sterile and bacteriostatic
• Supercritical CO2 is non polar
– Modifiers increase the polarity of CO2 for extraction of polar cpds
» Methanol(1-10%)- commonly used
• Can be added by:
– Adding directly to the sample in the extraction cell or by

mixing with the sample prior to loading in the extraction cell.
– Using CO2 gas cylinders containing premixed modifier.
– Adding in a continuous fashion with an external modifier

pump
• Advantages of using SFs for extractions
– inexpensive,
– contaminant-free,
– selectively controllable,
– Less costly to dispose safely than organic solvents, and
– Less likelihood of Oxidative and thermal degradation of active

compounds than conventional organic solvents
5. Infusion: powder is wetted with cold water in a pot .
✓ Immediately afterwards, boiling water is poured over it,
then covered wit lid and left to stand for a defined
period after which the tea is poured off.
✓ Unlike decoction, infusion doesn’t involve boiling
of the powder.
✓ Used for soft crude drugs for the penetration of

water and polar constituents
97
6. Decoction
– The crude drug is boiled in a specified volume of water for
a defined period
• The boiled mixture is cooled, filtered, concentrated, and
evaporated to dryness
– Suitable for extracting heat stable non volatile constituents
and hard plant parts (root and barks)
98
7. Soxhlet extraction
The powdered plant material is loaded in the thimble (porous
cellulose) which allow passage of solvent) of the soxhlet
apparatus.
The solvent is poured in the round bottom flux which is exposed
to direct heat by heating mantle/water bath.
Continuous and environmental friendly method
The solvent must be volatile solvents are used.
Pumice used to prevent bumping of the bubbles and loss of the
extract.
99
Condenser
Soxhlet apparatus
Extraction
chamber Thimble
Siphon arm
vapour
Round bottom flask

Heat source
Animation describing mechanism of Soxhlet extractor
100
The vaporized solvent condensed at the condenser drips back to the
powder in the thimble continuously.
The condensed solvent return into the round bottom flask from the
thimble through the siphon tube
This siphoned solvent contains soluble plant constituents.
Again the solvent vapor recycled through the powdered plant material
to dissolve the constituents.
The extraction process ends when clear solvent drips from the thimble
via the siphon arm
101
8. Digestion
✓ Form of maceration with warming during extraction.
✓ The temperature increase the efficiency of the solvent without altering the
active ingredients
✓ The most commonly used temperature is between 350c to 400c. It can raise
to 50 0c in some cases.
✓ Used for tougher plant parts or those with poorly soluble constituents.
✓ The plant material is placed preheated solvent and maintained for a
period of 30 min to 24hrs.
9. Hydrodistillation
• Used for extraction of volatile oils from dried plant part resistant to
decomposition by heat.
• Aromatic plant material is packed in a still with sufficient quantity of water
& subjected to boil
• The hot water ad seam generated freed the essential oil from the oil glands
evaporates to the condenser.
• The vapor mixture of water and oil is condensed by indirect cooling with
water.
• From the condenser, distillate flows into a separator, where oil separates
automatically from the distillate water.
Hydro-distillation Clevenger apparatus system

10. Water and steam distillation
➢ Employed with herb and leaf material.
➢ The plant material is immersed in water in a Still to which heat is

applied.
➢ Steam is fed into the main Still from outside

11. Steam distillation
• For fresh plant parts with thermolabile constituents
• Extraction procedure
– Steam passes through the organic matter that contains the
compounds for separation.
– The steam condenses against that matter to form a mixture.
– That mixture gets heated further by more incoming steam, which
continues to pass through the matter, evaporating the mixture.
– Due to the reduced vapor pressure, the required organic
compounds also evaporate as a part of the mixture and are thus
extracted from the organic matter.
Instrumentation of steam distillation

12. Enfleurage
➢ The oldest methods of essential oil extraction that implements the use of fat
➢ Used for preparation of perfumes and fragrances
Procedure
Cold Enfleurage
• Highly purified and odorless fat(lard or tallow) spread out over a chassis and
allowed to set.
• Fresh flower petals or whole flowers are then placed on the fat layer and pressed in.
They are allowed to set for 1-3 days or couple of weeks. During this time, their
scent seeps into the fat.
• Depleted petals replaced and the process repeated until the fat saturates
• The final product is the enfleurage pomade: the fat and the fragrant oil.
• The extract is separated from the fat by washing with alcohol. The fat is used to
make soap.
• Absolute is obtained by evaporating the alcohol
Cold enfleurage: cold fat is used
Hot enfleurage: hot fat is used
Botanical matter
Glass plate
Fat
Chassis (plate + wood frame)
Diagrammatic representation of Enfleurage

Isolation and purification of active ingredients
Methods.
A. Classical methods of separation
B. Chromatographic/Modern methods
A. Classical methods of separation
1. Filtration
2. Centrifugation
3. Crystallization
4. Solvent extraction
5. Distillation.
6. Fractional distillation
7. Sublimation
8. Chemical derivatization
111
Classical methods of separation….
The selection of a proper separation technique depends on
➔The differences in the physical and chemical
properties of components in the mixture.
112
1. Filtration
• To separate an insoluble solid from a liquid in a
suspension
The laboratory set-up of filtration

113
2. Centrifugation
✓ Separation based on the difference in the density of the mixtures.
• The suspension mixture is placed in centrifuge tube which is
plugged in to holders in a centrifuge
• The centrifuge is turned on to rotate the tubes.
• The denser solid is collected as a lump at the bottom of the tube with
the clear liquid above
114
Used When
➢ There is only a small amount of
suspension,
➢ Much faster separation is
required
A centrifuge
115
3. Crystallization
Crystals are solids that have
➔ a definite regular shape
➔smooth flat faces and straight edges
Crystallization is the process of forming crystals
116
I. Crystallization by Cooling a Hot Concentrated Solution
• To obtain crystals from an unsaturated aqueous solution
➔the solution is gently heated to make it more concentrated
• The solubility of most solids increase with temperature.
• When a hot concentrated solution is cooled
➔ the solution can’t hold all of the dissolved solutes
The “excess” solute separates out as crystals
117
Crystallization by cooling a hot concentrated solution
118
II. Crystallization by Evaporating a Cold Solution at Room
Temperature
As the solvent in a solution evaporates,
➔the remaining solution becomes more and more concentrated
➔eventually the solution becomes saturated
➔further evaporation causes crystallization to occur
However, it may take days or even weeks for crystals to form
119
Crystallization by slow evaporation of a solution (preferably

saturated) at room temperature
120
4. Solvent Extraction
• Involves extracting a component from a mixture with a
suitable solvent
• Water is the solvent used to extract salts from a mixture
containing salts and sand
• Non-aqueous solvents (e.g. 1,1,1-trichloroethane and diethyl
ether) can be used to extract organic products.
• Example: separation of organic compounds from aqueous
solution.
121
• The compounds will partition to the

solvent of similar polarity
– ‘like dissolves like’
– To remove the organic layer
repeated washing of the mixture
with small fresh portion of
diethyl ether is required.
The organic product in an aqueous solution can be extracted by solvent

extraction using diethyl ether
122
5. Distillation
• To separate a solvent from a solution containing non-volatile
solutes
• When a solution is boiled,
➔ only the solvent vaporizes
➔ the hot vapour formed condenses to liquid again on a
cold surface
• The liquid collected is the distillate
123
The laboratory set-up of distillation

124
Bumping
❖ vigorous movement of the liquid
❖ Leads to spurt out of unvaporized liquid into the

collecting vessel
❖ Prevented by adding anti-bumping granules into the

flask before boiling the solution.
125
6. Fractional Distillation
➢ To separate a mixture of two or more miscible liquids
➢ Separation based on boiling point difference.
❖ A fractionating column (column packed with glass beads)
❖ Attached vertically between the flask and the condenser
❖ provide a large surface area for the repeated condensation

and vaporization of the mixture.
126
❖ The thermometer measures the temperature of the escaping vapour
❖ When the temperature becomes steady, the vapour with the

lowest boiling point comes out from the column first.
❖ After all of the first liquid distilled off,
❖ The temperature reading rises again and becomes steady

leading to distillation of the second liquid
❖ Fractions with different boiling points can be collected separately
127
The laboratory set-up of fractional distillation

7. Sublimation
❖ Sublimation is the direct change of
➔ a solid to vapour on heating, or
➔a vapour to solid on cooling without going through the liquid

state
❖ When a mixture of two compounds is subjected to heating and

cooling one compound passes through these phases while the
other compound remains unaffected by heating and cooling.
129
A mixture of two compounds can be separated by

sublimation
130
B. Chromatography/modern methods
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B. Chromatography/modern methods
❖ Classical methods
❖There is no complete separation, and
❖Require large amounts of material
❖ Chromatography used when:
❖Small amount of material is available &
❖The components closely resemble each other
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Chromatography….
• Chromatography:
– Greek word
• Chroma- color
• Graphein- to write, chromatography means to write in

color
– First employed by Russian scientist Mikhail Tsyet
• Used for separation of pigments such as chlorophyll.
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Chromatography
• A technique of separating the components of a mixture based on their
relative partitioning characteristics between a
– Mobile phase (MP): the solvent moving through the column, and
– Stationary phase(Sp): a substance fixed inside the column.
• Components move through the column based on the difference in their

interaction towards MP & Sp.
• Small differences in a compounds’ partition coefficient result in

differential retention on the Sp
– Separation of components
look at the animation
7/10/2021 134
Chromatography…
• Chromatography may be analytical or preparative
• Analytical chromatography
– For smaller amounts of material,
– For measuring the relative proportions of analytes in a mixture.
– Provide the qualitative profile or finger print of the compound.
• Preparative chromatography
– To purify sufficient quantities of a substance for further use
– For isolation and purification of the components
7/10/2021 135
Classification
 Shape of Stationary phase  Separation mechanism

 Column chromatography  Adsorption chromatography
 Planar chromatography  Partition chromatography
 Paper chromatography  Affinity chromatography
 Thin layer chromatography  Ion exchange chromatography
 Physical state of mobile phase  Size-exclusion chromatography
 Gas chromatography
 Liquid chromatography
 Supercritical fluid chromatography

7/10/2021 136
Chromatography…
• Solute molecules move through the column only when they are
in the MP.
– The more the solute stays on the MP the faster it elutes.
– As the Mp velocity increases the velocity of the solute

particles also increase and vice versa.
• However, the longer the molecules of solute stay on Sp the

longer the retention time
– Elute from the column later
7/10/2021 137
Chromatography…
Chromatographic terms
• Analyte: substance to be separated during chromatography.
• Solute: the sample components in partition chromatography.
• Solvent: any substance capable of solubilizing another
substance/the mobile phase in liquid chromatography.
• Stationary phase: substance fixed in place for the
chromatography. Example the silica layer in TLC
• Bonded phase: Sp covalently bonded to the support particles
or to the inside wall of the column tubing.
• Immobilized phase: Sp that is immobilized on the support
particles, or on the inner wall of the column tubing.
• Retention time: characteristic time it takes for a particular
analyte to pass through the system (from the column inlet to
the detector) under set conditions.
7/10/2021 138
Chromatography…
• A chromatograph: equipment that enables a sophisticated
separation.
• Chromatography: physical method of separation that distributes
components to separate between stationary and mobile phases.
• A chromatogram: visual output of the chromatograph. peaks or
patterns on the chromatogram correspond to different components of
the separated mixture.
• Euate: is what is coming out of the column.
• Eluent: portion of the MP which carries the sample components
with in it. It is the solvent used as MP in LC & the carrier gas in GC.
• An eluotropic series is a list of solvents ranked according to their
eluting power.
7/10/2021 139
1. Adsorption Chromatography
• Separation based on the interaction of adsorbate with adsorbent.
➢ Hydrogen bonding occurs between the compound functional

groups and free hydroxyls on the sorbent.
• Separation occurs when one compound is more strongly adsorbed by

the sorbent than the other components.
– Least strongly adsorbed compounds are carried down the column

by the passage of solvent, & elute 1st.
7/10/2021 140
1. Adsorption Chromatography…
• When the sorbent is silica or alumina, polar natural products move
slowly compared to nonpolar ones.
• Adsorbent: adsorb substances on its porous surface. It is the Sp. Silica
gel, alumina, and cellulose microcrystalline are some of the adsorbents.

➢ Adsorption mechanism of separation is employed in
➢ Column chromatography,
➢ Thin layer chromatography,
➢ Ion exchange chromatography, and
➢ Gas – solid chromatography
7/10/2021 141
1. Adsorption Chromatography…
• Adsorption depends on
– Migration of solute
– Relative strengths molecular interactions of:
– solute – solute
– solute – solvent
– solvent – solvent
– solute and solvent affinities for active sites
– effect of molecules in adsorbed state
7/10/2021 142
2. Partition chromatography
• Separation based on the relative solubility of the compound between the
sorbent and the Mp.
• Partitioning between two phases
• Compounds that are more soluble in the MP migrate up the plate to

a greater extent than components that are more soluble in the Sp.
• Both the Sp and MP are liquids or MP is gas
• Partition coefficient (α) is the ratio in which the solute distributes itself
between the Sp &MP
• Constant at
– Constant temperature
– Over a limited range of concentration
7/10/2021 143
2. Partition chromatography…
• Types of partition chromatography
– Liquid-liquid Chromatography: paper chromatography
– Gas-liquid Chromatography
• Application of partition chromatography
– To separate and identify amino acids, tannins, alkaloids,

carbohydrates, and glycosides.
7/10/2021 144
2. Partition chromatography…
Diagrammatic representation of partition (paper) chromatography
7/10/2021 145
3. Ion exchange chromatography
• Consists of an insoluble matrix with chemically bound charged
groups and mobile counter ions.
• Involves reversible binding of charged sample molecules to the

insoluble matrix in exchange for the counter ions:
• Separation on the basis of differences in their net surface charge
• Eluted through displacement by stronger ionized species in the

eluent.
7/10/2021 146
3. Ion exchange chromatography…
• IEC consists of
– Mobile phases: an aqueous buffer system into which the

mixture to be resolved is introduced
– Stationary phases: an inert organic matrix bonded to

ionizable fg that carry a displaceable oppositely charged
counterion.
7/10/2021 147
1. Anion Exchangers/AX
• consists of polymeric cation and active anion
• Used to purify carboxylic acid natural products that ionized and

negatively charged at pH>6.0.
• Strong AX: Trimethyl benzyl ammonium, Quaternary ammonium,

Trimethyl benzyl hydroxyethyl ammonium
• Weak AX: Dimethyl amine, Diethyl amine, Diethyl amino ethyl
7/10/2021 148
2. Cation Exchangers/CX
• CX consists of a polymeric anion and active cation.
• To purify amine groups in natural products that can be protonated

and positively charged at pH<7.0.
• Strong CX: Sulfonic acid, Sulfopropyl
• Weak CX: Carboxylic acid, Carboxymethyl
7/10/2021 149
• Effectiveness of ion exchange depend on
• The affinity of the charged resin group toward ions and
• Their relative concentrations ([C] and [M]).
• Highly polarized ions are strongly attracted to the exchange

resin.
▪ Polyvalent ions have greater affinity for charged resin than

monovalent
– Due to multiple binding at more than one charged group on

the resin.
7/10/2021 150
4. Paper Chromatography
• The Sp is thin film of liquid supported by the paper
• Cellulose serve as binder due to interaction of its hydroxyl

group with water mole
• The solution to be separated is applied as a spot near one

end of a prepared filter-paper strip.
• As the solvent rises through the paper, it meets the sample

mixture, which starts to travel up the paper with the
solvent.
7/10/2021 151
4. Paper Chromatography…
• The paper is supported in an
airtight chamber which has an
atmosphere saturated with solvent.
• Separation based on partitioning of

solute between two immiscible
phases
7/10/2021 152
4. Paper Chromatography…
Application of paper chromatography
• Separating Colored Pigments
• Obtaining Pure Compounds
• Qualitative Analysis
• Pathology and Forensic Science
7/10/2021 153
5. Thin Layer Chromatography (TLC)
• The Sp is a thin layer of adsorbent (usually silica gel or alumina)
coated on a glass/ aluminum plate.
• Binder such as CaSo4 is spread on a glass plate & allowed to

dry.
• The plates are activated at 105oC for 30 minutes and used.
• Compared to paper, it has the advantage of faster runs, better

separations, and the choice between different adsorbents.
– TLC plates can withstand strong solvent and colour

forming reagents
7/10/2021 154
5. Thin Layer Chromatography (TLC)…
• The mixture to be separated is applied as a spot near the base
of the plate, which is then placed in a closed glass tank
containing a layer of developing solvent.
• The solvent moves up the plate by capillary action
– carrying the less strongly adsorbed components of the

mixture with it,
• The more strongly adsorbed compounds remain near

the base of the plate.
7/10/2021 155
Visualization
❖ If invisible, the spots may be made visible by
Heating for a specific period
Examining under U.V light (if substances are
florescent).
Spraying the finished chromatogram with a
suitable reagent
e.g. Dragendrof's reagent are used as sprays for
the general detection of alkaloids (although
they are not specific for alkaloids).
7/10/2021 156
• Each spot on TLC plate can be distinguished by its Retention
factor (Rf ) value on the surface of the stationary phase,
𝑅𝑓 value = Distance travelled by compound
Distance travelled by solvent front
• Rf value is a constant for a compound under constant

chromatographic conditions
– Used for identification of unknown substance.
7/10/2021 157
Advantages of TLC
- Simple, inexpensive
- Quick – results can be achieved from between 30 minutes to a

few hours
- Good separation of spots (compounds)
- Very sensitive
- The chromatogram is resistant to the action of chemicals used

for the visualization of compounds.
7/10/2021 158
Application of TLC
1. To determine the number of components in a mixture.
2. To determine the identity of two substances.
3. To monitor the progress of a reaction.
4. To determine the effectiveness of a purification.
5. To determine the appropriate conditions for a column

chromatographic separation.
6. To monitor column chromatography.
7/10/2021 159
6. Column chromatography
• The liquid MP passes over the Sp packed in a column tube.
• The column is either a glass or metal.
• Adsorbents– Starch, Calcium carbonate, Magnesia, Lime, Silica

gel, Alumina etc.
• Mobile phases–either singly or in combination of Pet. Ether,

dichloromethane, Cyclohexane, Chloroform, Acetone, Water,
and Organic acids
7/10/2021 160
6. Column chromatography…
Colum packaging
7/10/2021 161
6. Column chromatography…
Application of column chromatography
• Isolation of active ingredients
• Separating components of a mixture
• Drug estimation from drug formulation
• Purification of a sample from impurities
• Isolation of metabolites from biological fluids
7/10/2021 162
7. High Performance Thin Layer Chromatography
(HPTLC)
• The plates are similar to conventional TLC plates.
• Silica gel of very fine particle size is widely used a sorbent
in HPTLC.
• The use of smaller particle size helps in greater resolution
and sensitivity.
• HPTLC is applied for
– “Finger- print” patterns of herbal formulations,
– Quantification of active ingredients, and
– Detection of adulteration.
7/10/2021 163
Parameter TLC HPTLC
High Performance
Chromatographic plate used Thin
Hand Layer Chromatography
made /pre-coated Pre-coated
(HPTLC)….
Sorbent layer thickness 250 mm 100-200mm
Development distance 10-15 cm 3-5 cm
Particle size range 5-20 μm 4-8 μm
Pre-washing of the plate Not followed Must
Application of sample Manual/Semi automatic Semi automatic/Automatic
Shape Spot Spot/Band

Spot size 2-4mm 0.5-1mm
Sample volume 1-10 μl 0.2-5 μl
Application of larger volume Spotting leads to over loading applied as bands
No. of samples/plate (20X20) 15-20 40-50
Development time Depends on mobile phase 40% Less than TLC
Development chamber Require large quantity solvent Require lower quantity of
solvent
Reproducibility of results Difficult Reproducible
Resolution poor resolution High resolution.
Scanning Not possible Uses UV/vis/ fluorescence
scanner to scan the entire
7/10/2021 164
chromatogram quantitatively
8. Gas chromatography (GC)
• Gas-liquid chromatography, (GLC) have.
– Mobile phase: inert gas (helium, nitrogen)
– Stationary phase: supported liquid (SiO2 coated with polymer)
GC:
✓ Involves a sample being vaporized and injected onto the
head of the chromatographic column.
✓ The sample is transported through the column by the
flow of inert, gaseous mobile phase.
✓ The column itself contains a liquid Sp which is adsorbed
onto the surface of an inert solid
7/10/2021 165
Instrumentation of GC
Flow meter
Injector Septum Detector GC

Pressure Flow Chart
regulator controller 
Recorder
Oven
Gas
supply Column
7/10/2021 166
8. Gas chromatography (GC)…
• For GC analysis the sample
– Must be volatile, or
• Be able to be converted to volatile derivative.
• Form volatile metal chelate with trifluoroacetylacetone

(TFA) and hexafluoroacetylacetone (HFA) if it is
inorganic metals (aluminum, beryllium, and
chromium).
7/10/2021 167
8. Gas chromatography (GC)…
Application of GC
– Food Analysis
– Drug Analysis
– Environmental Analysis
– Forensic Analysis
7/10/2021 168
9. High Performance Liquid Chromatography (HPLC)
• Utilizes very small packing particles and a relatively high
pressure.
• Works by forcing the Mp through the column at much

higher rate increasing the resolution rate.
• It can be
– Normal phase HPLC
– Reverse phase HPLC
7/10/2021 169
9. High Performance Liquid Chromatography (HPLC)…
1. Normal phase HPLC
• Polar Sp: SiO2, Al2O3
• Non polar Mp: Hexane, heptane, cyclohexane…
• Polar compounds travel slower and eluted slowly due to higher
affinity to the Sp
1. Reverse phase HPLC
• Non polar Sp: n-octadecyl, n-octyl, phenyl diol,
• Polar Mp: methanol or acetonitrile/water
• Polar compounds elute faster than non polar compounds
7/10/2021 170
HPLC system
• Solvent Reservoir
• Degasser
• Solvent Delivery System

(Pump)
• Injector
• Column & oven
• Detectors
• Recorder (Data Collection)
7/10/2021 171
10. Supercritical fluid chromatography/SFC
• It is a normal phase chromatography using SCF as Mp
• Polar Sp: silica/Alumina
• Non polar Mp: commonly CO2
• SFC is not suitable for elution of polar compounds
– Overcomed by addition of modifiers (alcohols, acetonitrile,

chloroform)
• Modifiers increase the solvating power of CO2
7/10/2021 172
10. Size Exclusion Chromatography(SEC)
• Is non-adsorption and non-destructive method
• Applied to large molecules or macromolecular complexes

such as proteins and industrial polymers.
• Unlike IEC, SEC does not involve chemical interaction with

sample
7/10/2021 173
Mechanism of separation
• Separate molecules in solution by their hydrodynamic volumes
• Smallest molecules enter deep into the pores and elute last.
• Medium sized components penetrate some depth and elute separately

according to their size.
• Macromolecules, bigger than the pore size will not be retained.
• Exclusion limit is maximum molecular weight that can enter the pore
Animation
7/10/2021 174
Figure 2: schematic representation of the operation of
SEC column
7/10/2021 175
Types of size exclusion chromatography.
• Gel permeation chromatography (GPC),
– The column is packed with porous beads (Sephadex, agarose).
– Organic solvents like hexane & tolune used as MP
– To measure the molecular weight distribution of organic polymers.
– For polymer analysis.
• Gel filtration chromatography (GFC),
– The column is packed with porous beads (Sephadex, agarose).
– Aqueous solution used as Mp
– To separate, fractionate, or measure M.weight of polysaccharides

and proteins.
7/10/2021 176
........cont.
➢ Highly specialized gel filtration media
➢ composed of macroscopic beads synthetically
➢derived from the polysaccharide, dextran or

silica gel.
7/10/2021 177
Merits and demerits of SEC
• Merits
– Short analysis time
– Well defined separation

– Good sensitivity
– No sample loss, since no interaction with Sp
– Small MP required
7/10/2021 178
Merits and demerits of SEC…
• Demerits
– Prior filtration to prevent dust and other particulate
running in the column and interfering with the
detector
– Not applicable to similar-sized molecules, like isomers
– Limited number of bands accommodated since short
time scale
7/10/2021 179
Applications of SEC:
• Purification.
• Desalting.
• Protein-ligand binding studies.
• Protein folding studies.
• Concentration of sample.
• Relative molecular mass determination
7/10/2021 180
7/10/2021 181

Pharmacognosy: The Science of Natural Drugs

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Pharmacognosy: The Science of Natural Drugs

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Pharmacognosy

Dehnnet A.(Bpharm, MSc in Pharmacognosy)

❖Despite the variation in philosophical premise

❖The universal role of plants in the treatment of disease was

❖Egyptians medicine, Unani medicines, Allopathic

❖ Identify natural products with food, medicinal value, &

❖ This observations were handed from generation to

❖ Individuals like Hippocrates (460-370 B.C), Aristotle and others

❖ The term "pharmacognosy" was coined by the Johann Adam

❖ Pharmacognosy is the oldest of all pharmacy sciences.

❖ During the 19th C & the beginning of the 20th C

❖Pharmacognosy refers branch of medicine which deals with

• Medicinal (Ayurvedic) writings are:(Charaka Samhita, Susruta

• Most of medicines origin from plants & animals

• The collection of plant materials was done only by an

• The fresh plants were considered to be the most effective.

– Considered as ‘father of medicine’

– Wrote Hippocratic Corpus (oldest writings on medicine).

• Recommended a healthy diet & physical exercise

• Describes on the use of plants, repositioning of joints,

Dioscorides (70 A.D.) wrote De Materia Medica

– 600 plants described: Belladona, ergot, hyoscyamus, etc.

– considered as authoritative source of pharmacological info.

• 80% medicinal plants and 20% animals and minerals

Galen (131–A.D. 200)

• Devised methods of preparations of plant and animal drugs,

• He assumed that the only certain constituent not the herbs

– Prepare extracts using menstruum (water)

• Health care relied on magic and incantations.

• The drugs used were mainly of vegetable origin.

• They were known for Laws of Hamorabi (772 B.C.)

Biochemistry Pharmacognosy Phytochemistry

• Identification of the source of a drug

• Determination of its morphological character

• Investigation of the potency, purity and quality of the drug

• Planning on proper methods of cultivation of the medicinal plants

• Prescriptions of the details of process of collection and preparation.

• Detailed knowledge of the constituents and chemical nature of drugs.

Drug means any substances:

✓Recognized in the pharmacopeia

✓Used for diagnosis, mitigation, treatment, or prevention

➢Natural products that has not been advanced in value or

➢ By any process or treatment

➢Other than essential for Proper packing & prevention

✓ Entire organism (plant, animal, micro-organism)

✓ Part of an organism (a leaf or flower of a plant, an

✓ An extract or an exudate of an organism

✓ Isolated pure compounds

❖ Can be obtained from plants, animals, microorganism, and

Role of natural products in modern medicine:

➢ Supply basic compounds that may be modified slightly to

➢ Serve as prototypes or models for synthetic drugs

▪ Tigrigna: Acerkuka (Et.) (Araya, et al., 2015)

» : Hareg Temen (Er.) (Yemane, et al., 2017)

▪ Amharic: Aserkush (Birhanu, et al., 2015).

▪ Shinasha : Emen (Giday et al., 2007)

• Start with small initial,

▪ Piper nigrum (= black)

▪ Veratrum viride (= green)

▪ Citrus aurantium (= golden yellow)

▪ Digitalis purpurea (= purple)

▪ Digitalis lutea (= yellow)

3- An aromatic plant or certain aroma: