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Plants Based Natural products Extraction, Isolation and Phytochemical

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Plants Based Natural products
Extraction, Isolation and Phytochemical screening
methods
Vajira P. Bulugahapitiya
Plants Based Natural products
Extraction, Isolation and Phytochemical screening

methods
Vajira P. Bulugahapitiya
B.Sc (Ruhuna, SL), PhD (Fribourg, Switzerland)
Senior Lecturer
Department of Chemistry
Faculty of Science
University of Ruhuna
1st Edition
20th June 2013
ISBN 978-955-54456-1-0
Bar code 9 789555 445610
Printed by
Indika Graphics
Matara
Acknowledgement
I express my gratitude to Prof. Nobuhiro Kihara, Department of Chemistry, Kanagawa

University, Japan for his encouragement and the support given to me to complete this book. I
would like to express my sincere thank to Dr. Sriya Hemalika, Department of Chemistry,
University of Ruhuna for her support given to me with proof reading of the manuscript. I also
thank to my son, Hansika Madushan, for keeping my morality up always to end this task in the
middle of many work. Finally I thank to Mr. Indika, Indika Graphics Network, Matara for
making necessary steps to print this book.
Preface
This book “Plant based natural products; extraction, isolation and phytochemical screening
methods” provides the information about main classes of natural products, their extraction
methods from plants material, separation or isolation methods of the compounds present
in crude extracts and finally chemical screening methods to analysis the phytochemicals
present in plant material.
The goal of preparing of this book is to provide basic information of natural products,
necessary guidance for the extraction process and the novel techniques related to the
extraction, isolation and phytochemical analyses to the both undergraduates and
postgraduates research students who are involved in natural products research. This is the
first edition of the book after carrying out extensive references of this field and hope this
book would provide some help to the researches in the field.
The substances produced by living things which are having distinctive pharmacological or
biological effect on human and other animals are considered as natural products. The first
chapter of the book describes the categories of natural products, their roles on own
organism and the functions on human beings etc. Alkaloids, terpenoids, steroids,
glycosides, saponins, flavanoids and polyphenols are the main classes of natural products
which exert potential effects as medicine, other beneficiary effects on human and other
animal and sometime harmful effect on human as well. These compounds present in plants
are categorized as phytochemicals. Usually, one plant contains many of such phytochemical
classes and also many compounds in the same class. For example; the plant Catharanthus
roseas contains over 140 alkaloids and some of them are potential anti-cancer drugs.
The second chapter explain the extraction methods of plant based natural products.
Extraction is the separation of natural products from their source material. The separation
is based on the concepts of “ like dissolves like” . Therefore, the solvent extraction method
is the most suitable extraction method used in natural product research. Various types of
solvent extractions methods are available with novel techniques. These methods are
extensively described in this book. Depending on the natural product to be extracted, the
solvents and the extraction method can be selected. There are extraction method to extract
specific classes of compounds, for example: alkaloids fraction or terpenoids fraction or
steroid fraction or flavanoids fraction etc. The volatile oils from plant is mainly separated
by steam distillation method. These methods are explained in this book.
Usually a crude extract of natural product is a complex mixture of large number of different
compounds. Those compounds are vary according to their chemical structures and hence
the polarities. The fractionation of the crude mixture can be done based on their polarities
or based on their acid, base or neutral nature. This simplifies the isolation process of
desired compounds. Various fractionation methods are described here. Plants are the main
source for drug discovery. About 50% of drugs available today have been isolated from
plants. Testing of biologically activity ie carrying out bioassays is the important process in
drug discovery. The process called “Bioassay guided fractionation and isolation of
compounds is very important phenomena in drug discovery from natural sources and this
book explained how to carryout the bioassays guided fractionation and isolation of natural
compounds.
For the testing of biologically activity of natural compounds in drug discovery and for the
structure elucidation of natural potential compounds, very small quantity of the substance
in extreme pure stage is necessary. Therefore, isolation of compounds in very pure stage
from their extracts is the prime important phenomenon in natural product research.
Chromatography is the very useful technique in this regards and the methods of
performing the Thin Layer Chromatiography for analytical purpose and also the
preparative purpose (PTLC) and column chromatography for preparative purpose are
explained in details here.
The chemical analysis of phytochemicals present in plant extract or powdered plant
material is termed as phytochemical screenings. There are standard chemical tests to carry
out the analysis of phytochemicals. These methods are described here with the preparative
methods for the relevant reagents as well.
Dr. Vajira P. Bulugahapitiya

Department of Chemistry 1 / A3. Araliya Beach Road
Faculty of Science Meddawatta
University of Ruhuna Matara
Matara Sri Lanka
Sri Lanka.
Tel : +94 41 2227023 ext 44406 Tel: 01-2231205

Email : vajira@chem.ruh.ac.lk vp.bulugahapitiya@yahoo.com
20Th June 2013
Content
Title Page No
1.0 Introduction to Natural products 01
1.1 Plant based Natural products 02

1.2 Further reading 04
2.0 Extraction of plant based natural products 06
2.1 Collection, authentication and preparation of
plant material for the extraction purpose 07
2.3 Extraction methods of natural products 10
2.3.1 Solvent extraction 11
2.3.1.1 Isocratic and Gradient etraction 12
2.3.1.2 Choice of Solvents for extraction of
natural products as food additives or foods. 13
2.3.1.3 Use of water as an extracting solvent 14
2.4 Solvent extraction methods 14
2.4.1 Maceration (Steady-state extraction or cold extraction) 15
2.4.2 Solvent – Solvent partitioning method 16
2.4.3 Soxhlet Extraction 28
2.4.4 Plant tissue homogenization 20
2.4.5 Extraction of all the compounds present in plant material 21

2.4.6 Further reading 23
3.0 Extraction of specific classes of natural products 23
3.1 Essential oils (volatile oil) 23
3.1.1 Introduction to Essential oils (volatile oil) 23
3.1.2 Some examples for the essential oils
(monoterpenes and sesquiterpenes), their occurrence and uses 24
3.1.3 Extraction of essential oil (volatile oil) 26
3.1.3 .1 Distillation 26
3.1.3 .2 Extraction of essential oil using solvent extraction method 30

3.1.3 .3 Fat Extraction (enfleurage) of essential oil 30
3.1.3 .4 Essential oil extraction by expression 32
3.1.3 .5 Further reading 32
4.0 Alkaloids 35
4.1 Introduction to Alkaloids 35
4.1.1 Examples for some alkaloids, their structures

and physiological effects. 36
4.2 Extraction of alkaloids 38
4.3 Extraction of volatile liquid alkaloids 40

5.0 Flavonoids 43
5.1 Introduction to Flavanoids 43
5.1.1 Structure and examples for the flavonoids 43
5.2 Extraction of Flavonoids 44
6.0 Steroids 47
6.1 Introduction to Steroids 47

6.2 Extraction of steroids 48

7.0 Saponins 50
7.1 Introduction to Saponin 50
7.2 Extraction of Saponins 50
8.0 Sublimation, Sonication, Super critical Fluid extraction
and Microwave assited extraction of Natural Products 53
8.1 Extraction and isolation of Natural Product by Sublimation 53
8.2 Applying of Ultrasound (Sonication) method in Natural product
extraction 53
8.3 Super critical Fluid extraction 54
8.4 Microwave Assisted Extraction of Natural Products 57

9.0 Fractionation of crude plant extracts 60
9.1 Fractionating Techniques 60

9.1.1 Fractionation by Solvent – solvent partitioning method 60
9.1.2 Fractionation based on acid-base nature of the compounds 61
9. 1.3 Preparation of tannin free plant extract using fractionation 63
9.1.4 Preparation of pigment free plant extracts by fractionation 63
9.1.5 Bioassay guided fractionation and isolation of natural products 64

10.0 Isolation of compounds from natural products extracts 70
(purification of natural products extracts)
10.1 Use of chromatography for the purification of natural product
extracts 70
10.1.1 Thin Layer Chromatography in Natural product Research 71
10.1.2 Use of Column chromatography for isolation of compounds in
plant extracts 75
11.0 Phytochemical Analysis ( Phytochemical Screening) 83

11.1 Tests for alkaloids 83
11.2 Tests for glycosides 86
11.3 Test for Cyanogenic Glycosides 87
11.4 Tests for saponins 87
11.5 Tests for phytosterols and triterpenes 88
11.6 Tests for diterpenes 88
11.7 Test for phenols and Tannins 88
11.8 Tests for flavonoids 89
11.9 Test for coumarins 90
11.10 Test for Anthracene Derivatives 90
11.11 Tests for proteins and amino acids 90
11.12 Test for carbohydrates 91
11.13 Test for Betacyanin 93
11.14 Test for Quinones 93
Subject Index Cold extraction, 15
A Concentration of extracts ,17
Adsorption to cold fact, 10 Collection of plant material ,7
Active compounds,64 Column chromatography ,75
Agricultural application ,2 Column packing ,77
Alkaline reagent test ,89 Cosolvant ,54
Alkaloids ,1,35 Copper acetate test ,88
heterocyclic alkaloids ,35 Crystallization ,70
non-heterocyclic alkaloids ,35 Crude extracts ,16
structure of alkaloids ,36
examples for alkaloids ,36 D
extraction of alkaloids ,38 Diterpenoids ,24
tests for alkaloids ,83 Dragendroff’s test ,84
Amino acids ,90 Dragendroff’s reagent ,84
Analytical TLC ,72 Dry loading of column ,78
Authentication of plants ,8 Drugs ,1,65
Drying of plant material ,9
B E
Basic Organic compounds ,61 Ethnopharmacology ,3
Benedicts test ,92 Essential oil ,23
Benedict’s reagent ,92 Extraction of plant based natural products ,6
Biological effect ,64 Extraction of food additives ,13
Biological active agents ,64 Extraction of

alkaloids ,38
Bioassays ,64
volatile alkaloids ,40
Bioassay guided fractionation ,64
flavonoids ,44
Biuret test ,91
saponins ,50
C
sterols ,48
Carrotinoid ,24
volatile oil ,28
Classes of natural products ,23
Extraction by expression ,32
Cleaning of plant material ,9
F
Fat extraction (Enfleurage) ,30 Lead acetate test ,89
Fehling’s test ,91 Legal’s test ,87
Fehling’s reagent ,92 Libermann-Buchardt test ,88
Ferric chloride test ,88 Low polar solvents ,12
Flash chromatography ,75 M
Flavonoids ,1, 43 Maceration ,15
extraction of flavonoids ,44 Mayer’s test ,83
structure of flavonoids ,43 Mayer’s reagent ,83
Fractionation of crude extracts ,60 Medium polar solvents ,12
Fractionation based on acid-base nature ,61 Microwave assisted extraction ,57
Freeze dryer ,18 Millon’s test ,90
Froth test ,87 Millon’s reagent ,90
G Modified Borntrager’s test ,86
Gas chromatography ,71 Molisch’s test ,92
Gel-permeation chromatography ,71 Molisch’s reagent ,92
Gelatin test ,88 Monoterpenoids ,24
Gradient extraction ,12 N
Grinding of plant material ,10 Natural product isolation ,70
H Natural product ,1
Hager’s test ,85 Natural pharmaceutical industry ,3
High-performance liquid chromatography ,71 Ninhydrin test ,90
High polar solvents ,12 P
Hydro distillation ,28 Phytochemicals ,83
Phytochemical screening ,83
I Phtochemical analysis ,83
Intermediate polar solvents ,12 Pigments ,63
Ion-exchange chromatography ,71
Preparation of pigment free extracts ,63
Iodine test ,92
Plant based natural products ,2
Isocratic extraction ,12 Plant tissue homogenization ,20
Isolation of natural products ,70 Polyterpenoids ,24
Isoprene ,24 Powdered plant ,10
K Preparative thin-layer chromatography ,74
Keller-Killiani test ,86 Primary metabolites ,2
L Proteins ,90
Purification of natural product extracts ,70 alkaloids ,83
R cyanogenic glycosides ,87
Rf value ,73 saponins ,87
phytosterols and triterpenes ,88
S diterpenes ,88
Saponins phenols and tannins ,88
structure of saponins ,50 flavonoids ,89
extraction of saponins ,50 coumarins ,90
Salkowski test ,88 anthracene derivatives ,90
Secondary metabolites ,2 proteins and amino acids ,90
Sesquiterpenoids ,25 carbohydrates ,91
Sesterterpenoids ,24 betacynins ,93
Supercritical fluid ,54 quinones ,93
Size-exclusion chromatography ,71 Thin-layer chromatography
Solvent extraction ,11 analytical TLC ,72
Solvent polarity ,12 preparative TLC ,74
Solvent-solvent partitioning ,16,60 Thermo liable ,30
Sonication ,53 Traditional knowledge ,3
Soxhlet extraction ,18 U
Steam Distillation ,27 Ultrasound extraction ,53
Steroids V
structure of steroids ,47 Vacuum drying ,9
extraction of steroids ,48 Visualization of alkaloids ,86
Storing of plant material ,10 Voucher specimens ,8
Sublimation ,53 W
Supercritical carbon dioxide ,54 Wagner’s test ,84
Supercritical fluid ,54 Wagner’s reagent ,84
Supercritical Fluid Extraction ,54 Water distillation ,28
T Wet loading of column 77
Tannin ,63 X
Taxol ,4
Xanthprotic test ,91
Tests for
1
Natural products
1.0 Introduction to Natural products

The substances produced by living things which are having distinctive pharmacological or
biological effect are considered as natural products. These products are derived in plants,
animals and microorganisms, from both terrestrial and marines sources.
Natural product can be ;
1. Entire organism (plants, animal or microorganisms) that has not been subjected to
any kind of processing or treatment other than a simple process of preservation
(e.g., drying)
2. Part of an organism (e.g., leaves or flowers of a plant, an isolated animal organ)
3. An extract of an organism or part of an organism, and exudates
4. Pure compounds isolated from plants, animals, or microorganisms (e.g., alkaloids,
coumarins, flavonoids, glycosides, lignans, steroids, sugars, terpenoids, etc.)
Importance of Natural Products for human

As pharmaceutically active compounds (medicines)
As crude drugs
As health promotion compounds
As ointment
As food additives,
As perfumes
As herbicides
As pesticides etc.
1
The compounds which are produced by nature can be categorized into two classes: those
are primary metabolites and secondary metabolites.
Primary metabolites
The compounds produced in the cells that play central role in metabolism and
reproduction are called primary metabolites. Amino acids, fatty acids, nucleic acids and
glucose belong to the class of primary metabolites.
Secondary metabolites
The natural compounds with high-molecular weight which are derived from primary
metabolites belong to the class of secondary metabolites. Examples for such compounds :
Proteins, lipids, alkaloids, terpenoids, steroids, cellulose, starch, lignines,
phenylpropionides, glycocides, polyketids etc. Secondary metabolites are not important for
growth and reproduction of the cells but have responsibilities for some important
physiological activities of the organisms themselves and also for the forming of some
cellular structures of own organisms. The physiological activities related to secondary
metabolites in its own organism can be; maintaining good immunity system, carrying on
safety mechanism against infections, use as the source for some nutrients etc. On the other
hand, secondary metabolites present in plants have often attracted interest because of their
striking biological effect on humans and other organisms.
However, in most cases the term natural products refers to some secondary metabolites,
produced by an organism that are not strictly necessary for the survival of the organism
themselves but play important role for the well-being of other organisms including human.
1.1 Plant based Natural products

The whole plants, plants parts such as flowers, leaves, bark, seed or roots, plants extracts or
isolated compounds from the plants which possess potential uses for humans as medicines
(crude drugs or pure compounds) or as health care products or in agricultural applications
2
or as food additives etc. are considered as plant based natural products. Plant based natural
products have important uses in the ancient and modern societies.
Plant synthesizes a wide variety of chemical compounds, which can be sorted by their
chemical class, bio synthetic origin and functional groups. The biologically active
constituents present in medicinal or poisonous or commercial plants have been studied
throughout the development of Natural products chemistry and many of these compounds
are found as the secondary metabolites, belong to the classes of alkaloids, terpenoids,
steroids, glycosides, saponins and flavanoids etc. These classes of compounds are generally
called as phytochemicals. The traditional knowledge of plants and their mixtures or
decoctions, on medicinal and health care applications has been much useful in discovering
potent medicines and other potential compounds from the plants (Ethnopharmacology). It
has been estimated that over 57% of medicines have their origins from the natural
products, mainly from the plants. Many screening methods for investigating the bioactive
compounds present in plants exist and have led to discover many potential drugs such as
vincristine from Vinca rosea, morphine from Papaver somniferum, Taxol_ from Taxus.
brevifolia, etc. These are the top selling drugs in the world Today. Apart from natural
product-derived modern medicine, natural products are also used directly in the ‘‘natural’’
pharmaceutical industry.
Of the approximately 250,000 higher species of plants it is estimated that only 5-15% have
been investigated for natural products. Therefore, scientific exploration of the untouched
flora is a challenge and will lead to the discover many more potent medicines and many
wonderful applications for well-being of the society. Therefore, the ability to access the
natural products, understanding of their usefulness and derive the applications from those
is a need in natural product research in this era.
The following table (Table -1) shows some examples for the potential compounds isolated
from the plants and their pharmacological uses.
3
Plant Active compound(drug) Class of compound Medicinal value / potential use
Taxus brevifolia Taxol Terpenoids For cancer

((Pacific Yew )
(palitaxel)
Papaver somniferum (
Morphine Alkaloids As an analgesic
Opium poppy)
Quinine Alkaloids Malaria

Cinchona officinalis
(Cinchona bark)
Catharanthus roseus,
Vinblastine, Vincristine Alkaloids Anti-tumor
(mini mal)
As local anesthetic, refreshing

Menthol Terpenoids
Effect
Mentha arvensis (field
mint)
Table – 1 : Some examples for the plant derived medicines, their sources and
applications
1.2 Further reading
(1). Bruneton J. (1995) Pharmacognosy, Phytochemistry, Medicinal Plants. Springer-Verlag, Berlin
(2) Cragg, G. M., Newmann, D. J., and Snader, K. M. (1997) Natural products in drug discovery and
development. J. Nat. Prod. 60, 52–60.
(3) Cordell, G. A. (2000) Biodiversity and drug discovery—a symbiotic relationship

Phytochemistry 55, 463–480.
(4). Cordell, G. A. (2000) Biodiversity and drug discovery—a symbiotic relationships Phytochemistry 55,
463–480
(5). Cordell, G. A., Beecher, C. W. W., and Pezzuto, J. M. (1991) Can ethnopharmacology
contribute to the development of new anticancer drugs, J. Ethnopharmacol. 32, 117–133.
4
(6). Clardy, J. and Walsh, C. 2004, Lessons from natural molecules, Nature, 432(16):
829-837
(7). Faulkner, D. J. (2002) Marine natural products. Nat. Prod. Rep. 19, 1–48.
(8). Jonson, E. S., Feverfew; A traditional herbal remedy for migraine arthritis, Sheldon
press, London (1984).
(9). Heinrich M., Barnes J., Gibbons S., and Williamson E. M. (2004) Fundamentals of Pharmacognosy and
Phytotherapy. Churchill Livingstone, Edinburgh.
(10) . Kaufman, P. B., Cseke, L. J., Warber, S., Duje, J. A., and Brielman, H. L. (1999) Natural Products From
Plants, CRC Press, Boca Raton
(11). Natural products isolation. – 2nd ed. / edited by Satyajit D. Sarker, Zahid Latif, Alexander I.
Gray.2006, Humana Press Inc.
(12). Herbal medicine, Phytopharmaceuticals and other Natural Products: Trends and Advances, edited
by Lakshmi S.R Arambewela, Sukumal Wimalasena, Neelakanthi Gunawardhana (2006), published by
NAM S&T Centre, India and I.Chem, Ceylon.
(13). Samuelsson, G. (1999) Drugs of Natural Origin: A Textbook of Pharmacognosy.4th revised ed.
Swedish Pharmaceutical Press, Stockholm, Sweden.
(14). Trease and Evans (2009), Pharmacognosy,16th Ed, SAUNDERS, ELSVIER
(15). Sofowora A (1986). Medicinal plant and traditional medicine in Africa II, John Wiley Chiechester.
178.
(16). Stierle, A., Strobel, G., Stierle, D., Grothaus, P., and Bignami, T. (1995) The search for a Taxol_
producing microorganism among the endophytic fungi of the pacific yew Taxus brevifolia. J. Nat. Prod. 58,
1315–1324.
(17). Williamson E. M., Okpako D. T., and Evans F. J. (1996) Selection, preparation and pharmacological
evaluation of plant material, in Pharmacological Methods in Phytotherapy Research, vol. 1. John Wiley
and Sons, Chichester.
5
2
2.0 Extraction of plant based natural products
The extraction is the separation of the potential portion or substances from their sources
(plant or animal) in order to be used in required purpose using selective extraction
procedures. Extraction produces crude extracts which may contains active compound or
many potential compounds or many compounds (desired and undesired) in in-pure stage.
When plants are concerned, the potential compounds (active compounds) with some
exceptions, occur in amounts that are less than 0.01 % of the dry weight of the plant.
Extraction of 1 kg of dry plant material may yield less than 100 mg of a natural product.
Some of natural compounds may be unstable to be present as free form in plant. Therefore,
they may stay as part of a complex mixture in the cells such as slats form or forming
complex with other compounds etc. The isolation, separation and purification of these
natural products require considerable skill.
The required purposes for extraction of natural product can be:

a. To use as crude herbal drugs.
b. To isolate known mixture of compounds.
c. To isolate unknown compounds responsible for particular biological activity.
d. To isolate certain known compounds with biological interest.
e. To use in testing of biological activity of the secondary metabolites.
f. To isolate of active compounds in order to characterize them.
g. To isolate the group of compounds with some structural similarity.
h. To isolate all the secondary metabolites produced
i. To create a data base of natural products by isolating and characterizing of them
from natural sources.
6
The history of the extraction of natural products, goes back to Mesopotamian and Egyptian
times, where production of perfumes or pharmaceutically - active oils and waxes was the
major business.
As plants, contain large number of organic compounds with different polarities, the concept
“ like dissolves like” is mainly applied in the extraction process. It means, plant
compounds dissolve in solvents according to their polarity matching. Therefore the main
extraction technique used for the extraction process is solvent extraction. However, other
extractions methods are available depending of the need of extraction and the nature of the
source material and classes of the compounds to be isolated.
Prior to choosing an extraction method, it is necessary to establish the target of the

extraction.
Target of the extraction can be

1. To isolate unknown compounds or all the compounds present in a plant or a plant
parts
2. To isolate particular class of compounds or particular compound
Before the extraction process, proper care should be taken during the collection,
authenication and the preparation of plant material.
2.1 Collection, authentication and preparation of plant material for the extraction
purpose
Collection of plant material
The whole plant or a particular part of the plant is collected depending on where the
metabolites of interest are accumulated. The plant can be in wild or from cultivated land.
7
When collection from wild, it is good to get the support from known person about the
particular plant otherwise adulteration can takes place with other similar species of the
same family. The aerial parts ; such as leaves, stems, flowering tops, fruits, seeds, bark and
the underground parts ; such as bulbs, tubers, roots parts are necessary to be collected
separately. Only healthy specimens should be obtained as signs of contamination (fungal,
bacterial, or viral) may change metabolites present. Collection of plant material can also be
influenced by other factors such as the age of the plant and environmental conditions such
as temperature, rainfall, amount of daylight, soil characteristics, and altitude because
higher concentration of active metabolites in particular plant depends on above factors.
Therefore, it is good to get the support from skilled person for the collection.
In some cases, the targeted plant is a species of indigenous of a remotely accessible area. It
is important to take these issues into account for the recollection purposes to ensure a
reproducible profile (nature and amount) of metabolites.
Authentication of plant material and maintaining of voucher specimen

It should be noted that collected plant or plant parts must be identified correctly. A
specialized taxonomist should be involved in the detailed authentication of the plant (i.e.,
classification into its species, genus, family, order, and class). A dried specimen of plant or
part should be pressed between sheets of paper for drying and should be deposited in a
herbarium for future reference. Any features relating to the collection, such as the name of
the plant, the identity of the part(s) collected, the place and date of collection, should be
recorded as part of a voucher specimen.
Preparation of plant material

Cleaning and Drying
After returning to the laboratory, the collected plants should be washed or gently brushed
to remove soil and other debris. If the plant is known to contain volatile oils, It is necessary
to be distilled immediately after collection, if not, it is advisable to snap–freeze the material
as soon as possible. Frozen samples can be stored in a freezer (at - 200C) or freeze-dried
8
(lyophilized). After cleaning, the plant material should be dried as early as possible as dry
conditions are essential to prevent microbial fermentation and subsequent degradation of
metabolites. The duration of dying and the method of drying depend on plant material.
There are various ways of drying:

Open air Drying
Artificial Drying
Vaccum drying
Generally, open air drying is carried out. It is, however, a more common practice to leave
the sample to dry on trays at ambient temperature and in a room with adequate
ventilation. Plant material should be sliced into small pieces and distributed evenly to
facilitate homogenous drying. Open air drying is largely depends on weather. The duration
of drying varies from few hours to many days depend on plant. The plants spread on the
papers on wooden framework facilitates drying.
The artificial drying or drying with applying artificial heat is more rapid. This is selected
depending on plant material and climate. Usually it is necessary in tropical countries.
Vaccum drying is applied according to the plant and the climate.
Drying temperature is important factor. As a general rule, leaves, fruits, flowers maybe
dried between 30-40 C : barks and roots between 40-60C.
Grinding and storing

The dried plant material is cut into pieces ( if necessary) and then grinded into powdered
material using normal grinder or it is usual to grind them subsequently in a mortar with
liquid nitrogen. Extracting of the pulverized residue or powdered material should be done
immediately if not, storing in a freezer in a sealed container without exposing to sunlight is
necessary to prevent any changes in the profile of metabolites. Storage for prolonged
9
periods should be avoided, as some constituents may decompose. Sometimes the plant
extracts are stored till further use. In this case it is necessary to use clean dry sealed
container at low temperature. It is better if some anhydrous agents are used in the
container (placing anhydrous compounds bottom of the container) and separate the
extracts using perforated plate or paper. However, storing for long period may decompose
active constituents.
2.3 Extraction methods of natural products
(i). Solvent Extraction
• Maceration
• Solvent-solvent partitioning
• Soxhlet extraction
• Microwave assisted solvent extraction
Sonication assisted extraction
• Plant tissue homogenization
(ii). Steam distillation
(iii). Supercritical fluid extraction
(iv). Sublimation
(v). Absorption to cold fat
2.3.1 Solvent extraction
Solvent extraction is the main extraction method used in separation of natural substances
from their source material (plants of animals). Solvent extraction is based on polarities of
10
substances and solvents used. As plant consists of many compounds of wide range of
polarities, different polar solvents are used depending on the extracting substances.
General criteria for selecting of a solvent for the extraction
The following factors should be considered when selecting a solvent for the extraction:
a. Solvent power : Solvent should have enough salvation ability to extract the desired
constituents from the plant material.
b. Boiling temperature : The boiling point of the solvent is as low as possible in order
to facilitate removal of the solvent from the product after extraction.
c. Reactivity : The solvent should not react chemically with the substances and
should not readily decompose while heating or standing.
d. Viscosity : A low viscosity of the solvent is better. It leads good heat and mass
transfer.
e. Safety : The solvent should be non-flammable and non-corrosive, and should not
present toxic hazards.
f. Cost : The solvent should be readily available at low cost.
g. Vapor pressure : To prevent loss of solvent by evaporation, low vapor pressure at

operating temperature is required.
h. Recovery: After the extraction, the solvent should be easily removable from the
substances by distillation. Therefore the solvents can be reused after re-distillation.
11
However, it is necessary to purify the commercial solvents using simple distillation before
use them in extraction.
Categories of organic solvents according to polarity:
a. High polar solvents, eg: methanol, ethanol, butanol
b. Medium polar solvents , eg: ethyl acetate (ETOAC), dichlomethane etc.
c. Less polar solvents, eg: pet.ether, acetone etc.
d. Non-polar solvents : eg: n-hexane etc.
As plants are consisted of chemical compounds in wide range of polarity, extracting

solvents should be selected considering the compounds to be isolated. In a selective
extraction, the plant material is extracted using a solvent of an appropriate polarity
following the principle of ‘‘like dissolves like”.
Generaly, less polar solvents are used to solubilize less polar compounds (e.g., alkanes,
pigments, waxes, some terpenoids etc.). Medium-polarity solvents are used to extract
compounds of intermediate polarity (e.g., some alkaloids, steroids etc.), while more polar
solvents are used in extraction of more polar compounds (e.g., flavonoid glycosides,
tannins, polyphenols etc.).
2.3.1.1 Isocratic extraction and Gradient extraction
Isocratic extraction is the extraction using single solvent or same strength solvent mixture
(fixed- polarity solvent mixture) during the entire extraction process. This extraction
method extracts compounds in some polarity limit.
Gradient extraction is the extraction using solvents with increasing polarity eg: first
extracting solvent is less polar solvent (hexane, pet.ether etc.) and followed with
intermediate polar solvents (dichloromethane, ethyl acetate etc.) and followed with higher
12
polar solvent (methnol), finally extraction is with the most polar solvent or solvent mixture
(eg: alcohol-water mixture) etc. Gradient extraction divide the crude mixture or
constituents in powdered plant material into different fractions of compounds based on
their polarity.
Neither chemical composition of the plant nor the chemical nature of the desired
compounds is known, gradient extraction is the most suitable method of extraction for the
extraction of all constituents present in source material into different solvents.
2.3.1.2 Choice of Solvents for extraction of natural products as food additives or

foods.
The plants are rich with food additives. If the natural products going to be extracted is
intended to be used as food or food additives, only recommended solvents should be
utilized. Examples for such solvents: water, acetone, ethyl acetate, liquid CO2, propene and
ethanol etc. The toxic metals such as arsenic or lead contain should be minimized in
solvents used in food extraction.
Liquefied CO2 is preferable for extraction of food material from plants. It is used for
decaffeination of green coffee beans or tea, preparation of leaf extracts, extraction of spices,
herbs, essential oils, pungent constituents, natural colorants and antioxidants as well as
production of high – value fatty oils in industrial scale.
Some solvent like acetonitrile, benzene, chlorobenzene, carbon tetrachloride, 1,2-

dichloethane, pyridine, toluene etc. are known to have some toxicity, hence those are not
suitable for the extraction of natural products, specially natural food compounds or food
additives.
2.3.1.3 Use of water as an extracting solvent
13
Water belongs to inorganic solvent and it is the universal solvent. The polarity of water is
comparably high. As water has the highest heat of evaporation, it is more difficult to
remove by distillation compared to the organic solvents. Therefore, it is not often used as
an initial extracting solvents of natural products, even if the aim is to extract water-soluble
plant constituents (e.g., glycosides, quaternary alkaloids, tannins etc.). Water extracts polar
substances such as polyphenols, flavnoids etc.
In case of herbal medicines, the crude drug preparation is done in water. For example; in
Ayurveda or traditional medicine, a decoction of the plants is prepared by boiling of plants
or plant parts in water. For the analytical purposes, an aqueous extract of plant is prepared
by boiling of plant or plant parts in water using Soxhlet apparatus. As mentioned
previously water preferentially extracts most of polar compounds (e.g. plant pigments,
tannins) along with other compounds. Therefore, water extracts need some special type of
post treatment (e.g. use of ion exchange chromatography or caustic wash for further
purification etc.) to remove polar unwanted material from the aqueous extract.
In water extract or aqueous extract water is removed using freeze-drying method usually
as higher temperature may degrade the potential compounds.
2.4 Solvent extraction methods
The commonly used solvent extractions methods are:
a. Maceration
b. Solvent-solvent partitioning
c. Soxhlet extraction
d. Plant tissue homogenization
14
In addition to above solvent extraction method specific solvent extraction methods
such as sonication-assisted solvent extraction and microwave-assisted solvent
extraction are used in extraction of some natural products. These methods are
described in chapter 8.
2.4.1 Maceration (Steady-state extraction or cold extraction)
Maceration is an isocratic extraction method and cold extraction method. Maceration is

suitable for extraction of thermo liable compounds.
In this method, constituent in plant material is extracted into solvent by immersing the
plant material in a particular solvent. Maceration is done at room temperature and without
much shaking of the system (steady state extraction). The solvent for the maceration is
selected with considering the constituents to be extracted.
The procedure of maceration
1. Prepared plant material (cleaned and air dried crushed plant material or coarsely
powdered) is dipped in an appropriate solvent called menstrum in a closed
container and allowed to stand at room temperature for the period of 4-6 days with
occasional agitation, with opening the lid times to times to release any pressure
developed and shaking until the soluble matter has dissolved.
The solvents good for maceration : Methanol, methanol-water or any other organic
solvent according to the need.
2. Then, the remaining (the damp solid material) is filtered off using a funnel with a
cotton plug and then the marc is further pressed to recover as much occluded
solution as possible.
Note: The expressed liquid may be cloudy with colloidal and with small particles.
15
Therefore, sufficient time is necessary for coagulation and settling. The settled matter is
filtered through a filter paper.
3. The resultant extract is concentrated under reduced pressure to obtain the crude
extract of the plant.
4. The crude extract is used for further analysis.
If low polar solvent is used in maceration process, only less polar compounds dissolve in
that solvent. In most cases, higher polar solvents such as methanol or water-methanol
mixture are used in maceration, then almost all the compounds present in source material
dissolve in higher polar solvent. Maceration is good method in natural product isolation as
no higher temperature is applied.
When higher polar solvent is used in maceration, a gradient extraction method such as
solvent-solvent partitioning should be carried out for the resultant crude extract in order
to fractionate the compounds present in crude based on their polarities.
Note : As the system is static in maceration process, (except for occasional shaking) the
process of extraction works by molecular diffusion, which is very slow. Occasional shaking
assists diffusion and also ensures dispersal of the concentrated solution accumulating
around the surface of the particles, and bringing fresh menstruum to the particle surface
for further extraction. A closed vessel is used to prevent evaporation of the menstruum
during the extraction period.
2.4.2 Solvent – Solvent partitioning method
Solvent-solvent partitioning or liquid-liquid partitioning is a method used to separate or

extract organic compounds based on their relative solubility in two
16
different immiscible liquids, usually water and an organic solvent. This can be used as
gradient extraction method.
After maceration the compounds present in the crude methanolic extract, is suspended in
water (aqueous phase) and it is extracted into different organic solvent using solvent-
solvent partitioning method. Then the compounds move to organic phase from aqueous
phase according their affinities to organic phase which is based on their polarity.
Procedure for the solvent-solvent parttioning
1. The aqueous suspension prepared after maceration is transferred into a

seperatory funnel and less polar organic solvent (eg: hexane) is added to it as the
first extracting solvent. The content in the separatory funnel is mixed by swirling
the funnel and stand to be settled.
2. Hexane layer is decanted into separate flask and the extraction is repeated twice
with fresh hexane until all the less polar compounds move to hexane layer.
3. Then, next polar solvent (eg: ethyl acetate) is added to remaining aqueous phase
in the separatory funnel in order to extract next polar compounds present in
aqueous phase (extraction should be done three times for each solvent).
4. This procedure is continued with solvents with increasing polarity in order to

extract all the compounds into different polar solvents according to their
polarities matching.
5. Finally, the most polar solvent such as n-butanol is used as the extracting solvent.
6. Last fraction remaining in the separatory funnel contains highest polar

compounds in water (aqueous extract).
Concentration of solvent extracts

17
All the solvent extracts (fractions) obtained in solvent-solvent partitioning method are
concentrated by evaporating the solvent under reduced pressure. Usually, rotary
evaporator is used for the evaporation of the solvent under reduced pressure. Here
distilled and condensed solvents are recovered for future use after re-distillation. A freeze
dryer is used to remove water in aqueous extract.
The crude extract obtained in each case should be analyzed or subjected to planned
applications quickly as possible. If case of delaying the analyses or application, it is
necessary to store them in cool and dry conditions in a sealed container.
2.4.3 Soxhlet Extraction
This is a hot continuous extraction process using the solvents with different polarities and
can be done in laboratory scale. The compounds with particular polarities are extracted
into the solvents using a continuous extraction process at the boiling point of the solvent.
Special glass apparatus is used in this method named Soxhlet apparatus (Figure -1). The
extraction can be done using the solvents of increasing of polarity hence it is a sequential
extraction process. Soxhlet extraction is only required where the desired compound has a
limited solubility in a solvent at room temperature. The advantage of this system is that
instead of many portions of warm solvent being passed through the sample, just one batch
of solvent is recycled during four hours. This method cannot be used for thermo labile
compounds as prolonged heating may lead to degradation of compounds.
Soxhlet apparatus is consists of a flask, a Soxhlet extractor and a reflux condenser as shown
in Figure-1. Different sizes of Soxlet extractors can be used depending on the amount of
plant material.
The procedure for Soxhlet extraction
1. The finely ground plant material is placed in a porous bag or thimble made of
chromatographic paper or strong filter paper.
18
1. It is placed in the Soxhlet extractor as shown in the figure 1. Make sure not to block
the bottom outlet of the extractor.
2. First polar solvent (Less polar) is placed in the flask and brought to its boiling point
by heating (electrical heating).
3. According to Figure 1, the solvent vapors pass through the larger right hand tube
into the upper part of the extractor and then to the condenser where it condenses
and drops back onto the crude plant material.
4. During this period, the soluble constituents are extracted into the particular
solvent. When the level of the extract reaches the top of the siphon tube, the entire
volume of extract syphons flows over into the fl ask.
5. The process is continued (about 4 hours) until the components in particular polarity
are completely extracted. Then the extracted solvent in the flask is removed and
placed in a separate flask and labeled.
6. Then, next polar solvent is added to the flask in Soxhlet and the extraction is
continued another four hours while boiling the solvent as described above.
7. Extraction procedure is repeated using the solvents with increasing polarity until
the all the compounds in the plant material is extracted into different solvents based
on their polarities and extracts are placed in labeled flasks.
8. Excess solvent in each solvent extract is evaporated under reduced pressure (using
rotary evaporator) to obtain crude solvent extracts of the plant material.
9. Crude extracts should be subjected to further analyses immediately, if not they
should be stored in cold, dry place until further use.
19
Figure 1 : Schematic diagram for
Figure 2 : Soxhlet extraction
Soxhlet extraction
setup
2.4.4 Plant tissue homogenization
This is a special maceration method. In this method, dried or wet, fresh plant parts are
grinded in a blender to fine particles, and immense them in a certain quantity of desired
solvent with vigorous shaking for 5 - 10 min or left for 24 h. After this period, the extract is
filtered and the filtrate is then dried under reduced pressure. Sometimes centrifugation is
used to clarify the extract. The extracts are further fractionated if necessary or used as
medicine itself or further analysis and separation for discovering new drugs etc.
20
2.4.5 Procedure for extraction of all the compounds present in plant material
The required purpose for the extraction of compound from plant material can be
characterization of all the phytochemicals present in particular plant material or isolation
of specific unknown compounds having some potential effect. In such case all the
compounds present in plant material need to be extracted first. The gradient solvent
extraction method is the most suitable extraction method in this case.
This can be done in two ways;
Method -1 Plant material is subjected to maceration using methanol and

resultant extract is fractionated by solvent-solvent partitioning using solvent
gradient.
Method-2 Carrying out Soxhlet extraction using solvent gradient.
2.4.6 Further reading
1. Benthin, B., Danz, H., and Hamburger, M. (1999) Pressurized liquid extraction
of medicinal plants, J. Chromatogr. A 837, 211–219.
2. Banoti, A., 1980, Problems relating to the preparation and use of extracts from
medicinal plants, Fitoterapia, 51: 5-11.
3. Extraction technologies for medicinal and aromatic plants, International center for
Science and Higher technologies, Area Science Park, padriciano 99, 34012 Trieste,
Italy.
4. Farid Chemat et al (2012), Green Extraction of Natural Products: Concept and

Principles Int. J. Mol. Sci. 13
5. Lapkin, A.; Plucinski, P.K.; Cutler, M. (2006) Comparative assessment of technologies

for extraction of artemisinin. J. Nat. Prod., 69, 1653–1664.
21
6. Natural products isolation. – 2nd ed. / edited by Satyajit D. Sarker, Zahid Latif,
Alexander I. Gray.2006, Humana Press Inc.
7. Herbal medicine, Phytopharmaceuticals and other Natural Products: Trends and

Advances, edited by Lakshmi S.R Arambewela, Sukumal Wimalasena, Neelakanthi
Gunawardhana (2006), published by NAM S&T Centre, India and I.Chem, Ceylon.
8. Trease and Evans (2009), Pharmacognosy,16th Ed, SAUNDERS, ELSVIER
9. Trease, G. E. and Evans, W. C. (1996) Introduction and general methods, in

Pharmacognosy, 14 ed.,WB Saunders Company Ltd., London.
10. Samuelsson, G. (1999) Drugs of Natural Origin: A Textbook of Pharmacognosy.

4th revised ed. Swedish Pharmaceutical Press, Stockholm, Sweden.
11. Williamson E. M., Okpako D. T., and Evans F. J. (1996) Selection, preparation and
pharmacological evaluation of plant material, in Pharmacological Methods in
Phytotherapy Research, vol. 1. John Wiley and Sons, Chichester
22
3
3.0 Extraction of specific classes of natural products from plants
When a particular phytochemical constituent or compound class is to be the target of

investigation, different extraction procedures may be employed to produce the extracts
enriched with the specific constituents.
There are many ways to do this;
1) Use of specific solvent to exract particular class of compounds from the powdered
plants or from the initial extract or use of specific extraction procedure.
2) The polarity of the initial extract solution can be modified to cause particular
compound classes to be precipitated, leaving unwanted compounds in solution.
3) The compounds containing primary, secondary, or tertiary amines, carboxylic acids,
lactones, and phenols may be selectively extracted using pH modifications to
manipulate the polarity/solubility of the compounds of interest.
3.1 Essential oils (volatile oil)
3.1.1 Introduction to Essential oil (volatile oil)

The plant oil, the aroma compounds named as essential oil belongs to the class of
terpenoids, mainly mono and sesquiterpenoids (C-10 and C-15 units). Those are volatile
compounds of terpenoids and differ entirely in physical and chemical properties from fixed
oils. Large quantities of volatile oil are produced annually from plants and used in a wide
variety of consumer goods such as detergents, soaps, toilet products, cosmetics,
pharmaceuticals, perfumes, as starting compounds for the synthesis of other biologically
active compounds, confectionery food products, soft drinks, distilled alcoholic beverages
and insecticides etc.
All terpenoids are composed of isoprene units, C5 units (isoprene unit). Two or more
isoprene units are joined by head-to-tail manner to form tepenoids.
23
CH3
H2C C CH CH2
Isoprene
Generally terpenes are classified into different classes by the number of C-5 units that they
contain. The classes are: Monoterpenoids (C-10), Sesquiterpenoids (C-15), Diterpenoids (C-
20), Sesterterpenoids (C-25), Triterpenoids (C-30), Carotenoids ( C-40 ) and
Polyterpenoids ( > C-40). As mentioned above volatile oils belongs to the class of mono-
and sesqui terpenoids.
All the terpenoids are hydrocarbons and oxygenated hydrocarbons.
3.1.2 Some examples for the essential oils (monoterpenes and sesquiterpenes),
their occurrence and uses
Essential oils are derived from plants and they can be found in many plant parts including
flowers, leaves, stems, bark, seeds, fruits and rhizome.
Eg: flowers (e.g. rose, jasmine, carnation, clove, mimosa, rosemary, lavander), leaves (e.g.
mint, Ocimum spp., lemongrass, jamrosa), leaves and stems (e.g. geranium, patchouli,
petitgrain, verbena, cinnamon), bark (e.g. cinnamon, cassia, canella), wood (e.g. cedar,
sandal, pine), roots (e.g. angelica, sassafras, vetiver, saussurea, valerian), seeds (e.g fennel,
coriander, caraway, dill, nutmeg), fruits (bergamot, orange, lemon, juniper), rhizomes (e.g.
ginger, calamus, curcuma) etc.
Monoterpenes are low boiling compounds of essential oil. Some examples are: Geraniol
(3.1) is a major component of geranium oil (Pelargunium graveolens) and its isomer,
linalool (3.2) is found in the oil of a garden herb, clary sage. Citral (3.3) a constituent of
lemon oil, is obtained commercially from lemon grass oil (Cymbupugun flexuosus). Menthol
(3.4) is found in the essential oil of the field mint, Mentha arvensis, and possesses useful
physiological properties including local anaesthetic and refreshing effects. It is used to
flavour sweets, tobacco and toothpaste. Terpineol (3.5) and α-pinene (3.6) are found in
24
pine oil (terpentine). Camphor (3.7), which was isolated from the camphor tree,
Cinnamomum camphura, but is now made commercially from α -pinene, is used to protect
clothes from moths.
OH
OH CHO
3.1
3.2 3.3
OH
OH
3.7
3.4 3.5 3.6
Some sesquiterpenes are found in the higher boiling portions of essential oils. These
include caryophyllene (3.8) from oil of cloves, cedrene from cedar wood oil, longifolene
from Indian turpentine oil (Pinus ponderosa) and farnesol (3.9) from oil of rose, nerolidol
from oil of neroli, zingiberine from ginger oil etc.
H
CH2OH
3.8 25 3.9
3.1.3 Extraction of essential oil (volatile oil)
Plant containing essential oils usually have the greatest concentration at some particular
time of the day, eg: jasmine at sunset. Therefore, extraction should be done at the time that
higher concentration of oils are accumulated in particular plant part and it should be
performed immediately after collection.
The world production and consumption of essential oils and perfumes are increasing very
fast. Production technology is an essential element to improve the overall yield and quality
of essential oil. The traditional technologies used in essential oil production are of great
significance and are still being used in many countries.
Extraction methods for volatile oils

1. Distillation
a. Steam distillation
b. Water distillation
2. Solvent extraction using low boiling solvents
3. Adsorption on fat
3.1.3 .1 Distillation
Extraction of volatile oil from plant material by distillation has been long practiced. Almost
all constituents of essential oils are unstable at high temperature. To obtain the maximum
amount of oil in best quality, distillation must be done at low temperatures. Therefore,
distillation of volatile oils by means of water or steam ie : steam distillation or hydro
distillation, is carried out. This methods are widely applied in commercial production of
volatile oil Today. With modern apparatus and skills undesirable decompositions of the oil
in older methods of distillation is minimized.
26
The principle involves in steam / water distillation
Water or steam and the volatile oils are immiscible. Hence, they boil independent of each other
from their mixture. Therefore, boiling of the mixture occurs when the sum of the pure vapor
pressures of water and oils equals to the surrounding pressure:
According to Dalton Partial pressure Law; Total pressure equals to the sum of individual
pressures of the gases present in the mixture.
Ptotal = Pwat + Poil
Ptotal : total pressure
Pwat : vapor pressure of water
Poil : vapor pressure of essential oil
Thus, a mixture of two immiscible liquids boils at a temperature lower than the normal boiling
point of either component of the mixture. Because Pwat >> Poil, the mixture boils at a temperature
slightly less than the normal boiling point of water. Usually volatile oils have the boiling point
above 100 0C. Therefore, volatile oils can be extracted at lower temperatures using steam
distillation method.
Procedure for extraction through steam distillation or hydrodistillation

Extraction of volatile oils via steam or hydro distillation is consist of two steps.
a). Distillation of volatile oils with steam
b). Separation of volatile oils from water/volatile oils mixture (condensate)
a). Distillation of volatile oils with steam

Distillation can be done two ways :
i) passing direct steam through the plant material (live steam)
ii) distillation of the mixture of water and plant material (direct steam,water
distillation).
27
i) Steam distillations with live steam
In this method, steam is generated in external source and passed to the distilling pot (Figure 3).
The steam carries the oils vapor and water vapor into the distilling head and then into the
condenser, where the oil and water co-condense.
ii) Water distillation (hydro distillation)
In this method, a mixture of plant source and water is placed in the pot and get it boiled.
(Figure 3). This is a hydro distillation and undergo same principle as the steam distillation.
Figure 3: Live steam and direct steam generation
The whole set up for the steam distillation is shown in following figure (Figure 4).
28
Figure -4 : Set up for steam distillation
b). Separation of essential oils from water (condensate).

After distillation, next step is to recover the distilled oil from water/oil mixture.
This is done in following steps
1. The distillate (the mixture of water and oil) is transferred to a separatory funnel and
diethyl ether is added to it.
2. The funnel is swirled and allowed to stand
3. Down water layer is drained off to a Erlenmeyer flask and ether layer is poured to
another flask from the top of the funnel.
4. The aqueous phase is transferred to separatory funnel again and the extraction process is
repeated twice using fresh diethyl ether in each case and all the ether layers are combined
together
5. The ether layer is dried shaking with anhydrous MgSO4
6. MgSO4 is filtered off and the ether is distilled off under reduced pressure or if the amount
is less, evaporation is carried out on a steam bath under fume-hood.
7. The oil is subjected to further analysis or used in required application. If further use is
delayed, it is stored in a sealed vials at low temperature.
29
Note: Essential oil obtained after steam distillation is in crude form. It may have
suspended impurities and appreciable moisture content. It might even contain some
objectionable constituents which degrade its flavor quality. The presence of moisture and
impurities adversely affects the keeping quality of oil and accelerates polymerization and
other undesirable reactions. Addition of a drying agent like anhydrous sodium sulphate to
the oil, standing overnight followed by filtration will remove the moisture and free the oil
of suspended impurities. Use of high-speed centrifugation to clarify the essential oils is
common method.
Parameters acting yield and quality of essential oils obtained by distillation

The yield and the quality of essential oil from steam distillation is affected by the various
process parameters. It is advisable to keep them in mind while designing such systems.
Some of the important parameters are below.
Mode of distillation
Proper design of equipment
Material of fabrication of equipment
Condition of raw material
Loading of raw material and steam distribution
Operating parameters
3.1.3 .2 Extraction of essential oil using solvent extraction method

In the perfume industry, most of modern essential oil production is accomplished by
extraction, using volatile solvents such as petroleum ether or hexane.
The extraction can be performed in two ways;

i. Maceration : plant material is immersed in low boiling point solvent such as
hexane or low boiling petroleum ether in a closed container and stand for 24 hours
with occasional shaking
30
j. Using Soxhlet extractor: plant material is heated under reflux condition below 50°
C or at 50° C using soxhlet extractor. This is mainly used in laboratory scale.
The chief advantages of extraction over distillation is that uniform temperature (usually
50° C) can be maintained during the process, As a result, extracted oils have a more natural
odor that is unmatched by distilled oils, which may have undergone chemical alteration by
the high temperature. This feature is of considerable importance to the perfume industry;
however, the established distillation method is of lower cost than the extraction process
and distillation can be performed in very large scale.
3.1.3 .3 Fat Extraction (enfleurage) of essential oil

Certain high-quality odor-producing flowers such as jasmine, tuberose and gardenia yield
small quantities of volatile oils and cannot be distilled by steam distillation. Furthermore,
the oil components are thermo liable and such flowers, even after plucking, continue to
emit small quantities of perfume. In this case oil is extracted by enfleurage, by digestion in
melted fats. This is specially done in France in manufacturing of perfumes.
Fat possesses a high power of absorption of volatile oil and, if brought in contact with
fragrant flowers, it absorbs the perfumes (volatile oils). The fat used in the extraction
process is specially a mixture of lard and tallow.
Procedure for enfleurage
1. The chassis shown below (Figure 5) is prepared with fat and it is charged with fresh
flower petals
2. It is let for 24 hours to absorb the volatile oil
3. Using spatula , flower petals are removed and chassis is recharged with fresh petals
4. This procedure is continued till the fat in the chassis is saturated with volatile oil.
5. Then, the fat is immersed in absolute Ethanols or Methanol and the essential oil is
dissolved in alcohol
31
6. The alcohol is evaporated under reduced pressure and pure oils are obtained.
Note: sometimes, alcoholic mixture of volatile oils is marketed as perfumes
Figure 5 : Chassis of fats with flower petals
Pile of chassis
3.1.2 .4 Essential oil extraction by expression

Expression or cold pressing, as it is also known, is only used in the production of citrus oils.
The term expression refers to any physical process in which the essential oil glands in the
peel are crushed or broken to release the oil. The oil release in this process is absorbed by
sponge and it was recovered back by squeezing the sponge. It is reported that oil produced
this way contains more of the fruit odor character than oil produced by any other method.
32
3.1.2 .5 Further reading
1. Bohra, P. M., Vaze, A. S. and Pangarkar, V. G., 1994, Adsorptive recovery of water
soluble essential oil components, Journal of Chemical Technology & Biotechnology,
60: 97-102.
2. Brown, G. D., Liang, G.-Y., and Sy, L.-K. (2003) Terpenoids from the seeds
of Artemisia annua. Phytochemistry 64, 303–323.
3. Dung N. X. and Dinh, T., 2005, Extraction and Distillation of essential Oils,
Processing, Analysis and Application of Essential Oils, 1st Edition, Har Krishan
Bhalla & Sons Book Company.
4. Khan, M. R., Gray, A. I., and Waterman, P. G. (1990) Clerodane diterpenes

from Zuelania guidonia stem bark. Phytochemistry 29, 2939–2942.
5. Ganga, A., Pinaga, A., Quero, A., Valles, S. and Ramon, D., 2001, Cell wall degrading
enzyme in release of grape aroma precursors, Food Science and Technology
International, 7: 83-87.
6. Ghisalberti, E. L., Jefferies, P. R., Skelton, B. W., White, A. H., andWilliams, R. S. F.

(1989) A new stereochemical class of bicyclic sesquiterpenes from Eremophila
virgata W. V. Fitzg. (Myoporaceae). Tetrahedron, 45, 6297–6308
7. Gibbons, S.; Gray, A. I.; Waterman, P. G. Clerodane diterpenes from the bark of
Casearia tremula. Phytochemistry 1996, 41, 565-570.
8. Kahol, A. P., 1984, Distillation Technology. In: Practical Manual on: The Essential Oils
Industry, Wijesekera, R. O. B (Ed.), UNIDO, Vienna.
9. Lawrence, B. M., 1995, The Isolation of Aromatic Materials from Natural Plant
Products. In: A Manual on the Essential Oil Industry, Tuley De Silva (Ed.), United
Nations Industrial Development Organization (UNIDO), Vienna, Austria
33
12. Oberlies, N. H.; Burgess, J. P.; Navarro, H. A.; Pinos, R. E.; Fairchild, C. R.; Peterson, R.
W.; Soejarto, D. D.; Farnsworth, N. R.; Kinghorn, A. D.; Wani, M. C.; Wall, M. E. Novel
Bioactive Clerodane Diterpenoids from the leaves and twig of Casearia sylvestris. J.
Nat. Prod. 2002, 65, 95-99.
13. Scheffer, J. J. C., 1997, Various methods for the isolation of essential oils,
Phytotherapy Research, 10: S6-S7.
14. Trease, G. E. and Evans, W. C. (1996) Volatile oils and resins, in Pharmacognosy,
14 ed., W B Saunders Company Ltd., London, pp. 255–292.
15. Trease and Evans (2009), Pharmacognosy,16th Ed, SAUNDERS, ELSVIER.
16. Terpenes as Green Solvents for Extraction of Oil from Microalgae Celine Dejoye
Tanzi, Molecules 2012, 17.
17. Sawamura, M. Citrus Essential Oil: Flavor and Fragrance; Wiley: Weinheim, Germany,
2010.
34
4
4.0 Alkaloids
4.1 Introduction to Alkaloids
The classes of the natural products that was first isolated from medicinal plants were
alkaloids. The alkaloids are the most diverse group of secondary metabolites found in living
organisms. Although alkaloids have been traditionally isolated from plants, an increasing
number are to be found in animals. For example : Frogs, insects, marine vertibrates etc.
Alkaloids exert striking pharmacological effects on human and other animals. Many
alkaloids have neuroactive properties and interact with the receptors at nerve endings.
Alkaloids have been used as medicines for hundreds of years and some are still prominent
drugs today. Many alkaloids serve as models for the synthesis of analogues with better
physiological properties.
Alkaloids are basic organic compounds, they contain one or more Nitrogen atom, usually in
a heterocyclic ring. Typical alkaloids are derived from plants. The alkaloids which are called as
“proto-alkaloids”or “amino-alkaloids” are applied to the compounds which have lost one or more
character of typical alkaloids, for example Ephedrin is an alkaloid which has amine group in the
side chain. Alkaloids show great variety in chemical structure, biochemical origin and
pharmaceutical action. Therefore, many system of classification are available. Example: alkaloids
may be grouped according to their plant sources, e.g. Aconitum, Amaryllidaceae, Cinchona,
Curare, Ergot, Opium, Senecio and Vinca. The best classification is the classification based
on the chemical structure. According to that alkaloids are categorized into two main
groups
(i). Non-heterocyclic or atypical alkaloids (proto-alkaloids or biological amine
Ex: Hordenine, Ephedrine etc.
(ii). Heterocyclic alkaloids or typical alkaloids
Heterocyclic alkaloids are grouped into 14 groups according to their the nature of
nitrogen containing ring.
35
Ex: Pyrrol and pyrrolidine group, Tropane alkaloids, pyridine and pipperidne group
etc.
This classification also reflects their biosynthetic origin. Alkaloids are originated in cells
from amino acids such as ornithine, lysine, phenylalanine, tyrosine and tryptophan etc.
4.1.1 Examples for some alkaloids, their structures and physiological effects.
Examples for the group of alkaloids which possess piperidine or pyrrolidine rings : the
hemlock plant (Conium maculatum) alkaloids; coniine (4.1) which causes paralysis of
nerve endings. The alkaloid piperine (4.2) and its cis,cis isomer chavicine are found in the
fruit of the pepper, Piper nigrum, and are responsible for the sharp taste of pepper. The
fruit of the betel palm, Areca catechu, produces a mild stimulant, arecoline (4.3). The coca
plant, Erythroxylon coca, is notorious for the production of cocaine (4.4), which has a
paralysing effect on sensory nerve endings and produces a sense of euphoria. The roots,
leaves and berries of a number of poisonous plants of the Solanaceae, including deadly
nightshade (Atropa belladona), henbane (Hyoscyamus niger) and thorn apple (Datura
stramoniurn), are rich source of therapeutically important tropane alkaloids. These plants
provided some of the hallucinogenic “sorcerer’s drugs’’ of the Middle Ages. Atropine (4.5)
and the related epoxide, scopolamine, are two examples with a powerful biological activity.
Atropine dilates the pupils of the eye and its derivatives are used in opthalmology. The
tobacco plant, Nicotiana tabacum, produces the toxic alkaloid. Nicotine (4.6), which is the
major neuroactive component of tobacco smoke. It is also used as an insecticide.
36
o
O
H O
OMe
N
N Me O N
H H
4.2 4.3
4.1
Me N Me N
CO2Me CO2Me OH
H N
O Ph O Ph Me
O O N
4.4 4.5 4.6

The benzylisoquinoline alkaloids have been found in various plants from the Annonaceae,
Lauraceae, Rhamnaceae, Ranunculaceae and Papaveraceae families. The best known source
of these alkaloids is the opium poppy, Papaver somniferum. The pain-relieving and narcotic
properties of opium were known in ancient times. Morphine is the crystalline alkaloid (4.7 )
from opium. Other examples in morphine series is papaverine (4.8), which has been used
as a muscle relaxant. Berberine (4.9) is a yellow pigment from Berberis and Mahonia
species which was used as a mild antibiotic in the treatment of sores. Quinine (4.10) was
isolated from Cinchona ledgerianu bark which is potent anti-malaria drug even for today.
Another big family of alkaloids from nature is the indole alkaloids These alkaloids include
poisons compounds such as strychnine (4.11) and other compounds in this series have
useful medicinal properties. Examples: Reserpine from Rauwolfia bark is used in the
treatment of mental disease and as anti-hypertensive drug. The purine carbon skeleton is
found in alkaloid, caffeine (4.12) is a stimulants that occur in coffee and tea. The plant
Cathranthus roseus is a medicinal plant and produce about 150 alkaloids: For examples,
Vinblastine (4.13), Vincristine, Vindesine, Vinorelbine etc. Many of these compounds are
currently used in the treatment of cancer (treatment of Hodgkin’s disease, leukaemia,
small-cell lung cancer etc.) as they show superior anticancer activity.
37
N CH
3 O
MeO
N
N O
MeO
OMe
O
HO OH
OMe OMe
OMe
4.7 4.8 4.9
O
N Me
Me N N
H H
HO N
N H
H O N N
R
O H O Me
N
4.11 4.12
4.10
4.13
4.2 Extraction of alkaloids
Alkaloid are basic organic compounds and generally they exist as the salts of organic acids
or phenolic compounds or tannin compounds in plants and some are present as free amine
form. Therefore the large scale extraction method is applied based on their alkaline nature
Extraction methods for alkaloids from plants vary with the scale, purpose of the operation,
and also with the raw material used. If an appreciable quantity (relatively large amount) of
38
alkaloid is required, general methods described below ( Extraction-A and Extraction –B)
are applied.
Extraction process - A
The powdered plant material is moistened with water and mixed with lime. This releases
free alkaloids (free amines) from their salts. Extraction of free alkaloids in the above
mixture is carried out by shaking the paste with organic solvents such as ether or
petroleum spirit. Extraction is repeated three times and combine organic layers is
concentrated under vaccum. The concentrated organic phase is transferred into separatory
funnel and it is then shaken with aqueous mineral acid (dil HCl) and allowed to be settled.
Alkaloid salts are now in the aqueous phase while many impurities remain behind in the
organic phase. The aqueous phase is transferred into a flask. The neutralization of aqueous
phase by adding NaOH may precipitate free alkaloids or if an oil appeared, again extraction
is carried out into ether and followed by evaporation yields free alkaloids mixture.
Extraction process – B
The powdered plant material is placed in a container and aqueous alcohol containing dilute
acid (acidic methanol) is added. The mixture is shaken well. Alkaloids salts are extracted
into aqueous alcohol in this stage. The solution is filtered and the filtrate is transferred into
separatory funnel and it is washed with petroleum ether in order to remove pigments and
other unwanted organic materials. The aqueous phase is transferred into a flask and it is
neutralized by adding excess sodium bicarbonate or ammonia. Then, free alkaloids may
precipitate or separate as an oil. If precipitated, it is separated by filtration, if not it is
extracted with organic solvents. After evaporation of organic solvent, free alkaloids are
obtained as a mixture.
Separation / purification of alkaloids from its mixture
39
The separation and purification of a mixture of alkaloids may sometimes be done by
fractional precipitation or by fractional crystallization of salts such as oxalates, tartrates or
picrates. Chromatographic methods are particularly suitable when there is a complex
mixture of alkaloids and if small quantities of alkaloids is sufficient.
For many research purposes chromatography specially column chromatography gives both
speedy and accurate results for the isolation of alkaloids from crude plant extracts.
Extraction of small quantities of alkaloids

When small amount of alkaloids are going to be isolated from plant or plant parts, it is
better to follow normal solvent extraction procedure described in chapter 2 and isolation
of compounds from the crude by carrying out chromatography.
The steps can be outlined as follows:

1. Maceration of plant in methanol
2. Fractionation of crude extract by solvent-solvent partitioning method. As alkaloids
are somewhat polar compounds, it will be extracted into polar solvent such as ethyl
acetate.
3. Carrying out chromatography (column chromatography) for the crude extract.
4.3 Extraction of volatile liquid alkaloids
Volatile liquid alkaloids such as nicotine is most conveniently isolated by distillation. An

methanolic extract prepared as above is made alkaline with caustic soda or sodium
carbonate and free alkaloid distilled off in steam. Nicotine is an important insecticide, and
large quantities of it is prepared from the parts of the tobacco plant which cannot be used
for tobacco manufacture.
4.4 Further reading
40
1. Akhtar, M. N., Atta-ur-Rahman, Choudhary, M. I., Sener, B., Erdogan, I., and Tsuda, Y.
(2003) New class of steroidal alkaloids from Fritillaria imperialis. Phytochemistry
63, 115–122.
2. Alkaloids, Biochemistry, Ecology and Medicinal applications edited by Margeret F.

Roberts and Michae Wink, premium Press, New York and London, 1998
3. Cseke , L.J. , et al. (ed.) ( 2006 ) Natural Products from Plants , 2nd edn , CRC Press ,
Boca Raton, USA .
4. Diaz, J. G., Ruiz, J. G., and de la Fuente, G. (2000) Alkaloids from Delphinium
staphisagria. J. Nat. Prod. 63, 1136–1139.
5. Kumarasamy, Y., Cox, P. J., Jaspars, M., Nahar, L., and Sarkar, S. D.
(2003) Isolation, structure elucidation and biological activity of two unique
alkaloids, hederacine A and B, from Glechoma hederaceae. Tetrahedron 59, 6403–
6407.
5. Jones , W.P. and Kingkorn , A.D. ( 2006 ) Extraction of plant secondary metabolites ,
in Natural Products Isolation , (eds S.D. Sarker , Z. Latif and A.I. Gray ), Methods in
Biotechnology , Vol. 20 , 2nd edn , Humana Press , Totowa, NJ, USA ,

8. Salim, A. A., Garson, M. J., and Craik, D. J. (2004) New Alkaloids from
41
Pandanus amaryllifolius. J. Nat. Prod. 67, 54–57.
9. Sarker, S. D., Armstrong, J. A., and Waterman, P. G. (1995) An alkaloid, coumarins

and a triterpene from Boronia algida. Phytochemistry 39, 801–804
42
5
5.0 Flavonoids
5.1 Introduction to Flavanoids

Flavonoids are the most important secondary metabolism products mainly polyphenoilc
compounds which are present in most of plants, concentrating in seeds, fruit skin or peel,
bark, and flowers. There are several types of flavanoids, which are broken down into six
subgroups: chalcones, flavones, flavonols, flavanones, anthocyanins, and isoflavonoids.
They all serve a variety of functions in plants, and are thought to be associated with a
number of health benefits for humans. Examples : Flavonoids purifies blood, strengthens
immune system, monitors cholesterol level, regulates blood pressure, suppresses acid
secretion, prevents thrombus, suppresses cytophy, act as antibacterial, antifungal,
antiallergic and anticancer, promotes metabolism and vasodilatory activity etc. A great
number of plant medicines contain flavonoids. Flavonoids have been shown in a number of
studies to be potent antioxidants, capable of scavenging hydroxyl radicals, superoxide
anions, and lipid peroxy radicals.
There are many different sources of flavonoids, including berries, tea, wine, beer, chocolate,
many vegetables, and most fruits.
5.1.1 Structure and examples for the flavonoids

The flavonoids are mainly polyphenolic compounds possessing 15 carbon atoms. The
structural components common to these molecules include two benzene rings on either
side of a 3-carbon ring. Multiple combinations of hydroxyl groups, sugars, oxygens, and
methyl groups attached to these structures create the various classes of flavonoids:
flavanols (5.1), flavanones, flavones, flavan-3-ols (catechins), anthocyanins, and
isoflavones.
43
Flavones Flavonols
Flavonones Anthocyanine
Isoflavonoids
5.2 Extraction of Flavonoids

Maceration followed by solvent-solvent partitioning method is used. Crude plant extract
obtained after maceration in Methanol or 70% aqueous methanol for 4-6 days followed by
evaporation, is washed with petroleum ether or hexane to remove any pigment or less
polar organic moities. Then the resultant crude is dissolved in water and flavonoids are
extracted into ethyl acetate and n-butanol successively.
44
The mixture of flavonoids obtained after evaporation of solvent under vaccum is subjected
to flash column chromatography or Sephadex column chromatography for purification.
5.3 Further reading
1. Dae-Young Lee et al (2007), Isolation of Flavonoids from the Fruits of Cornus kousa
Burg. J. Appl. Biol. Chem. 50(3), 144-147.
2. Cristiane P. Victório1,et al, (2009), Flavonoid extraction from Alpinia zerumbet

(Pers.) Burttet Smith leaves using different techniques and solvents Ecl. Quím., São
Paulo, 34(1): 19-24.
3. DRAGAN T. VELI^KOVI] et al (2007), Extraction of flavonoids from garden (Salvia

officinalis L.) and glutinous (Salvia glutinosa L.) sage by ultrasonic and classical
maceration DRAGAN T. VELI^KOVI] et al J. Serb. Chem. Soc. 72 (1) 73–80 .
4. Han JT, Bang MH, Chun OK, Kim DO, Lee CY, and Baek NI (2004) Flavonol glycosides
from the aerial parts of Aceriphyllum rossii and their antioxidant activities. Arch
Pharm Res. 27, 390-395.
6. Shafaghat et al (2008), Extraction and Determining of Chemical Structure of

Flavonoids in Tanacetum parthenium (L.) Schultz. Bip. from Iran A. J. Sci. I. A. U
(JSIAU), Vol 18, No. 68.
45
8. Yan X, Murphy BT, Hammond GB, Vinson JA, and Neto CC (2002) Antioxidant
activities and antitumor screening of extracts from cranberry Fruit (Vaccinium
macrocarpon). J Agric Food Chem 50, 5844-5849.
9. Zhang XF, Hung TM, Phuong PT, Ngoc TM, Min BS, Son KS, Seong YH, and Bae KH
(2006) Anti-inflammatory activity of flavonoids from Populus davidiana. Arch
Pharm Res 29, 1102-1108.
46
6
6.0 Steroids
6.1 Introduction to Steroids
Steroids are type of organic compounds that contain a characteristic arrangement of

four cycloalkane rings that are joined to each other. Examples : the dietary fat cholesterol,
the sex hormones and the anti-inflammatory drugs etc. The core of steroids is composed of
twenty carbon atoms bonded together that take the form of four fused rings:
three cyclohexane rings (designated as rings A, B, and C in the figure to the right) and
one cyclopentane ring (the D ring). The steroids vary by the functional groups attached to
this four-ring core and by the oxidation state of the rings. Sterols are special forms of
steroids, with a hydroxyl group at position-3 and a skeleton derived from cholestane
CH3
H3 C CH CH2 CH2 CH2 CH
CH3 CH3
CH3
HO
Cholestan Cholesterol
Plants produce a wide array of steroid molecules which can be divided into three groups,
based on their biological relevance (1) Substances which have physiological roles in the
plant itself, as hormones or pheromones. Eg: brassinosteroids are growth-promoting. (2)
Allelochemical substances related to animal hormones: ecdysteroids are analogues of
insect moulting hormones, whereas androgens, oestrogens, corticosteroids and
cholecalciferols are related to vertebrate hormones. (3) Plant-specific allelochemical
substances, which often display protective (repellent, antifeedant, toxic) actions towards
47
phytophagous animals or parasitic substances fungi: these are e.g., cucurbitacins and
cardenolides
Phytosterols, which encompass plant sterols and stanols, are steroid compounds similar
to cholesterol which occur in plants and vary only in carbon side chains and/or presence or
absence of a double bond. Stanols are saturated sterols, having no double bonds in the
sterol ring structure. More than 200 sterols and related compounds have been
identified. Free phytosterols extracted from plants are insoluble in water, relatively
insoluble in oil, and soluble in alcohols. The seed of soya bean contain appreciable
quantities of phytosterols called, stigmasterol and sitosterol. Some phytosterols are found
in cotton-seed oil, sugarcane wax etc.
Plants produce steroidal alkaloids, steroidal glycosides and steroidal saponins that make
important physiological effects on humans. Most of drugs contain steroidal alkaloids,
steroidal glycosides and steroidal saponins.
Ex:
Veratrum alkaloids, Kurchi alkaloids (steroidal alkaloids)
Cardiac glycosides (steroidal glycosides)
Diosgenin ( steroidal saponins)
6.2 Extraction of steroids
Steroids are polar substances. The powdered plant is first extracted with petroleum ether
(60-80ºC) using Soxhlet apparatus to remove pigments matters, the marc is dried and
extracted with methanol using Soxhlet apparatus for four hours. The methanol is
evaporated and the extract is dried. It is partitioned between water and chloroform. The
chloroform is evaporated and the extract is dried. Dried crude extract which contain
48
steroids is subjected to Silica gel chromatography and Sephadex chromatography to obtain
steroids.
6.3 Further reading

1. Atta-ur-Rahman, R.A. Ali, M.I. Choudhary, B. Sener and S. Turkoz (1992), New Steroidal
Alkaloids from Rhizomes of Veratrum album, J. Nat. Prod., 55 (5), 565-70.
2. Atta-ur-Rahaman and M.I Choudhary (1993), Metods in Plant biochemistry 9ed

P.G.Waterman) Vol. 8, academic press, London.
3. Bhutani K K, et al (1998), Three new alkaloids from the bark of Holarrhena

antidysenterica, Phytochemistry,; 27: 925-928.
4. Dinan, L., Harmatha, J., and Lafont, R. (2001) Chromatographic procedures

for the isolation of plant steroids. J. Chromatogr. A 935, 105–123.
5. M.S. Al-Said et al, (1996), New Sulfides from Ferula rutabensis, Int. J. Pharmacognosy,
Vol. 34, No.3, 189-193.
6. I.A. Abdulmalik et al, Isolation of Steroids from Acetone Extract of Ficus iteophylla Br. J.
Pharmacol. Toxicol., 2(5): 270-272, 2011.
9. XU, S.H. and L.M. Zeng, 2000. The Identification of Two New Sterols from Marine
Organism. Chinese Chem. Let., 11(6): 531-534.
49
10. Yun-Song, W., Y.S. Jing-Hua, Z. Hong-Bin and L. Liang, 2006. New Cytotoxic Steroid from
Stachyurus himalaicus. Molecule, 11: 536-542.
50
7.
7.0 Saponin
7.1 Introduction to Saponin
Plant containing saponins have long been used in many years of the world for their
detergent properties. Example : In Europe plant Saponaria officinalis etc. These plants
containing steroidal saponins known as saponins. These are responsible for producing
frothing.They also have heamolytic properties, when saponin injected into blood stream,
makes highly toxic. Diosgenin, a steroidal saponin from the Mexican yam, and hecogenin,
from sisal, are used as starting materials for the partial synthesis of the steroidal hormones.
Steroidal saponins have great pharmaceutical value due to their relationships to sex
hormones and other important compounds etc.
HO
Diosgenin
Some plants contain triterpenoid saponins.
7.2 Extraction of Saponins
Saponins have higher molecular weight and higher polarity. Therefore extraction needs to
use very polar solvents and isolation of saponins in very pure stage is somewhat difficult.
The scheme 1.0 shows a procedure for the extraction of saponins (ie. steroidal or triterpene
glycosides). First, the plant material is defatted with n-hexane, and then it is extracted with
MeOH in a Soxhlet apparatus about four hours. The MeOH extract is concentrated under
vacuum, and suspended in de-ionized water. Then it is partitioned with n-butanol. Diethyl
51
ether is added to the butanol partition (fraction) to precipitate the saponins.
Chromatographic method such as Sephadex column is used to separate individual saponin
from the mixture.
Ground plant material
Defat with hexane or pet.ether
Marc hexane / pet.ether

fraction
Extract with methanol
(or ethnol/water)
Methanol extract Marc

(discard)
i. Concentrate
ii. Dissolve in water
iii. Partition with n-butanol
n-Butanol Aqueous
fraction fraction
Add diethyl ether
to precipitate
saponin
Crude saponin
Scheme 1.0 : Separation of saponins from the plant material
52
73 Further reading
1. Klyne, W. (1957) The Chemistry of the Steroids. Wiley, New York.Natural products
isolation. – 2nd ed. / edited by Satyajit D. Sarker, Zahid Latif, Alexander I. Gray.2006,
Humana Press Inc.
3. Ding, Y. et al (1993), Two new steroidal saponins from dried fermented residues of
leaf-juices of Agave sisalana forma Dong, Chem. Pharm. Bull. 1993;41(3):557-560.
53
8
Sublimation, Sonication, Super Critical Fluid Extraction and Microwave assisted

extraction of natural products
8.1 Extraction and isolation of Natural Product by Sublimation

Sublimation is the process that the transition of a solid phase substance to its vapor phase
without passing through the liquid phase. Sublimation can be used for the extraction of
some natural products from plants. For example: extraction of caffeine from tea leaves.
Sublimation is also used for purification of compounds from its crude extracts. Ex: isolation
of caffeine from crude extract of tea leaves.
8.2 Applying of Ultrasound method (Sonication) in Natural product extraction
This is a novel technique used in natural product extraction. The procedure involves the
use of ultrasound with frequencies ranging from 20 kHz to 2000 kHz in solvent extraction
process. This increases the permeability of cell walls and produces cavitations. Although
the process is useful in some cases, Ex: extraction of rauwolfia root, and extraction of bio
active compounds form sage, its large-scale application is limited due to the higher costs.
The disadvantage of the procedure is occasional but known that harmful effect of
ultrasound energy (more than 20 kHz) on the active constituents of medicinal plants
through formation of free radicals and consequently undesirable changes in the drug
molecules. Ultrasound assisted extraction is effective than classical maceration but less
efficient than Soxhlet extraction.
Procedure for Ultra sound extraction

The dried plants and the extracting solvent are placed in an Erlenmayer flask about the
ratio of plant material and extracting solvent is 1:10 w/V. A series of flaks with different
solvent are prepared and immersed into an ultrasonic cleaning bath operating at 40 kHz
54
frequency and sonicated at 40°C till maximum concentration of extractable substances in
the liquid extracts is achieved (about 20 min). Then the residue is filtered off and each
extract is concentrated under reduced pressure to obtain crude extract of the plant
material.

1. Mason, T.; Chemat, F.; Vinatoru, M. (2011),The extraction of natural products using
ultrasound and microwaves. Curr. Org. Chem., 15, 237–247.
2. Sargenti, S. R. and Vichnewski, W. (2000), Sonication and liquid chromatography as

a rapid technique for extraction and fractionation of plant material, Phytochemistry
Annals, 11: 69-73.
8.3 Super critical Fluid extraction of Natural product
Supercritical fluid extraction (SFE) is an alternative extraction method with the goals of
using less amount of organic solvents. Generally, liquid CO2 is used in SFE. The factors to
consider in this method are temperature, pressure, sample volume, analyte collection,
modifier (co-solvent) addition, flow and pressure control and restrictors. Generally,
cylindrical extraction vessels are used for SFE.
There are many advantages for the use of CO2 as the extracting fluid. In addition to its
favorable physical properties, carbon dioxide is inexpensive, safe and abundant. But while
carbon dioxide is the preferred fluid for SFE, it possesses several polarity limitations.
Solvent polarity is important when extracting polar solutes. Organic solvents are frequently
added to the carbon dioxide extracting fluid to alleviate the polarity limitations. Instead of
carbon dioxide, argon is being used in SEF because it is inexpensive and more inert. The
component recovery rates generally increase with increasing pressure or temperature: the
highest recovery rates in case of argon are obtained at 500 atm and 150° C.
55
The SEF procedure possesses important advantages:
i) The extraction of constituents at low temperature, which strictly avoids damage to
bioactive constituents from heat and some organic solvents.
ii) No solvent residues remains
iii) Environmentally friendly extraction procedure.
SFE finds extensive application in the extraction of pesticides, environmental samples,

foods and fragrances, essential oils, polymers and other natural products. The major
restriction in the commercial application of the extraction process is its higher capital
investment.
Example for application of SEF in isolation of drug:

Taxol, one of the most effective anticancer natural product drugs, is a complex diterpene
isolated from the Pacific yew tree (Taxus brevifolia). The SFE protocol for the extraction of
Taxol from the bark was introduced recently. About 50% of the Taxol present in the bark
was selectively extracted using a CO2–EtOH mixture in contrast to 25% extraction with
supercritical CO2 alone.
1. Ashraf-Khorassani, M. and Taylor, L. T. (2004) Sequential fractionation of grape

seeds into oils, polyphenols, and procyanidins via a single system employing CO2
based fluids J. Agric. Food Chem. 52, 2440–2444.
2. Bevan, C. D. and Marshall, P. S. (1994) The use of supercritical fluids in the isolation
of natural products Nat. Prod. Rep. 11, 451–466.
3. Bruno, T. J. and Ely, J. F. (1991) Supercritical Fluid Technology: Reviews in Modern

Theory and Application. CRC Press, Boca Raton, FL.
56
4. Ferreira, S. R. S. and Meireles, M. A. A. (2002) Modelling the supercritical fluid
extraction of black pepper (Piper nigrum L) essential oil. J. Food Eng. 54, 263–269.
5. Jennings, D. W., Deutsch, H. M., Zalkow, L. H., and Teja, A. S. (1992) Supercritical
extraction of taxol from the bark of Taxus brevifolia J. Supercrit. Fluids 5, 1–6.
6. Herrero, M.; Cifuentes, A.; Ibañez, E. (2006), Sub- and supercritical fluid extraction
of functional ingredients from different natural sources: Plants, food-by-products,
algae an microalgae: A review. Food Chem. 98, 136–148.
7. Hamburger, M., Baumann, D., and Adler, S. (2004) Supercritical carbondioxide

extraction of selected medicinal plants effects of high pressure and added ethanol on
yield of extracted substances. Phytochem. Anal. 15, 46–54.
8. Louli, V., Folas, G., Voutsas, E., and Magoulas, K. (2004) Extraction of parsley seed oil
by supercritical CO2 J. Supercrit. Fluids 30, 163–174.
9. Martino, K. G. and Guyer, D. (2004) Supercritical fluid extraction of quercetin from

onion skins J. Food Process Eng. 27, 17–28.
11. Salgin, U., Calimli, A., and Uysal, B. Z. (2004) Supercritical fluid extraction of jojoba
oil J. Am. Oil Chem. Soc. 81, 293–296.
12. Venkat, E. and Kothandaraman, S. (1998) Supercritical fluid methods, in Natural

Products Isolation. 1st ed. (Cannell, R. J. P., ed.), Humana Press, New Jersey.
57
8.4 Microwave Assisted Extraction (MAE) of Natural Products
The development of microwave assisted extractions was first reported by Ganzler and co-
workers
In microwave assisted extraction, the advantage of microwave heating is based on
disruption of weak hydrogen bonds promoted by the dipole rotation of the molecules. A
higher viscosity of the medium lowers this mechanism by affecting molecular rotation
Furthermore, this makes solvent penetration into the matrix and thus facilitates the
solvation of the analyte. The effect of microwave energy is strongly dependent on the
nature of both the solvent and the solid matrix. Solvents generally is used in wide range of
polarities, from heptane to water. Most of the time, the chosen solvent possesses a high
dielectric constant and strongly absorbs microwave energy, however, the extracting
selectivity and the ability of the medium to interact with microwaves can be modulated by
using mixtures of solvents. This method has been applied to extract many types of natural
products such as essential oils and steroids etc.
There are two types of approaches possible in microwave extraction. The most common
procedure involves extraction in a closed vessel under controlled pressure and
temperature, whilst an alternative approach uses an open extracting vessel under
atmospheric pressure. It must be strongly stressed that using a domestic microwave oven
for laboratory purposes should not be considered. Application of microwave energy to
highly flammable organic solvents may cause serious hazards. Furthermore,
reproducibility may be poor with a domestic device because of the lack of homogeneity of
the microwave field. It is therefore seriously recommended that only equipment approved
for MAE be used.
58
1. Abert Vian, M.; Fernandez, X.; Visinoni, F.; Chemat, F. Microwave hydro-diffusion and
gravity: A new device for extraction essential oils. J. Chromatogr. A 2008, 1190, 14–
17.
2. Be´atrice Kaufmann and Philippe Christen (2002), Recent Extraction Techniques for
Natural Products: Microwave-assisted Extraction and Pressurised Solvent
Extraction., Phytochem. Anal. 13: 105–113.
3. Craveiro, A. A., Matos, F. J. A., Alenkar, J. W. and Plumel, M. M., (1989), Microwave for
extraction of essential oil, Flavour and Fragrance Journal, 4: 43-44.
4. Ganzler K. et al., (1990), Effective sample preparation method for extracting of

biologically active compounds from different matrices using microwave techniques,
J.Chromatogr., 520, 257-262.
5. Kosar, M., Özek, T., Göger, F., Kürkcüoglu, M. and Baser, K. H. C., 2005, Comparison of
microwave-assisted hydrodistillation and hydrodistillation methods for the analysis
of volatile secondary metabolites, Pharmaceutical Biology, 43(6): 491-495
6. Kaufmann, B. and Christen, P., (2002), Recent extraction techniques for natural
products: microwave-assisted extraction and pressurized solvent extraction,
Phytochemical Analysis, 13(2): 105-113.
7. Longares-Patron, A. and Canizares-Macias, M. P., 2006, Focused microwaves-

assisted extraction and simultaneous spectrophotometric determination of vanillin
and p-hydroxybenzaldehyde, Talanta, 69: 882-887
8. V.F. P´eres et al. (2006), Comparison of soxhlet, ultrasound-assisted and pressurized
liquid extraction of terpenes, fatty acids and Vitamin E from Piper gaudichaudianum
Kunth J. Chromatogr. A 1105 115–118.
59
9
9.0 Fractionation of crude plant extracts
A crude natural product extract is an extremely complicated mixture of several compounds

with various chemical and physical properties. The fundamental strategy for separating of
these compounds is based on their physical and chemical properties. The process that
divides the crude mixture of compounds into different chemical classes is defined as
fractionation. Different techniques are used for fractionation of natural product extrcats
9.1 Fractionating techniques
9.1.1 Solvent – solvent partitioning method

This can be successfully applied in the separation (fractionating) of different classes of
natural products from the initial extract. The separation technique using solvent
partitioning involves primarily the use of two immiscible solvents in a separating funnel,
and the compounds are distributed between two solvents according to their different
partition coefficients. This method is relatively easy to perform and highly effective as the
first step of the fairly large-scale separation of compounds from crude natural product
extracts. Solvents with different polarities can be applied in order to separate different
polar compounds or particular compounds from the initial plant extract.
However, in some cases, from the literature of the related genera and families, it is possible
to predict the types of compound classes that might be present in a particular extract. This
tentative prediction on the possible identity of the classes of compounds may help to
choose suitable partitioning methods. For example, for separation of phenolics compounds,
saponin compounds, alkaloids etc.
60
The solvent partitioning method is explained in detail under the solvent extraction
procedures.
9.1.2 Fractionation based on acid-base nature of the compounds

Organic compounds are classified into acidic, basic and neutral compounds. For example:
carboxylic acids, phenolic, amines and amides or aldehyde, ketones etc.. The compounds in
the initial solvent extract can be divided into acidic, basic and neutral fractions based on
their acidic basic or neutral nature.
The procedure for fractionation based on acid-base nature

A typical fractionation procedure on acid-base nature is shown in Scheme 2.
A plant extract in an organic solvent (ex: ethyl acetate) is shaken with an inorganic base
such as aqueous sodium hydrogen carbonate to separate the carboxylic acids as their
water-soluble sodium salts. The more weakly acidic phenolic compounds may only be
extracted with strong base solution such as sodium hydroxide solution. Extraction of the
original solution with an acid such as dilute hydrochloric acid will remove the bases such as
the alkaloids, amines as their salts. The neutral compounds remain behind in the organic
phase. The acids and the phenols may be recovered from the aqueous solution of their
sodium salts by treatment with dilute hydrochloric acid and re-extraction with an organic
solvent, and the bases may be recovered by treatment of their salts with ammonia and re-
extraction with an organic solvent.
61
Plant material
i. maceration in methanol
ii. Evaporation
Methanol extract
Shaken with aqueous NaHCO3

solution
Organic acidic compounds Other compounds (basic, neutral

as water soluble salts and phenolic
Treated with Aqu. HCl

Treated with aq. NaOH
solution
Organic acidic compounds
are extracted to ethyl acetate
Phenolic compounds Basic and neutral

as sodium sals in water Organic compounds
i. Neutralization as residue
with HCl
Phenolic c ii. Extraction with
ethyl acetate Treated with aq. HCl
ompounds
Basic organic compounds Neutral Organic

as salts in water compounds
i.Acedification with Extraction

dil HCl with CHCl3
ii. Extraction
with ethyla acetate
Neutral Organic
compounds
Basic Organic compounds
Scheme 2: Separation of the extract into

acidic, basic, phenolic and neutral nature
naturefractions
62
Fractionating techniques are important to prepare tannin free plant extracts and to remove
unwanted matters such as pigments, protein etc. from the plant extracts which make easy
to proceed with isolating of desired compounds.
9.1.3 Preparation of tannin free plant extract using fractionation
Presence of tannin may disturb the isolation of desired compounds. Fractionating of the
crude methanolic extract can be done according to the procedure shown in scheme 3 to
remove tannin from crude extract.
Ground plant material

i. maceration with methanol
ii concentration under vaccum
Methanolic extract
i. suspension with 90% methanol
ii. partitioning with hexane or pet.ether
Methanol fraction hexane fraction

i. concentration
ii. suspension with water
extraction with CHCl3
chlorofoam fraction Aqueous extratct

i. Washing with 1% NaCl in water
to remove tannin
ii. evaporation of chlorofoam
Organic matters free NaCl fraction

of tannin (waste)
Scheme 3 : Removing of tannin from crude extracts by fractionation
63
9.1.4 Preparation of pigment free plant extracts by fractionation
Presence of plant pigment such as Chlorophyll. Carotinoids etc. disturb the isolation of
target compounds from the extracts and also may interfere with chemical or biological
assays. Therefore, it is good to remove pigments at the beginning of extraction.
Carotinoids can be removed by filtering the initial extract over silver nitrate-impregnated
alumina. Chlorophyll can be removed by Solvent–solvent partition between n-hexane or
petroleum ether and 90% MeOH.
9.1.5 Bioassay guided fractionation and isolation of natural products
Introduction to Bioassay
Bioassay is the assay used for the testing of biological activity of natural product extracts or
pure compounds. In bioassay methods, in vitro or in vivo studies are done using animals or
microorganisms or cell lines or enzymes in order to determine the active plant extract or
active compounds against the particular disease conditions or infection. Therefore,
bioassays detect the compounds with particular biological activities.
Bioassay-guided fractionation and isolation of natural products

Testing of particular biological activity of different solvent extracts of the plant material
and identification of bioactive extract based on their response to that biological activity is
known as bioassays guided fractionation of natural product extracts. The extract which
gives positive response to particular biological activity is known as active fraction or active
extract. With continuing the bioassay process using the separated compounds (using
chromatography) from the bioactive extract, active compounds from the particular source
(plant) can be identified and isolated. This process is practiced in natural product based
drug discovery. The recommended bioassay methods for many diseases conditions have
64
been reported. It is necessary to follow recommended methods if it is not a new situation.
The general procedure bioassay guided fractionation is descibed below;
At first, biological agent (animal or cell lines or enzymes) is needed to be selected for the
particular studies according to the recommended procedure.
Note: If animals (eg: mouse) are selected for particular bioassays, animals should be
maintained under the attention of skilled person and the guidance and support should be
taken from expertise. The animals with same age and same growing conditions should be
selected for the bioassays.
Particular animals are induced with a particular disease conditions and some incubation
period should be given to develop the disease or pathological state. Then, the affected
animals are treated with plant extracts (orally or intravenously) with known dosages and
regular intervals for defined days. After that, the animals are tested for its recovering
ability using defined parameters (without being scarified or not). The testing period is
considered according to the literature method. If particular plant extract is active against
induced disease condition, that extract is considered as an active extract. Then the active
extract is subjected to column chromatography or other suitable separation technique in
order to isolate the compounds present in. Then, the same bioassay is repeated with
isolated compounds. This will identify the active (leading) compounds present in the plant
extract against particular disease. This method is summarized in scheme 4.
65
Plant extracts in diferent solvents
Bioassays (in vitro or in vivo)
Extracts with negative Extract with positive

response to bioassays resposee to bioassays
( active extract)
Separation of compounds
in active extract using
chromatography
Compound with +ve response Compound with -ve response

to bioassays to bioassays
(potential compounds / drug)
Characterization
using spectroscophy
Drug
Scheme 4 ; Bioassay guided fractionation and isolation of natural product
Note: If a natural product, particularly a pure compound isolated from crude extract is
going to be tested for biological activity, it is important to know at least the degree of purity
and, preferably, the nature of the impurities present in. It is always possible that the
impurities may give to all or part of the biological activities in question. If a compound is to
66
be used to collect pharmacological or pharmacokmetic data, it is usually important that the
compound is extremely pure stage (generally >99% pure).

1. Farnsworth, N. R. (1966) Biological and phytochemical screening of plants. J. Pharm.
Sci. 55, 225–276.
2. Clark, A. M. (1996), Natural Products as a Resource for New Drugs. Pharm. Res., 13,
1133-1141
3. Donadio, S.,Monciardini, P., Alduina, R. et al. (2002) Microbial technologies

for the discovery of novel bioactive metabolites. J. Biotechnol. 99, 187–198.
4. Kumarasamy, Y., Cox, P. J., Jaspars, M., Nahar, L., and Sarker, S. D. (2003) Bioactivity
of secoiridoid glycosides from Centaurium erythraea. Phytomedicine 10, 344–347
5. Houghton, P. J. and Raman, A. (1998) Laboratory Handbook for the Fractionation of

Natural Extracts, Chapman & Hall, London, UK.
6. Houghton, P. J. Use of small scale Bioassays in the Discovery of novel drugs from
Natural Sources. Phytotherapy Research 2000, 14, 419-423
7. Mez-Mangold, L. (1971) A History of Drugs. F. Hoffmann-La Roche, Basle,

Switzerland
67
9. Newman, D. J.; Cragg, G. M.; Snader, K. M.( 2000) The Influence of Natural Products
upon Drug Discovery. Nat. Prod. Rep., 17, 215-23.
10. Perdue, R. E., Jr. (1976) Procurement of plant materials for antitumor
screening. Cancer Treat. Rep. 60, 987–998.
11. Phillipson, J. D. Phytochemistry and Medicinal Plants. Phytochemistry 2001, 56,

237-243.
12. Ramsey, E. D. (1998) Analytical Supercritical Fluid Extraction Techniques.

Kluwer Academic Publishers, Dordrecht
13 Statz, D. and Coon, F. B. (1976) Preparation of plant extracts for antitumor

screening. Cancer Treat. Rep. 60, 999–1005.
14 Trease and Evans (2009), Pharmacognosy,16th Ed, SAUNDERS, ELSVIER.
15. Tulp, M. Th. M. The Use of Receptor Binding, a Very Specific, High Capacity Screening
Method, in the Identification of Biologically Active Components from Natural
Sources. In Bioassay Methods in Natural Product Research and Drug Development;
Bohlin, L., Bruhn, J. G., Eds.; Proceedings of the Phytochemical Society of Europe;
Kluwer Academic Publishers: Boston, 1999; 53-65.
16. Vlietenck, A. J. Screening Methods for Detection and Evaluation of Biological

Activities of Plant Preparations. In Bioassay Methods in Natural Product Research and
Drug Development; Bohlin, L., Bruhn, J. G., Eds.; Proceedings of the Phytochemical
Society of Europe; Kluwer Academic Publishers: Boston, 1999; 37-52.
17. Viletinck, A. J. and Apers, S. (2001) Biological screening methods in the search for
pharmacologically active natural products, in Bioactive Compounds from Natural
Sources (Tringali, C., ed.), Taylor and Francis, New York, USA, pp. 1–30.
68
18. Vogel, A. I., Furniss, B. S., Hannaford, A. J., Rogers, V., Smith, P. W. G.,
and Tatchell, A. R. (1978) Vogel’s Textbook of Practical Organic Chemistry.
4 ed., Longman, New York.
19. Wall,M. E., Taylor, H., Ambrosio, L., and Davis, K. (1969) Plant antitumor
agents. III. A convenient separation of tannins from other plant constituents.
J. Pharm. Sci. 58, 839–841.
69
10
10.0 Isolation of compounds from natural products extracts OR Purification of

natural products extracts
Natural product extracts are in most cases highly complex and comprise mixtures of
neutral, acidic, basic, lipophilic, hydrophilic, or amphiphilic compounds. For the testing of
biologically activity specially in drug discovery and for the structure elucidation of natural
potential compounds, very small quantity of the substance in extreme pure stage is
necessary. Therefore, isolation of compounds from their extracts in pure stage is the prime
important phenomenon in natural product research. There are many techniques applied
for isolation / purification depending on the nature of the extract and the compounds
desired.
Examples for isolation / purification techniques ;

Chromatography
Distillation
Crystallization
Sublimation
10.1 Use of chromatography for the purification of natural product extracts
Chromatography is the commonly used technique for the analysis and for the purification
of crude extracts or impure substances in natural product research as well as in organic
synthesis. This is a physical method.
Principle of chromatography : Distribution of components in a mixture based on their

relative affinities to the mobile phase and stationary phase. Stationary phase is the non-
moving phase and the mobile phase is the moving phase. The chemical entities used in
these two phases are different in their polarities. As plant extract contains compounds with
70
wide polarity range, their affinities is different to mobile and stationary phase, hence the
components can be separated. Further, this technique can be used for the analysis of a
extracts or crude mixture in order to find the number of components and their relative
concentration, finally for the separation of individual compound from the mixture.
Examples for the chromatographic methods used in Natural product analysis and
isolation process.
Many chromatographic techniques are available.

1. Thin Layer Chromatography (TLC)
2. Column Chromatography
3. High Performance Liquid Chromatography (HPLC)
4. Gas-liquid chromatography etc.
All the above chromatographic method follow the same principle as described. In addition
to above chromatographic methods, some other methods are available. Ex: Gel permeation
chromatography, Size exclusion chromatography and Ion exchange chromatography. These
methods involve other mechanisms and not much applied in natural products isolation
process.
10.1.1 Thin Layer Chromatography in Natural product research

Thin-layer chromatography (TLC) is the most commonly used planar chromatographic
method in natural product research. This is the easiest and cheapest technique and can be
applied in the analysis, isolation and setting the parameters for column chromatography in
natural products research. Usually, silica or alumina (more polar) is used as the stationary
71
phase and organic solvents (less polar) are used as the mobile phase. This situation is
categorized as normal phase chromatography. In contrast to this, reverse phase TLC is
available, in which stationary phase is alkyl bonded silica or alumina (less polar) and
mobile phase is polar solvent like water, alcohol etc. Usually normal phase
chromatography is applied as it is less expensive.
Thin Layer Chromatography is two types:

Analytical Thin Layer Chromatography (Analytical TLC)
Preparative Thin Layer Chromatography (Preparative TLC)
10.1.1.1 Analytical TLC

Analytical TLC is used to analysis the plant extract in order to get an idea on the number of
components present in the mixture and to set the conditions for purification process by
column chromatography etc. This is easy and cheapest method to be performed in the
laboratory.
The procedure for analytical TLC
Analytical TLC plates are used, which are commercially available from suppliers such as
Merck. The commonest analytical silica gel plate is the 6 cm X 2.5 cm, plastic or aluminum
having a 0.2 mm thickness of silica sorbent (stationary phase) with florescent material, F254
or the plates can be easily prepared in the laboratory using silica gel; F254 and with
microscopic slides. The plate is spotted with the dilute solution of plant extract on a line
drawn using pencil about 1cm up from one edge (Figure 1). Then, the plate is placed into a
tank (TLC tank or closed glass container) with sufficient amount of suitable mobile phase
(solvent or mixture of solvent) just to wet the lower edge of the plate/sorbent but not
adequate to wet the part of the plate where the spots were applied. The solvent front then
migrates up the plate through the sorbent by capillary action, and this process is known as
development (Figure 6).
72
Figure 6 : Carrying out analytical TLC
After development, separated spots can be seen by exposing them to UV lamp or by

spraying the reagents such as phosphomolybdic acid or dipping in I2 bath. The spots are
distinguished using Rf value which is the ratio between the distance to the spot and the
distance to the solvent front from the spotted line.
Distance travelled by the analyte

Rf =
Distance travelled by the solvent front
The ideal solvent system for TLC is one that moves the compound of interest in the mixture
to the Rf of 0.15-0.35.
Selection of solvents for mobile phase in chromatography

The solvent can be selected depending on polarity of the mixture. As different polar
solvents are available in the laboratory, single solvent or solvent system prepared by
mixing of two or three solvents can be applied.
73
Polarity order of common solvents:
Hexane
Toluene
Diethyl ether
Dichloromethane
Acetone
Increasing polarity
Tetrahydrofuran
Ethyl acetate
Acetonitrile
Isopropanol
Ethanol
Methanol
Water
10.1.1.12 Preparative TLC (PTLC)
This is the techniques used in small-scale isolation of natural products from its extract.
Many natural products are isolated by conventional preparative TLC (PTLC) in small scale.
PTLC is still a useful isolation method in many cases because of its simplicity, cost, speed,
and ability to separate compounds in the 1 mg –20 mg range.
The steps for PTLC
1. 20 x 20 cm sized silica plates are used to run the preparative TLC.
74
2. A suitable mobile phase (eluent) for the PTLC is selected by carrying out analytical
TLC for the particular extract.
3. A pencil line is drawn about 1 cm height from one edge of the PTLC plate and a very
concentrated solution of the mixture is spotted on that line as a line (not as the
spots) using a capillary tube. (Note: a mixture should be dissolved in minimum
amount of mobile phase or other polar solvent for spotting)
4. The plate is then developed by inserting it in the container having suitable mobile
phase.
5. After running the mobile phase (about 2/3 of the plate), it is taken out from the
developing tank and separated components are visualized by exposing the plate to
UV lamp (Note: no other visualizing method is suitable as the purpose is to recover
the compounds without any chemical changes).
6. The separated components can be seen as bands and two margin of the each band is
marked using a pencil.
7. The separated bands are scratched out along with silica gel and placed them in the
separate flasks (separate compound bands in separate flasks).
8. Some amount of methanol is added to each flask and resultant solutions are shaken
well. Finally, each solution is filtered off to remove silica gel and pure compound is
obtained as the methanolic extract.
9. After evaporation methanol under vaccum , the solvent free pure compound is isolated.
10.1.2 Use of Column chromatography for isolation of compounds in plant extracts
Column chromatography is the most effective technique used in separation of crude plant
extracts into its components in pure form. This is a preparative chromatographic method
and the stationary phase (silica gel) is packed in a column and then the mobile phase
(eluent) is passed through the column after loading the sample (extract) on the top of the
stationary phase. The mobile phase carries the compounds present in the mixture at
75
different rate based on their affinities to the stationary and mobile phase. Separated
compounds can be collected along with the mobile phase.
Column chromatography can be performed in two ways;

Normal column chromatography and flash column chromatography.
Flash column chromatography, also known as medium pressure liquid chromatography

(MPLC), is a rapid form of column chromatography that uses packed columns, through
which a solvent is passed at a high flow rate, ie: external pressure is applied to the top of
the column. This is convenient method for the isolation of compounds in plant extracts as
plant extract is a complex mixture of many compounds.
In normal column chromatography, column is run without applying external pressure.
Two types of solvent systems are used in column chromatography:

a). Isocratic system
b). Gradient system
Isocratic system : It means “same solvent strength”. A single-strength solvent or solvent

mixture is used throughout the column running.
Gradient system : It means “ different solvent composition during the course of elution”.
The polarity of the mobile phase increases gradually during the elution by using different
solvent or mixing different ratios of different polar solvents. As plant extracts contain
mixture of complex compounds and they differ greatly in polarities, natural product
chemists usually use gradient solvent system for isolation of compounds.
76
Procedure for the column chromatography
• The columns are available varying the diameter and the length.
• The bottom of the column should have either a sintered glass frit or a plug of glass wool
to support the stationary phase .
• As shown in Figure 7 , the column is packed with the stationary phase (silica) using first
developing solvent, least polar solvent (Note: 50 g of silica is good for 1g of the crude
extract). While vibrating the column (or tapping), carefully pour the stationary phase in
and open the outlet. Allow the stationary phase to settle while the liquid is flowing.
Continue adding stationary phase until the bed is complete.
• Fill the column with the first developing solvent, taking care to ensure that the bed is
not disturbed.
• Open the column outlet to allow the solvent to flow naturally under gravity.
• Continue the equilibration stage until the stationary phase bed has a uniform
appearance, i.e., no visible dry areas or air pockets.
• Reduce the solvent level to just above the stationary phase. Stop the flow by closing the
outlet valve.
Note: After the packing, the column should not be allowed to dry, therefore the
solvent layer should be maintained above the upper limit of the stationary phase all
the time.
• It is good to add layer of white sand (about 4 nm thickness) to the top of the stationary
phase, which supports to keep uniform of the upper limit of the stationary phase.
• Next is the loading of the sample (analyte) to the column.

There are two popular approaches of sample loading to the column. Those are wet
loading and dry loading.
77
10.1.2.1 Wet Loading
Wet loading is the placing of the liquid sample directly onto the top of the column using a
paster pippet, and allow it to percolate through the top of the sorbent bed. If the sample/
plant extract is a solid material it should be dissolved in minimum amount of less polar
solvent (If it is not soluble in low polar solvent better to use small portion of high polar
solvent).
Open the outlet valve, and allow the sample to flow into the bed. When the sample has been
adsorbed completely, the glass column sides can be washed carefully with a little amount of
the mobile phase and then allowed to flow again. Care must be taken to ensure the bed is
not disturbed and is not allowed to become dry. Therefore, it is always necessary to
maintain solvent layer above the sorbent limit after loading as well.
10.1.2.2 Dry Loading

Dry loading is the method of choice for placing plant extracts or the reaction mixtures
consisting of polar substances onto normal phase columns.
The procedure for dry loading is ; The plant extract is dissolved in a smallest amount of
suitable solvent (polar solvent) which can be easily removed is better (ensure that
complete dissolution if possible). Then, some amount of absorbent (silica gel) is added to
the above solution. The ideal proportion of sample to silica gel ranges from 1:1 to 1:3 by
volume. The resultant mixture is well mixed and the solvent let to be evaporated under
fume-hood (or rotary evaporator if the amount is larger). The resultant blend (dry powder)
is loaded to the top of the stationary phase of the column using a spatula. Dry loading is
preferable for natural product separation.
it is always necessary to maintain solvent level above the sorbent. Then, add carefully first
developing solvent while opening the outlet slowly. Then, analyte will percolate to the
sorbent and make sure the solvent level to be above the upper limit of the sorbent. Then,
carefully fill the column with first developing solvent.
78
The next step is to run the column (development of the column) in order to separate the
components.
• Development of column by gradient elution method and fraction collection
All separation processes involve the division of a mixture into a number of discrete
fractions. Gradient elution is recommended for natural product isolation as plant extracts
contain compounds with polarity range.
At first, less polar solvent (first developing solvent) is used for the running of column and
the fractions are collected in a separate tubes. For complex separations, small fractions can
be collected (there are automated fraction collectors as well). Then, the next polar solvent
or solvent mixture is applied and fractions are collected. This procedure is repeated by
increasing of the polarity of mobile phase gradually and the fractions are collected
accordingly. The fraction size and the flow rate is adjusted according to the result of TLC
analysis of the particular mixture.
• Detection of compounds in the fractions
TLC analysis is the more convenient way to detect the elution of compounds. If the
compounds are difficult to detect in UV, other chromatographic method such as GLC or
HPLC can be carried out.
• Pooling of the fractions and evaporation of the solvent
After running the column, the final step is the pooling /combination of the fractions having
same compound (same Rf). This is done according to the results of TLC analysis or other
chromatographic analyses. The fractions containing similar compounds are pooled
79
together and then the solvents are evaporated under reduced pressure. If the separated
compounds are not enough pure after the column, it is necessary to run another column for
the mixture after evaporating solvent.
Normal Column set up Flash Column set up
Figure 7. Normal column and Flash column set up.
80
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Academic Publishers.
2. Cannell, R. J. P. (1998) How to approach the isolation of a natural product, in

Methods in Biotechnology, vol. 4: Natural Products Isolation (Cannell, R. J. P., ed.),
Humana, Totowa, NJ, pp. 1–51.
3. Merck Handbook—Dyeing Reagents for Thin Layer and Paper Chromatography

(1980) E. Merck, Darmstadt, Germany.
4. Eurby, M. R. and Petersson, P. (2003) Chromatographic classification and

comparison of commercially available reversed-phase liquid chromatographic
columns using principle component analysis. J. Chromatogr. A 994, 13–26.
5. Glasl, S., Gunbilig, D., Narantuya, S., Werner, I., and Jurenitsch, J., 2001, Combination
of chromatographic and spectroscopic methods for the isolation and
characterization of polar guaianolides from Achillea asiatica, Journal of
Chromatography A, 936: 193-200
6. Jiang, Y., Lu, H. T. and Chen, F., 2004, Preparative purifi cation of glycyrrhizin
extracted from the root of liquorice using high-speed counter-current
chromatography, Journal of Chromatography, 1033(1): 183-186.
7. Kery, A. Turiak, Gy. and Tetenyi, P., 1988, Isolation of parthenolide by droplet
countercurrent chromatography, Journal of Chromatography A, 446: 157-161.
81
9. Poole, C. F., 1999, Planar chromatography at the turn of the century, Journal of
Chromatography A, 856: 399-427.
10. Poole, C. F., 2003, Thin-layer chromatography: challenges and opportunities, Journal
of Chromatography A, 1000: 963-984.
11. Sherma, J., 2000, Thin-layer chromatography in food and agricultural analysis,
Journal of Chromatography A, 880: 129-147.
12. Scott, R. P. W. (1993). Silica Gel and Bonded Phases: Their Production, Properties
and use in LC. Wiley, New York.
13. Salituro, G. M. and Dufresne, C. (1998) Isolation by low-pressure column

chromatography, in Natural Products Isolation, 1 ed. (Cannell, R. J. P.,ed.), Humana
Press, New Jersey.
14. Stead, P. (1998) Isolation by preparative HPLC, in Methods in Biotechnology, vol. 4:

Natural Products Isolation (Cannell, R. J. P., ed.), Humana, Totowa, NJ, pp. 165–208.
15. Touchstone, J. C. and Dobbins, M. F. (1982) Practice of Thin Layer Chromatography.

John Wiley and Sons Publishers.
16. Unger, K. K. (1990) (ed.) Packings and stationary phases in chromatographic

techniques, in Chromatographic Science Series. vol. 47, Dekker, New York.
17. Wagner, H. and Bladt, S. (1996) Plant Drug Analysis—A Thin Layer Chromatography
Atlas. Springer-Verlag, Berlin.
82
18. Zygmunt, B. and Namiesnik, J. (2003) Preparation of samples of plant material
for chromatographic analysis. J. Chromatogr. Sci. 41, 109–116.
11
11.0 Phytochemical Analysis (Phytochemical Screening)
Plants chemicals which can make some biological or pharmacological effects on human or
other animals are generally considered as phytochemicals. Mostly the secondary
metabolites; such as : alkaloids, terpenoids, saponins, glycosides, steroids, polyphenols,
flavanoids, polyketides etc. are included in phytochemicals (the chemical nature and
extraction of those phytochemicals have been explained in the previous chapters). The
standard chemical screening methods described in literature for identification of
phytochemicals are named as phytochemical screening. The Knowledge of the chemical
constituents of plants is important, not only for the discovery of therapeutic agents, but
also such information be of value in disclosing new resources of such chemical substances
(ie: preparing data base).
Powdered plant material or crude solvent extracts of the plant material (in methanol or
other solvents) can be used for phytochemical screening analyses. The standard chemical
tests reported in literature and preparation of relevant reagents are described here.
11.1 Tests for Alkaloids:
About 500 mg of crude methanolic extract (or about 5 g of powdered plant material) is
mixed with 6-8 mL of 1% Hydrochloric acid and the mixture is boiled in a water bath for
five minutes. After cooling, the solution is filtered and following tests are carried out for the
filtrate to identify the presence of alkaloids.
83
a) Mayer’s Test:
A portion of the filtrate (1 mL) is treated with few drops of Mayer’s reagent.
Formation of turbidity or yellow coloured precipitate indicates the presence of
alkaloids. When the amount of alkaloid is less in the particular extract, it may give
turbidity with Mayer’s reagent.
Preparation of Mayer reagent : This is solution of Potassium Mercuric Iodide. About

0.355 g of mercuric chloride is dissolved in 60 ml of distilled water and 5.0 g of
potassium iodide is dissolved in 20 ml of distilled water. Both solutions are mixed
and volume is raised to 100 ml with distilled water
b) Wagner’s Test:
A portion of filtrate (1 mL) is treated with Wagner’s reagent. Formation of brown
or reddish precipitate indicates the presence of alkaloids. When the amount of
alkaloid present is less in the particular extract, presence of alkaloids may
indicate forming turbidity with Wagner’s reagent.
Preparation of Wagner’s reagent : This is an aqueous solution of Iodine in

Potassium Iodide. About 2.0 g of KI is dissolved in 5 ml of distilled water and 1.27 g
of I2 is added to it. Then the solution is diluted up to 100.00 ml with water in a 100.00ml
volumetric flask.
c) Dragendroff’s Test:
A portion of filtrate (1 mL) is treated with Dragendroff’s reagent. Formation of red
precipitate indicates the presence of alkaloids. When the amount of alkaloid present
is less in the particular extract, presence of alkaloids may indicate forming turbidity
with Dragendroff’s reagent
84
Preparation of Dragendroff’s reagent: This is a solution of Potassium Bismuth Iodide.
Two different protocols are given;
Procedure -I :
Solution A: 1.7 g of bismuth nitrate and 20 g of tartaric acid are dissolved in 80 ml of
distilled water.
Solution B: 16 g of potassium iodide is dissolved in 40 ml of distilled water.
Just before the use, the solutions, A and B are mixed in 1:1 ratio.
These two solutions (A and B) are stable for some period when they are stored in a closed
container without exposing to sun light.
Dragendroff’s spraying reagent can be prepared as a solution of 10 g tartaric acid, 50 ml water & and
5 ml mixed solution of A and B.
Procedure II :
Solution – A : 0.85 g of Bi(NO3)3 is dissolved in 40 ml of distilled water, and 10 ml of glacial
acetic acid.
Solution – B : 8 g of KI is dissolved in 40 ml of distilled water and 10 mL of glacial acetic
acid.
Just before the use, equal volumes of A and B are mixed together.
d). Hager’s Test:

A portion of filtrate (1 mL) is treated with Hager’s reagent. Presence of alkaloids is
indicated by the formation of yellow colored precipitate.
Preparation of Hager’s reagent : This is a saturated solution of picric acid. Picric acid is
dissolved in distilled water till it get saturated.
e) Confirmatory test for alkaloids
85
About 6 g of powdered plant material or 2 g of crude methanolic extract is moistened
with water and then mixed with 1g of Ca(OH)2. The paste is mixed well and free
alkaloids are extracted into diethyl ether or petroleum sprite (5 mL). Ether is
evaporated (inside the hood) and the residue is treated with 5 mL of 1% of sulphuric
acid. The resultant solution is filtered and the filtrate is tested with Dragendroff’s and
Mayer’s reagents to see the formation of turbidity or precipitates.
f). Visualizing of alkaloids on TLC

Diethyl extract of the plant material prepared above can be used for TLC analysis. A
little portion of the extract is diluted and it is subjected TLC analysis using suitable
solvent system ( relatively polar solvent system is necessary as alkaloids are polar
substances). The spots are visualized by spraying Dragondroff’s reagent. This gives
an indication about number of alkaloids present and their relative concentrations.
11.2 Test for Glycosides:

The following tests indicate the presence of glycosides in plants.
a). To 100 mg of methanol extract ( or other solvent extracts), 5ml of chloroform and 10%
ammonia solution was added. Pink color formation indicates the presence of glycosides.
b). Keller-kilani test

About 200 mg of methanolic extract (or other solvent extracts) is dissolved in 3ml of glacial
acetic acid and then few drops of 5% ferric chloride are added. The mixture is then poured into
the test tube containing 2 mL of concentrated sulphuric acid. Brown ring formation at the
interface indicates the presence of cardiac glycosides. A violet ring may appear beneath the
brown ring, while in the acetic acid layer, a greenish ring may also form just gradually
throughout the layer.
c). Modified Borntrager’s Test

About 200 mg of methanolic extract is treated with 5% Ferric Chloride solution and
immersed in a boiling water bath for about 5 minutes. The mixture is cooled and extracted
86
with equal volume of benzene. The benzene layer is separated and treated with aqueous
ammonia solution. Formation of rose-pink colour in the ammonical layer indicates the
presence of anthranol glycosides.
d). Legal’s Test

About 200 mg of plant extract is treated with sodium nitropruside in equal amounts of
pyridine and sodium hydroxide. Formation of pink to blood red colour indicates the
presence of cardiac glycosides.
11.3 Test for Cyanogenic Glycosides
2 g of the powdered plant material is placed in a test tube and it is mixed with little
quantity of water Sodium picrate test paper is placed in the test tube and it is stoppered
immediately. The test tube is placed in boiling water bath. A change in colour from yellow
to red of the sodium picrate test papers after 10 minutes confirms as positive result. The
same test can be carried out at room temperature and also with dry powder in order to
compare the results.
11.4 Tests for Saponins

The following tests indicate presence of saponins
a). Froth Test:
About 500 mg of methanolic extract (or other solvent extracts) is shaken with 2 ml of
distilled water. If foam produced persists for ten minutes it indicates the presence of
saponins. Saponin content in different solvent extracts can be compared by measuring of
heights of the froth
b). About 2.0 g of the powdered plant material is placed in a test tube and distilled water
(15 mL) is added. The mixture is boiled in boiling water bath and filtered. 10 ml of the
filtrate is mixed with 5 ml of distilled water and shaken vigorously to the formation of
87
stable persistent froth. The frothing is mixed with 3 drops of olive oil and shaken
vigorously for the formation of emulsion thus a characteristic of saponins.
11.5 Tests for Phytosterols and Triterpenes

About 500 mg of methanolic plant extract is shaken with petroleum ether in order to
remove the colouring material. The residue is extracted with 10 ml chloroform and the
chloroform layer is dried over anhydrous sodium sulphate. Following tests are carried out
for the chloroform layer.
a). Libermann-Burchardt test

5 ml of chloroform layer is mixed with 0.25 ml of acetic anhydride and then two drops of
concentrated sulphuric acid is added. Formation of different colours indicate the presence
of sterol or terpenes as following. Green colour indicates the presence of sterols and pink
to purple indicates the presence of terpenes and triterpenes.
b). Salkowski test

To 5 ml of chloroform extract, few drops of concentrated (H2SO4) is added. Appearance of
golden yellow colour indicates the presence of triterpenes.
11.6 Test for Diterpenes

Copper acetate Test: About 500 mg of methanol extract (or other extracts) is dissolved in
water and treated with 3-4 drops of copper acetate solution. Formation of emerald green
colour indicates the presence of diterpenes
11.7 Test for Phenols and Tannins.

a). Ferric Chloride Test: About 100 mg of crude methanolic extract (or other solvent
extracts) is treated with 1 mL of aqueous ferric chloride solution (2%). Formation of
bluish- green or black colouration indicates the presence of phenols and tannins
88
b). Gelatin Test: About 500 mg of crude methanolic extract (or 5 g of powdered plant
material) is dissolved in10 mL of 1% NaCl solution. To this solution gelatin solution
containing sodium chloride is added. Formation of white precipitate indicates the presence
of tannins.
11.8 Tests for Flavonoids

a). Shinoda test: About 200 mg Methanolic Crude extract is dissolved in 2 mL of 50%
methanol solution. After adding of few pieces of magnesium ribbon to this solution, few
drops of concentrated HCl is added. Appearance of red colour indicates the presence of
flavonoids.
b). Alkaline Reagent Test: About 100 mg of methanol extract or other solvent extracts is
treated with few drops of dilute sodium hydroxide solution. Formation of intense yellow
colour, which becomes colourless on addition of dilute acid, indicates the presence of
flavonoids.
c). Lead acetate Test: About 100mg of the crude methanolic extract (or other solvent
extracts) is treated with few drops of lead acetate solution. Formation of yellow colour
precipitate indicates the presence of flavonoids.
d). 0.5 g of the methanolic plant extract is shaken with petroleum ether to remove the fatty
materials . The defatted residue is dissolved in 20 ml of 80% ethanol and filtered. The
filtrate is used for the following tests:
(i) 3 ml of the filtrate is mixed with 4 ml of 1% aluminium chloride in methanol in a test

tube. Formation of yellow colour indicates the presence of flavonols, flavones and
chalcones.
(ii) 3 ml of the filtrate is mixed with 4 ml of 1% potassium hydroxide in a test tube. A dark
yellow colour indicates the presence of flavonoids
89
(iii) Few drops of 1% aluminium solution are added to the filtrate, a yellow colouration is
observed indicating the presence of flavonoids.
e). About 5g of powdered plant is heated with 10 ml of ethyl acetate in a test tube over a
steam bath for 3 min. The mixture is filtered and 4 ml of the filtrate is shaken with 1 ml of
dilute ammonia solution. Yellow coloration indicates the presence of flavonoids.
11.9 Test for Coumarins

About 500 mg of the moistened methanolic plant extract is taken in a test tube. The mouth
of the tube is covered with filter paper treated with 1 N NaOH solution. Test tube is placed
for few minutes in boiling water and then the filter paper is removed and examined under
the UV light. Appearance of yellow fluorescence indicates the presence of coumarins
11.10 Test for Anthracene Derivatives

About 5 g of the powdered plant material is boiled with 20 ml 10% H2SO4 and filtered
while hot. The filtrate is shaken with 10 ml chloroform. The chloroform layer is separated
and 5 mL of 10% ammonium hydroxide solution is added. Formation of a pink colour in
the aqueous layer confirms the presence of anthracene derivatives
11.11 Tests for Proteins and Amino acids
Test for proteins

a). Millon’s test
A portion of crude extract is mixed with 2ml of Millon’s reagent. Formation of white
precipitate which turn red upon gentle heating confirm the presence of soluble protein.
Specially Millon’s reagent gives positive results for protein containing tyrosine.
Preparation of Millon’s reagent
90
10 grams of mercury is dissolved in 20 mL of hot concentrated nitric acid, and the resulting
solution is diluted with 30 mL of distilled water.
b). Ninhydrin test

A portion of crude extract is boiled with 2ml of 0.2% solution of Ninhydrin. Appearance of
violet colour indicates the presence of amino acids.
c). Xanthoprotic test
About 50 mg of plant extract in a boiling tube is treated with few drops of concentrated HNO3
and it is heated over a flame for 2minutes. The mixture is cooled and 40% NaOH is added until
the solution become strongly alkaline. Deep orange color indicates the presence of proteins with
aromatic ring containing amino acids.
d). Biuret test
100 mg of plant extract in a test tube is treated with 5-6 drops of dilute CuSO4. Formation of
violet or pink coloration with addition of 40% NaOH solution indicates the presence of proteins.
11.12 Test for Carbohydrates
a). Tollen’s test.
To 100 mg of plant extract, few drops of Tollen’s reagent is added and the mixture is
warmed at 40∘C for 3-4 minutes. Formation of silver mirror inside the wall indicates the
presence of reducing sugars.
Preparation of Tollen’s reagent: 2-3 drops of dilute NaOH is added to 5 mL of AgNO3 and
followed by adding of sufficient amount of NH4OH until silver oxide precipitate is dissolved.
91
b). Fehling’s test
Equal volume of Fehling A and Fehling B reagents are mixed together and 2ml of it is added
to 50 mg of crude extract and gently boiled. A brick red precipitate appeared at the bottom
of the test tube indicates the presence of reducing sugars.
Preparation of Fehling ‘s reagents :
Fehling’s A: 17.3 g of CuSO4 in 250 ml H2O containing few drops of conc. H2SO4.
Fehling B: 8.65g of sodium potassium tartarate (Rochelle salt) and 35 g of NaOH in 250 ml
H2O.
c) Benedict’s test
About 50m mg of crude extract is mixed with 2ml of Benedict’s reagent and boiled, a
reddish brown precipitate formed indicates the presence of the carbohydrates.
Preparation of Benedicts Reagent:
Benedict A: Dissolve 8.65g of CuSO4.5H2O in 75 ml of H2O.
Benedict B: Dissolve 86.5 g of sodium citrate and 50 g of anhydrous Na2CO3 in 400 ml H2O.
Benedicts Reagent: Pour Benedicts solution A slowly into solution B and make up the volume
to 500 ml . Mix well.
d). Molisch’s test

About 50 mg of crude extract is mixed with 2ml of Molisch’s reagent in a test tube and
shaken well. 2ml of concentrated H2SO4 is poured carefully along the side of the test tube.
Appearance of a violet ring at the inter-phase indicates the presence of carbohydrate.
Molisch’s reagent : The reagent is prepared by dissolving 5g of alpha napthol in 100 ml of 95

% ethanol.
92
e). Iodine test
A portion of crude extract is mixed with 2ml of iodine solution. A dark blue or purple
coloration indicates the presence of carbohydrate (starch).
11.13Test for Betacyanin

To 100 mg of crude extract in 2 mL of methanol, 1ml of 2N sodium hydroxide is added and
heated for 5 minutes at 100ºC. Formation of yellow color indicates the presence of betacyanin.
11.14Test for Quinones

To 100 mg of crude extract in 2 mL of methanol, 1ml of concentrated sulphuric acid is added.
Formation of red color indicates the presence of quinones
11.15 Further reading
1. Amir Muhammad Khan et al (2011), Phytochemical analysis of selected medicinal

plants of Margalla Hills and surroundings, J. Med. Plants Res. 5(25), pp. 6017-6023.
2. A. Das Talukdar et al, (2010) Phytochemical screening and TLC profiling of plant
extracts of Cyathea gigantea (Wall. Ex. Hook.) Haltt. and Cyathea brunoniana. Wall.
ex. Hook. (Cl. & Bak.) Assam University Journal of Science & Technology : Biological
and Environmental Sciences Vol. 5 Number I, 70-74.
3. Diaz, J. G., Ruiz, J. G., and de la Fuente, G. (2000) Alkaloids from Delphinium
staphisagria. J. Nat. Prod. 63, 1136–1139.
4. Harborne, J. B. (1984) Methods of plant analysis, in Phytochemical Methods,

2 ed., Chapman and Hall, London, pp. 1–36.
5. Harborne, J. B. (1998) Phytochemical Methods: A Guide to Modern Techniques
93
of Plant Analysis. 3 ed., Chapman & Hall, New York.
6. Hostettmann, K., Wolfender, J.-L., and Terreaux, C. (2001) Modern screening

techniques for plant extracts. Pharmaceut. Biol. 39 (supp), 18–32.
7. Harborne, J. B at al, (1984), phytochemical methods a guide to modern techniques of

plant analysis
8. Harborne J. B., Heywood V. H., Saleh N. A. M. (1970). Chemosystematics of the

Compositae: flavonoid patterns in the Chrysanthemum complex of the tribe
Anthemideae. Phytochemistry, 9: 2011
9. Markham, K. R. (1982) Techniques of Flavonoid Identification, Academic Press, New

York.
10. Njoku PC, Akumefula MI (2007). Phytochemical and Nutrient Evaluation of Spondias
mombin Leaves. Pak. J. Nut., 6: 613-615.
11. S. De et al. (2010), phytochemical investigation and chromatographic evaluation of

the different extracts of tuber of Amorphallus paeonifolius (Araceae), International
Journal on Pharmaceutical and Biomedical Research (IJPBR) Vol. 1(5), 150-157.
12. Pandith Javid Iqbal (2012), Phytochemical screening of certain plant species of Agra
city, Journal of Drug Delivery & Therapeutics; 2(4), 135-138.
13. Prashant Tiwa (2011), Phytochemical screening and Extraction: A Review

Internationale Pharmaceutica Sciencia Jan-Mar 2011 Vol 1 Issue 1.
14. Sofowora, A., (1993). Medicinal Plants and Traditional Medicines in Africa.
Chichester John Wiley and Sons New York, pp: 97-145.
94
15. R. Harisaranraj et al (2009), Evaluation of the Chemical Composition Rauwolfia
serpentina and Ephedra vulgaris, Advan. Biol. Res., 3 (5-6): 174-178.
16. Trease, G. E. and Evans, W. C. (1996) Phenols and phenolic glycosides, in

Pharmacognosy, 14 ed.,WB Saunders Company Ltd., London, pp. 218–254.
……………………………………………………………………..
95
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Plants Based Natural products Extraction, Isolation and Phytochemical

Book · June 2013

undergraduate project View project

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Extraction, Isolation and Phytochemical screening

20th June 2013

Bar code 9 789555 445610

I express my gratitude to Prof. Nobuhiro Kihara, Department of Chemistry, Kanagawa

Dr. Vajira P. Bulugahapitiya

Tel : +94 41 2227023 ext 44406 Tel: 01-2231205

1.0 Introduction to Natural products 01

1.1 Plant based Natural products 02

2.0 Extraction of plant based natural products 06

2.1 Collection, authentication and preparation of

plant material for the extraction purpose 07

2.3 Extraction methods of natural products 10

2.3.1 Solvent extraction 11

2.3.1.1 Isocratic and Gradient etraction 12

2.3.1.2 Choice of Solvents for extraction of

natural products as food additives or foods. 13

2.3.1.3 Use of water as an extracting solvent 14

2.4 Solvent extraction methods 14

2.4.1 Maceration (Steady-state extraction or cold extraction) 15

2.4.2 Solvent – Solvent partitioning method 16

2.4.3 Soxhlet Extraction 28

2.4.4 Plant tissue homogenization 20

2.4.5 Extraction of all the compounds present in plant material 21

3.0 Extraction of specific classes of natural products 23

3.1 Essential oils (volatile oil) 23

3.1.1 Introduction to Essential oils (volatile oil) 23

3.1.2 Some examples for the essential oils

(monoterpenes and sesquiterpenes), their occurrence and uses 24

3.1.3 Extraction of essential oil (volatile oil) 26

3.1.3 .2 Extraction of essential oil using solvent extraction method 30

4.1 Introduction to Alkaloids 35

4.1.1 Examples for some alkaloids, their structures

4.3 Extraction of volatile liquid alkaloids 40

6.1 Introduction to Steroids 47

6.3 Further reading 48

8.3.1 Further reading 55

8.4 Microwave Assisted Extraction of Natural Products 57

9.1 Fractionating Techniques 60

9.1.5 Bioassay guided fractionation and isolation of natural products 64

11.0 Phytochemical Analysis ( Phytochemical Screening) 83

Biological active agents ,64 Extraction of

1.0 Introduction to Natural products

Importance of Natural Products for human

1.1 Plant based Natural products

Taxus brevifolia Taxol Terpenoids For cancer

Quinine Alkaloids Malaria

As local anesthetic, refreshing

1.2 Further reading

(1). Bruneton J. (1995) Pharmacognosy, Phytochemistry, Medicinal Plants. Springer-Verlag, Berlin

(3) Cordell, G. A. (2000) Biodiversity and drug discovery—a symbiotic relationship

(14). Trease and Evans (2009), Pharmacognosy,16th Ed, SAUNDERS, ELSVIER

2.0 Extraction of plant based natural products

The required purposes for extraction of natural product can be:

Prior to choosing an extraction method, it is necessary to establish the target of the

Target of the extraction can be

2. To isolate particular class of compounds or particular compound