Prof. N. Raaman is the Emeritus Professor and Former Director of the Centre for Advanced Studies in Botany at University of Madras, Chennai, India. He obtained his Ph.D. and D.Sc. degree from University of Madras. He is working on Natural products for the past 35 years and he has guided 41 Ph.D. scholars, of which 30 theses are on Phytochemistry.
He has published five books and 175 research articles in National and International Journals to his credit. He is the joint Inventor of “AYUSREM” the Herbal remedy for Swine Flu. He is a joint Inventor of a new compound, Mazapotin which has anticancer as well as antidiabetic effects for which Madras University obtained 2 patents.
Prof. Raaman is accomplished with 22 awards such as UNESCO Young Scientist Award 1988, Bharatha Rathna Sir M.Vishvesvaraya Memorial National Parisara Rathna Award, 1999, Leading Scientists of the World Award 2005, Tamil Nadu Scientist Award 2006, Bharat Gaurav Award 2009, International Achiever’s Award 2009, The International Einstein Award for Scientific Achievement 2009, National Citizen Excellence Award 2009, Academic Achievement Award 2012, Mother Teresa Excellence Award 2012, Bharat Excellence Award 2014, Rastriya Ratan Award 2014, Indian Award for Excellence in Education 2014, Hindustan Gagan Gaurav Award 2015 etc. He has attended several conferences in many Universities and delivered Lectures in India, besides visiting several countries including UK, USA, Canada, France, Germany, Spain, Turkey, Japan, Singapore, Malaysia, Hong Kong, Thailand and Indonesia.
Prof. Raaman has carried out 25 research projects sponsored by University Grants Commission, Department of Science and Technology, Department of Biotechnology, Council for Science and Industrial Research, Indian Council for Agricultural Research, Indian Council for Forest Research, Tamil Nadu Pollution Control Board, Tamil Nadu Forest Department and industries such as Retort Pharmaceuticals and Sakthi Masala.
Introduction
Plants are used as medicine since time immemorial. One of the area of Ethnobotany, Ethnopharmacology is considered as the scientific evaluation of traditional medicinal plants (Cotton, 1996). Cox (1994) has suggested that the ethno-directed sampling is most
likely to succeed in identifying drugs used in the treatment of gastrointestinal, inflammatory and dermatological complaints.
The Plant kingdom is a virtual goldmine of potential drug targets and other active molecules awaiting to be discovered. It has been estimated that only 10-15 per cent of the 7,50,000 existing species of higher plants have been surveyed for biologically active compounds. Natural products produced by plants, fungi, bacteria, insects and animals have been isolated as biologically active pharmacophores. Approximately one-third of the top-selling drugs in the world are natural products or their derivatives often with ethnopharmacological background. Moreover, natural products are widely recognized in the pharmaceutical industry for their broad structural diversity as well as their wide range of pharmacological activities.
New medicines have been discovered with traditional, empirical and molecular approaches (Harvey, 1999). The traditional approach makes use of material that has been found by trial and error over many years in different cultures and systems of medicine (Cotton, 1996). Examples include drugs such as morphine, quinine and ephedrine that have been in wide spread use for a long time, and more recently adopted compound such as the antimalarial artemisinin. The empirical approach builds on an understanding of a relevant physiological process and often develops a therapeutic agent from a naturally occurring lead molecule (Verpoorte, 2000). Examples include tubocurarine and other muscle relaxants, propranolol and other adrenoreceptor antagonists, cimetidine and other histamine H, receptor antagonists. With the development of molecular biological techniques and the advances in genomics, the majority of drug discovery is currently based on the molecular approach.
The major advantage of natural products for random screening is the structural diversity provided by most available combinatorial approaches based on heterocylic compounds (Claeson and Bohlin, 1997; Harvey, 1999). Bioactive natural products often occur as a part of a family of related molecules so that it is possible to isolate a number of homologues and obtain structure-activity information. Of course, lead compounds found from screening of natural products can be optimised by traditional medicinal chemistry or by application of combinatorial approaches. Overall, when faced with molecular targets in screening assays library seems more likely to provide the chemical diversity to yield a hit than a library of for which there is no information about low molecular weight leads, use of a natural products similar numbers of compounds made by combinatorial synthesis.
Plants are viable for modern medicine in 4 basic ways:
They are used as sources of direct medicinal agents.
They serve as a raw material base for elaboration of more complex semi-synthetic chemical compounds.
The chemical structure derived from phytoconstituents can be used as models for new synthetic compounds.
Plants can be used as taxonomic markers for discovery of new therapeutic compounds. Important plant secondary metabolites such as glycosides, flavonoids, lignins, terpenoids and alkaloids have been isolated from plants.
Since the use of botanicals and herbal remedies has increased dramatically in the last several years, the number of researchers on the isolation and identification of compounds from plants have also been increased.
To help those who involved in the isolation of compounds from plants, some of the essential phytochemical techniques are included in this book.
Chapter 2 deals with the production processes for herbals and botanicals. Selection of plant and plant parts for phytochemical analysis are included in
Chapter 3. Different methods of extraction are given in
Chapter 4. Qualitative phytochemical screening is presented in
Chapter 5. Various methods for separation of phytochemicals, which include paper and thin layer chromatography and column chromatography are given in
Chapter 6. Qualitative and quantitative estimation of phytochemicals using gas chromatography, high performance liquid chromatography and high performance thin layer chromatography are described in
Chapter 7. The various methods of identification including the physical characteristics and spectroscopy are included in
Chapter 8. The ultraviolet spectroscopy, infrared spectroscopy, near infrared spectroscopy, mass spectroscopy, nuclear magnetic resonance spectroscopy and crystallography are included in this chapter. The categories of phytochemicals are given in
Chapter 9. A case study of isolation and identification of compounds in the laboratory of the author of this book is included in
Chapter 10. The most obvious changes in this second revised edition are the 4 new chapters. i. Isolation of alkaloids is given in
Chapter 11. ii. Extraction and isolation of phenolic compounds is described in
Chapter 12. iii. Isolation of anthocyanin compounds is included in
Chapter 13. iv. Extraction and analysis of essential oils are described in Chapter 14.
Preface to the First Edition
Various techniques are available in different disciplines of Science such as Botany, Chemistry, Physics, Mathematics etc. The isolation, characterization and identification of phytochemicals from plants involve the knowledge of all disciplines of Science. With the growing interest in the field of medicinal plants by researchers and industries, it is necessary to have a book which will give information on different interdisciplinary techniques. The aim of this book is to give a self- contained detail of all aspects of phytochemical techniques. It is designed as a textbook for those students who use phytochemical techniques and for researchers working on medicinal plants. Important plant secondary metabolites such as glycosides, flavonoids, lignins, terpenoids and alkaloids have been isolated over a period of time from natural sources. To encourage many students and researchers to carry out work on phytochemicals and to find out some novel compounds from plants, various techniques are given in a nutshell. This book contains 10 chapters. Chapter I provides a general introduction. In chapter 2, the production processes for herbals and botanicals are given. The selection of plant parts for extraction is briefed in chapter 3. The chapter 4 provides the various methods of extraction such as organic solvent extraction, extraction with supercritical gases, steam distillation, purification and concentration of miscella and schemes for extraction.
Different procedures of qualitative phytochemical screening are presented in chapter 5. The methods for separation of phytochemicals including paper chromatography, thin layer chromatography and column chromatography are discussed in chapter 6. In chapter 7, qualitative and quantitative estimation of phytochemicals with gas and liquid chromatography, HPTLC and OPLC are given. The methods of identification using physical characteristics and spectroscopy which includes UV, IR, NIR, Mass and NMR are presented in chapter 8. In chapter 9, details and structures of different categories of phytochemicals are considered. Finally in chapter 10, a case study of isolation and identification of compounds from Clansarena dentata in my laboratory is discussed.I would like to thank all those who helped me in the preparation of the text. A special mention is to Prof. Muraleedharan G Nair, Bioactive Natural Products and Phytoceuticals, National Food Safety and Toxicology Center, Michigan State University, USA who extended the facilities to me during my visit to USA. My sincere thank to Prof. R. Raghunathan, Department of Organic Chemistry, University of Madras, Chennai, India, Dr.A. Banerji (Former Head), Dr.S.Chattopadhyay (Head) and Dr.GJ. Chintalwar of Bio-Organic Division, Bhabha Atomic Research Centre, Mumbai, India for their help in identification of some of our compounds. Prof. P.T. Manoharan (Former Head) and Prof. A.K. Mishra (Head) of
Sophisticated Analytical Instrument Facility, Indian Institute of Technology, Chennai, India were kind enough to allow us to use the facility. The help rendered by Prof. D. Velmurugan, Department of Crystallography & Biophysics, University of Madras, Chennai, India for crystallographic studies and ANCHORM Mumbai, India for HPTLC studies is of worth mentioning. I am thankful to the staff of New India Publishing Agency, New Delhi, India for bringing out this book in time.
Preface to the Second Edition
The First Edition of the book which was published in 2006 received overwhelming response from students and researchers. In the fifteen years since the first edition of this book was published in 2006 with 10 chapters, I have received numerous
email messages and phone calls from several readers commenting on the book and requesting for more information on Alkaloids, Phenolic compounds, Anthocyanin compounds, Essential oils etc.
I have also built up a large file of ideas based on my own experiences in reading, writing, and editing and in examining and supervising theses. With the aid of all this information, I have revised this Second Revised Edition of the book. The most obvious changes in this second revised edition are the 4 new chapters.
(i) isolation of alkaloids is given in Chapter 11.
(ii) Extraction and isolation of phenolic compounds is described in Chapter 12.
(iii). Isolation of anthocyanin compounds is included in Chapter 13.
(iv) Extraction and analysis of essential oils are described in Chapter 14.
It is hoped, therefore, that the book will continue to appeal to those with an interest on Phytochemistry and Phytochemical Techiques. I would like to thank my student Dr.C.Sivaraj who helped me in the preparation of the text. I wish to thank my wife Mrs.Swarna Raaman for her enthusiasm and support. I am thankful to the staff of New India Publishing Agency, New Delhi, India for bringing out this Second Revised Edition of the book in time.
June, 2022 RAAMAN Chennai, India
Contents
1. Introduction
2. Production Processes for Herbals and Botanicals
2.1 Introduction
2.2 Cultivation
2.3 Collection from the Wild
2.4 Cleaning
2.5 Drying
2.5.1 Natural Drying Artificial Drying
2.6 Packaging of Dried Plants
3.Selection of Plant and Plant Parts for Phytochemical Analysis.
4.Methods of Extraction
4.1.Introduction
4.2 Organic Solvent Extraction
4.2.1 Percolation
4.2.2 Cold Percolation
4.2.3 Countercurrent Extraction
4.3 Extraction with Supercritical Gases
4.4 Steam Distillation
4.5 Maceration Other Minor Extraction Methods
4.6 Extraction of Essential Oil
4.7 Soxhlet Extractor.
4.8 Accelerated Solvent Extractor
4.9 Purification and Concentration of Miscella
4.10 Schemes of Procedure for Extracting Plant Tissues
5.Qualitative Phytochemical Screening
5.1 Introduction
5.2 Detection of Alkaloids
5.3 Detection of Carbohydrates and Glycosides
5.4 Detection of Saponins.
5.5 Detection of Proteins and Amino Acids.
5.6 Detection of Phytosterols.
5.7 Detection of Fixed Oils and Fats
5.8 Detection of Phenolic Compounds and Tannins.
5.9 Detection of Gum and Mucilages.
5.10 Ltection of Volatile Oil
6.Methods for Separation of Phytochemicals
6.1 Introduction
6.2 Chromatography
6.2.1 Introduction
6.2.2 Theoretical Principles
6.3 Paper Chromatography
6.3.1 Introduction
6.3.2 Theoretical Principles
6.6.3 Instrument
6.6.4 Sample Handling
6.6.5 Detection and Identification of Components
6.4 Thin layer Chromatography
6.6.1 Introduction
6.6.2 Theoretical Principles
6.6.3 Instrument
6.6.4 Sample Handling
6.6.5 TLC Plate Visualization by Spraying and Dipping
6.5 Column Chromatography
6.6.1 Introduction
6.6.2 Instrument
6.6.3 Sample Handling
7.Qualitative and Quantitative Estimation of Phytochemicals
7.1 Introduction
7.2 Gas Chromatography
7.2.1 Introduction
7.2.2 Theoretical Principles
7.2.3 Instrument
7.2.4 Sample Handling
7.2.5 Chromatogram
7.3 Liquid Chromatography
7.3.1 Introduction
7.3.2 Preparative Chromatography
7.4 High Performance Liquid Chromatography
7.4.1 Introduction
7.4.2 Principles High Performance Liquid Chromatography,
7.4.3 HPLC Instrument
7.4.4 Sample Handling
7.4.5 Identification, Qualitative and Quantitative Analysis
7.5 High Performance Thin Layer Chromatography
7.5.1 Introduction
7.5.2 Theoretical Principles
7.5.3 Instrument
7.5.4 Sample Handling
7.5.5 Chromatogram Evaluation
7.5.6 HPTLC Identification of Astragalus and Hedysaram
7.6 Optimum Performance Laminar Chromatography (OPLC)
7.6.1 Introduction
7.6.2 Theoretical Principles
7.6.3 Instrument
7.6.4 Sample Handling
8. Methods of Identification
8.1 Introduction
8.2 Physical Characteristics
8.2.1 Introduction
8.2.2 Colour
8.2.3 Odour
8.2.4 Acid or Base character Solubility
8.2.5 Melting Point
8.2.6 Boiling Point
8.3 Spectroscopy
8.3.1 Introduction
8.4 Ultra Violet Spectroscopy
8.4.1 Introduction
8.4.2 Theoretical Principles
8.4.3 Instrument
8.4.4 Sample Handling
8.4.5 Interpretation of Spectrum
8.5 Infrared Absorption Spectroscopy
8.5.1 Introduction
8.5.2 Theoretical Principles
8.5.3 Instrument
8.5.4 Sample Handling.
8.5.5 Interpretation of Spectrum
8.6 Near-Infrared Absorption Spectroscopy (NIR)
8.6.1 Introduction
8.6.2 Instrument
8.7 Mass Spectroscopy
8.7.1 Introduction
8.7.2 Theoretical Principles
8.7.3 Equipment
8.7.4 Sample Handling
8.7.5 Mass Spectrum
8.7.6 Interpretation of a Mass Spectrum
8.7.7 Determination of Molecular Formula
8.7.8 Calculation of Molecular Weight
8.7.9 Combinations of Mass Spectrometry
8.8 Nuclear Magnetic Resonance Spectroscopy
8.8.1 Introduction
8.8.2 Theoretical Principles
8.8.3 Carbon-13 NMR (“C NMR)
8.8.4 Hydrogen-1 NMR (‘H NMR)
8.8.5 Instrument
8.8.6 Interpretation of NMR Spectra
8.8.7 Different Techniques of NMR
8.9 CHN Analysis
8.9.1 Introduction
8.9.2 Estimation of Elements
8.9.3 Determination of Molecular Weight
8.9.4 Calculation of Empirical and Molecular Formula
8.10 X-Ray Crystallography
8.10.1 Introduction
8.10.2 Theoretical Principles
8.10.3 Instrument
8.10.4 Sample Handling
9.Categories of Phytochemicals
9.1 Introduction
9.2 Terpenoids
9.2.1 Introduction
9.2.2 General Properties of Terpenoids
9.2.3 Classification of Terpenoids
9.2.4 Determination of Structure of Terpenoids
9.2.5 Monoterpenoids
9.2.6 Sesquiterpenoids
9.2.7 Diterpenoids
9.2.8 Triterpenoids
9.2.9 Pentaterpenoids
9.2.10 Essential Oils
9.3 Steroids
9.3.1 Introduction
9.3.2 Stereochemistry of Steroids
9.3.4 Sterols
9.3.5 Steroidal Alkaloids
9.4 Phenolic Compounds
9.4.1 Introduction
9.4.2 Phenylpropanoids
9.4.3 Flavonoids
9.5 Alkaloids
9.5.1 Introduction
9.5.2 Distribution
9.5.3 Properties.
9.5.4 Test for Alkaloids
9.5.5. Extraction of Alkaloids
9.5.6 Structure and Classification
9.6 Glycosides
9.7 Carbohydrates
9.7.1 Introduction
9.7.2 Classification
9.7.3 Monosaccharides
9.7.4 Disaccharides
9.7.8 Trisaccharides
9.7.9 Polysaccharides
9.7.10 Structural Polysaccharides
9.7.11 Food Storage Polysaccharides
9.8 Lipids and Related Compounds
9.8.1 Introduction
9.8.2 Classification of Lipids
9.8.3 Fats and Oils
9.8.4 Waxes
9.8.5 Phospholipids
9.9 Plant Pigments
9.9.1 Introduction
9.9.2 Chlorophylls
9.9.3 Carotenoids
9.9.4 Phycobilins
9.9.5 Anthocyanins
9.10 Vitamins
9.10.1 Introduction
9.10.2 Classification of Vitamins
9.10.3 Sources of Vitamins
9.10.4 Structures of Vitamins
9.11 Amino Acids
9.11.1 Introduction
9.11.2 Essential and Non-essential Amino Acids
9.11.3 Classification of Amino Acids
9.11.4 Structures of Amino Acids
9.11.4 Introduction
9.12 Proteins
9.12.1 Peptides
9.12.2 Primary Structure of Peptides
9.12.3 Secondary and Tertiary Structures of Large Peptides and Proteins
9.13 Nucleic Acids
9.13.1 Introduction
9.13.2 DNA
9.13.3 RNA
9.13.4 Base Pairing
9.15.5 Double Helix structure of DNA
10. A Case Study in Prof. N. Raaman’s Laboratory
10.1 Introduction
10.2 Preparation of Extracts from Clausena dentate
10.3 Qualitative Phytochemical Screening
10.4 Isolation of Compounds
10.4.1 Thin Layer Chromatography
10.4.2 Column Chromatography
10.5 Quantitative Estimation of Isolated Compounds in Extracts by High Performance Thin Layer Chromatography (HPTLC).
10.6 Characterisation of Compound CD-1
10.6.1 Physical Characteristics.
10.6.2 as Chromatogram
10.6.3 UV and Visible Spectrum.
10.6.4 Infra Red Spectrum.
10.6.5 H-NMR and “C-NMR Spectra
10.6.6 GC-MS Spectrum
10.7 Identification of Compound CD-1
10.8 Identification of a Compound from Plant Using X-ray Crystallography.
10.9 Ph.D. Theses Under the Supervision of Prof. N. Raaman on Natural Products
11. Isolation of Alkaloids
11.1 Introduction
11.2 Extraction of Alkaloids
11.2.1 Soxhlet Extraction Process
11.3 Tests for Alkaloids
11.4 Estimation of Alkaloids
11.4.1 Preparation of reagents.
11.4.2 Preparation of standard curve
11.4.3 Preparation of plant extract
11.5 Liquid Chromatography-Mass Spectrometry (LC-MS) or HPLC-MS
11.6 Identification and Quantification
12.Extraction and isolation of Phenolic Compounds
12.1 Introduction
12.2 Sample preparation
12.2.1 Physical Treatments
12.2.2 Hydrolysis
12.3 Extraction
12.3.1 Solvent Extraction
12.3.2 Ultrasound-Assisted Extraction.
12.3.3 Microwave-Assisted Extraction
12.3.4 Pressurized Liquid Extraction.
12.3.5 Solid-Phase Extraction
12.3.6 Molecularly Imprinted Polymers.
12.3.7 Supercritical Fluid Extraction
12.4 Isolation of Phenolic compounds
12.4.1 Column Chromatography
12.4.2 Counter-Current Chromatography
13. Isolation of Anthocyanin Compounds
13.1 Introduction
13.2 Preparation of Extract
13.3 Acetone extraction and chloroform partition of Anthocyanins
13.3.1 Materials
13.3.2 Procedure
13.4 Isolation of Anthocyanins
14.Extraction and Analysis of Essential Oils
14.1 Introduction
14.2 Extraction methods
14.2.1 Steam distillation
14.2.2 Hydro distillation or water-steam distillation
14.2.3 Solvent extraction methods
14.2.4 Maceration.
14.2.5 Enfleurage
14.2.6 Solvent Extraction
14.2.7 Supercritical CO2 extraction
14.2.8 Cold-pressing or Expression
