Thin Layer Chromatography
in Phytochemistry
CHROMATOGRAPHIC SCIENCE SERIES
A Series of Textbooks and Reference Books Editor: JACK CAZES
1. Dynamics of Chromatography: Principles and Theory, J. Calvin Giddings
2. Gas Chromatographic Analysis of Drugs and Pesticides, Benjamin J. Gudzinowicz
3. Principles of Adsorption Chromatography: The Separation of Nonionic Organic Compounds, Lloyd R. Snyder
4. Multicomponent Chromatography: Theory of Interference, Friedrich Helfferich and Gerhard Klein
5. Quantitative Analysis by Gas Chromatography, Josef Novák 6. High-Speed Liquid Chromatography, Peter M. Rajcsanyi
and Elisabeth Rajcsanyi
7. Fundamentals of Integrated GC-MS (in three parts), Benjamin J. Gudzinowicz, Michael J. Gudzinowicz, and Horace F. Martin
8. Liquid Chromatography of Polymers and Related Materials, Jack Cazes 9. GLC and HPLC Determination of Therapeutic Agents (in three parts),
Part 1 edited by Kiyoshi Tsuji and Walter Morozowich, Parts 2 and 3 edited by Kiyoshi Tsuji
10. Biological/Biomedical Applications of Liquid Chromatography, edited by Gerald L. Hawk
11. Chromatography in Petroleum Analysis, edited by Klaus H. Altgelt and T. H. Gouw
12. Biological/Biomedical Applications of Liquid Chromatography II, edited by Gerald L. Hawk
13. Liquid Chromatography of Polymers and Related Materials II, edited by Jack Cazes and Xavier Delamare
14. Introduction to Analytical Gas Chromatography: History, Principles, and Practice, John A. Perry
15. Applications of Glass Capillary Gas Chromatography, edited by Walter G. Jennings
16. Steroid Analysis by HPLC: Recent Applications, edited by Marie P. Kautsky
17. Thin-Layer Chromatography: Techniques and Applications, Bernard Fried and Joseph Sherma
18. Biological/Biomedical Applications of Liquid Chromatography III, edited by Gerald L. Hawk
19. Liquid Chromatography of Polymers and Related Materials III, edited by Jack Cazes
20. Biological/Biomedical Applications of Liquid Chromatography, edited by Gerald L. Hawk
21. Chromatographic Separation and Extraction with Foamed Plastics and Rubbers, G. J. Moody and J. D. R. Thomas
22. Analytical Pyrolysis: A Comprehensive Guide, William J. Irwin 23. Liquid Chromatography Detectors, edited by Thomas M. Vickrey 24. High-Performance Liquid Chromatography in Forensic Chemistry,
edited by Ira S. Lurie and John D. Wittwer, Jr.
25. Steric Exclusion Liquid Chromatography of Polymers, edited by Josef Janca
26. HPLC Analysis of Biological Compounds: A Laboratory Guide, William S. Hancock and James T. Sparrow
27. Affinity Chromatography: Template Chromatography of Nucleic Acids and Proteins, Herbert Schott
28. HPLC in Nucleic Acid Research: Methods and Applications, edited by Phyllis R. Brown
29. Pyrolysis and GC in Polymer Analysis, edited by S. A. Liebman and E. J. Levy
30. Modern Chromatographic Analysis of the Vitamins, edited by André P. De Leenheer, Willy E. Lambert, and Marcel G. M. De Ruyter 31. Ion-Pair Chromatography, edited by Milton T. W. Hearn
32. Therapeutic Drug Monitoring and Toxicology by Liquid Chromatography, edited by Steven H. Y. Wong
33. Affinity Chromatography: Practical and Theoretical Aspects, Peter Mohr and Klaus Pommerening
34. Reaction Detection in Liquid Chromatography, edited by Ira S. Krull 35. Thin-Layer Chromatography: Techniques and Applications,
Second Edition, Revised and Expanded, Bernard Fried and Joseph Sherma
36. Quantitative Thin-Layer Chromatography and Its Industrial Applications,edited by Laszlo R. Treiber
37. Ion Chromatography, edited by James G. Tarter
38. Chromatographic Theory and Basic Principles, edited by Jan Åke Jönsson
39. Field-Flow Fractionation: Analysis of Macromolecules and Particles, Josef Janca
40. Chromatographic Chiral Separations, edited by Morris Zief and Laura J. Crane
41. Quantitative Analysis by Gas Chromatography, Second Edition, Revised and Expanded,Josef Novák
42. Flow Perturbation Gas Chromatography, N. A. Katsanos 43. Ion-Exchange Chromatography of Proteins, Shuichi Yamamoto,
Kazuhiro Naka-nishi, and Ryuichi Matsuno
44. Countercurrent Chromatography: Theory and Practice, edited by N. Bhushan Man-dava and Yoichiro Ito
45. Microbore Column Chromatography: A Unified Approach to Chromatography, edited by Frank J. Yang
46. Preparative-Scale Chromatography, edited by Eli Grushka 47. Packings and Stationary Phases in Chromatographic Techniques,
edited by Klaus K. Unger
48. Detection-Oriented Derivatization Techniques in Liquid
Chromatography, edited by Henk Lingeman and Willy J. M. Underberg 49. Chromatographic Analysis of Pharmaceuticals, edited by
John A. Adamovics
50. Multidimensional Chromatography: Techniques and Applications, edited by Hernan Cortes
51. HPLC of Biological Macromolecules: Methods and Applications, edited by Karen M. Gooding and Fred E. Regnier
52. Modern Thin-Layer Chromatography, edited by Nelu Grinberg 53. Chromatographic Analysis of Alkaloids, Milan Popl, Jan Fähnrich,
and Vlastimil Tatar
54. HPLC in Clinical Chemistry, I. N. Papadoyannis
55. Handbook of Thin-Layer Chromatography, edited by Joseph Sherma and Bernard Fried
56. Gas–Liquid–Solid Chromatography, V. G. Berezkin 57. Complexation Chromatography, edited by D. Cagniant
58. Liquid Chromatography–Mass Spectrometry, W. M. A. Niessen and Jan van der Greef
59. Trace Analysis with Microcolumn Liquid Chromatography, Milos KrejcI
60. Modern Chromatographic Analysis of Vitamins: Second Edition, edited by André P. De Leenheer, Willy E. Lambert, and Hans J. Nelis 61. Preparative and Production Scale Chromatography, edited by
G. Ganetsos and P. E. Barker
62. Diode Array Detection in HPLC, edited by Ludwig Huber and Stephan A. George
63. Handbook of Affinity Chromatography, edited by Toni Kline
64. Capillary Electrophoresis Technology, edited by Norberto A. Guzman 65. Lipid Chromatographic Analysis, edited by Takayuki Shibamoto 66. Thin-Layer Chromatography: Techniques and Applications:
Third Edition, Revised and Expanded, Bernard Fried and Joseph Sherma
67. Liquid Chromatography for the Analyst, Raymond P. W. Scott 68. Centrifugal Partition Chromatography, edited by Alain P. Foucault 69. Handbook of Size Exclusion Chromatography, edited by Chi-San Wu 70. Techniques and Practice of Chromatography, Raymond P. W. Scott 71. Handbook of Thin-Layer Chromatography: Second Edition,
Revised and Expanded, edited by Joseph Sherma and Bernard Fried 72. Liquid Chromatography of Oligomers, Constantin V. Uglea
73. Chromatographic Detectors: Design, Function, and Operation, Raymond P. W. Scott
74. Chromatographic Analysis of Pharmaceuticals: Second Edition, Revised and Expanded, edited by John A. Adamovics
75. Supercritical Fluid Chromatography with Packed Columns: Techniques and Applications, edited by Klaus Anton and Claire Berger
76. Introduction to Analytical Gas Chromatography: Second Edition, Revised and Expanded, Raymond P. W. Scott
77. Chromatographic Analysis of Environmental and Food Toxicants, edited by Takayuki Shibamoto
78. Handbook of HPLC, edited by Elena Katz, Roy Eksteen, Peter Schoenmakers, and Neil Miller
79. Liquid Chromatography–Mass Spectrometry: Second Edition, Revised and Expanded, Wilfried Niessen
80. Capillary Electrophoresis of Proteins, Tim Wehr, Roberto Rodríguez-Díaz, and Mingde Zhu
81. Thin-Layer Chromatography: Fourth Edition, Revised and Expanded, Bernard Fried and Joseph Sherma
82. Countercurrent Chromatography, edited by Jean-Michel Menet and Didier Thiébaut
83. Micellar Liquid Chromatography, Alain Berthod and Celia García-Alvarez-Coque
84. Modern Chromatographic Analysis of Vitamins: Third Edition, Revised and Expanded, edited by André P. De Leenheer, Willy E. Lambert, and Jan F. Van Bocxlaer
85. Quantitative Chromatographic Analysis, Thomas E. Beesley, Benjamin Buglio, and Raymond P. W. Scott
86. Current Practice of Gas Chromatography–Mass Spectrometry, edited by W. M. A. Niessen
87. HPLC of Biological Macromolecules: Second Edition, Revised and Expanded, edited by Karen M. Gooding and Fred E. Regnier 88. Scale-Up and Optimization in Preparative Chromatography:
Principles and Bio-pharmaceutical Applications, edited by Anurag S. Rathore and Ajoy Velayudhan
89. Handbook of Thin-Layer Chromatography: Third Edition,
Revised and Expanded, edited by Joseph Sherma and Bernard Fried 90. Chiral Separations by Liquid Chromatography and Related
Technologies, Hassan Y. Aboul-Enein and Imran Ali
91. Handbook of Size Exclusion Chromatography and Related Techniques: Second Edition, edited by Chi-San Wu
92. Handbook of Affinity Chromatography: Second Edition, edited by David S. Hage
93. Chromatographic Analysis of the Environment: Third Edition, edited by Leo M. L. Nollet
94. Microfluidic Lab-on-a-Chip for Chemical and Biological Analysis and Discovery, Paul C.H. Li
95. Preparative Layer Chromatography, edited by Teresa Kowalska and Joseph Sherma
96. Instrumental Methods in Metal Ion Speciation, Imran Ali and Hassan Y. Aboul-Enein
97. Liquid Chromatography–Mass Spectrometry: Third Edition, Wilfried M. A. Niessen
98. Thin Layer Chromatography in Chiral Separations and Analysis, edited by Teresa Kowalska and Joseph Sherma
99. Thin Layer Chromatography in Phytochemistry, edited by
Monika Waksmundzka-Hajnos
Medical University of Lublin Lublin, PolandJoseph Sherma
Lafayette College Easton, Pennsylvania, U.S.A.Teresa Kowalska
University of Silesia Katowice, PolandThin Layer Chromatography
in Phytochemistry
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Library of Congress Cataloging-in-Publication Data Thin layer chromatography in phytochemistry / editors, Monika
Waksmundzka-Hajnos, Joseph Sherma, Teresa Kowalska. p. cm. -- (Chromatographic science series)
Includes bibliographical references and index.
ISBN 978-1-4200-4677-9 (hardback : alk. paper) 1. Plants--Analysis. 2. Thin layer chromatography. I. Waksmundzka-Hajnos, Monika. II. Sherma, Joseph. III. Kowalska, Teresa. IV. Title. V. Series.
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Contents
Preface... xiii Editors ... xv Contributors ... xix
Part I
Chapter 1 Overview of the Field of TLC in Phytochemistry
and the Structure of the Book ... 3 Monika Waksmundzka-Hajnos, Joseph Sherma,
and Teresa Kowalska
Chapter 2 Plant Materials in Modern Pharmacy and Methods
of Their Investigations... 15
Krystyna Skalicka-Wozniak, Jarosław Widelski, and Kazimierz
Głowniak
Chapter 3 Medicines and Dietary Supplements Produced
from Plants... 37 Anita Ankli, Valeria Widmer, and Eike Reich
Chapter 4 Primary and Secondary Metabolites
and Their Biological Activity ... 59 Ioanna Chinou
Chapter 5 Plant Chemosystematics ... 77
Christian Zidorn
Chapter 6 Sorbents and Precoated Layers for the Analysis
and Isolation of Primary and Secondary Metabolites ... 103 Joseph Sherma
Chapter 7 Chambers, Sample Application, and Chromatogram
Development... 119 Tadeusz H. Dzido and Tomasz Tuzimski
Chapter 8 Derivatization, Detection (Quantification),
and Identification of Compounds Online ... 175
Bernd Spangenberg
Chapter 9 Biodetection and Determination of Biological Activity
of Natural Compounds ... 193
Ern Tyihák, Ágnes M. Móricz, and Péter G. Otto
Chapter 10 Forced-Flow Planar Layer Liquid Chromatographic
Techniques for the Separation and Isolation
of Natural Substances ... 215 Emil Mincsovics
Part II
Primary Metabolites
Chapter 11 TLC of Carbohydrates ... 255
Guilherme L. Sassaki, Lauro M. de Souza, Thales R. Cipriani, and Marcello Iacomini
Chapter 12 TLC of Lipids ... 277
Svetlana Momchilova and Boryana Nikolova-Damyanova
Chapter 13 Amino Acids... 299
Ravi Bhushan
Secondary Metabolites—Shickimic Acid Derivatives
Chapter 14 Sample Preparation and TLC Analysis
of Phenolic Acids ... 331 Magdalena Wójciak-Kosior and Anna Oniszczuk
Chapter 15 Application of TLC in the Isolation and Analysis
of Coumarins ... 365
Chapter 16 Application of TLC in the Isolation and Analysis
of Flavonoids ... 405
Marica Medic-Šaric, Ivona Jasprica, Ana Mornar,
andŽeljan Maleš
Chapter 17 TLC of Lignans ... 425
Lubomír Opletal and Helena Sovová
Secondary Metabolites
—Isoprenoids
Chapter 18 TLC of Mono- and Sesquiterpenes ... 451
Angelika Koch, Simla Basar, and Rita Richter
Chapter 19 TLC of Diterpenes ... 481
Michał Ł. Hajnos
Chapter 20 TLC of Triterpenes (Including Saponins) ... 519
Wieslaw Oleszek, Ireneusz Kapusta, and Anna Stochmal
Chapter 21 TLC of Carotenoids ... 543
George Britton
Chapter 22 TLC of Sterols, Steroids, and Related Triterpenoids ... 575
Laurie Dinan, Juraj Harmatha, and Rene Lafont
Chapter 23 TLC of Iridoids... 605
Gra_zyna Zgórka
Secondary Metabolites—Amino Acid Derivatives
Chapter 24 TLC of Indole Alkaloids ... 623
Peter John Houghton
Chapter 25 TLC of Isoquinoline Alkaloids... 641
Monika Waksmundzka-Hajnos and Anna Petruczynik
Chapter 26 TLC of Tropane Alkaloids ... 685 Tomasz Mroczek
Chapter 27 TLC of Alkaloids from the Other Biosynthetic Groups ... 701
Jolanta Flieger
Secondary Metabolites—Compounds Derived
from Acetogenine (Acetylocoenzyme A)
Chapter 28 Polyacetylenes: Distribution in Higher Plants,
Pharmacological Effects, and Analysis ... 757 Lars P. Christensen and Henrik B. Jakobsen
Chapter 29 Quinone Derivatives in Plant Extracts ... 817
Gra_zyna Matysik, Agnieszka Skalska-Kaminska, and Anna
Matysik-Wozniak
1
Overview of the Field
of TLC in Phytochemistry
and the Structure
of the Book
Monika Waksmundzka-Hajnos, Joseph Sherma,
and Teresa Kowalska
CONTENTS
1.1 Survey of Phytochemistry... 3
1.2 Procedures of Thin Layer Chromatography ... 5
1.3 Organization of the Book ... 9
1.1 SURVEY OF PHYTOCHEMISTRY
Phytochemistry is a broad area, generally termed ‘‘plant chemistry.’’ Investigations
in thefield of phytochemistry are important for numerous research disciplines, such
as plant physiology, plant biochemistry, chemosystematics (which is often referred to as chemotaxonomy), plant biotechnology, and pharmacognosy.
Plant physiology focuses on the life processes occurring in plants. Especially
important are the investigations on the influence of various external factors, such as
ultraviolet–visible (UV–Vis) radiation, temperature, the nature of soil, the climate,
etc., on the composition of active compounds contained in plants. One part of this discipline is known as allelopathy. Within the framework of allelopathy, the responses of the plant organisms to external pathological factors (e.g., environmental pollution, the presence of pathogens, insects, etc.) are investigated.
Plant biochemistry focuses on biochemical transformations that play a funda-mental role in the biosynthesis of active compounds contained in plants, which are referred to as primary and secondary metabolites.
Chemosystematics involves the classification of plants on the basis of their
biochemistry and chemistry. It proves to be of special importance when searching
for and collectingfloral specimens. Within the framework of chemosystematics, the
relations are investigated between the classes of plants and the occurrence of
the specific substances or substance groups in the plant tissues.
The most important application of phytochemical investigation methods is to the field of pharmacognosy. Pharmacognosy is a part of the pharmaceutical sciences and is focused on natural products (mainly on plant materials) and the components thereof that show biological activity and are, therefore, used in therapy.
The history of phytotherapy is almost as long as the history of civilization. The
term ‘‘pharmacognosy’’ has been in use for little more than a century, but its
foundations were laid out by early civilizations. The Assyrian, Egyptian, Chinese, and Greek records of great antiquity make reference to the nature and use of herbs and herbal drugs. Knowledge of medicinal plants spread in West Europe and then in the whole Western World, to a large extent through the monasteries and their schools
of medicine. In 16th century, early botanists published herbals—usually illustrated
with the woodcut pictures—describing the nature and use of an increasing number of
plants. In modern science, phytotherapy appeared in the 19th century, when thefirst
biologically active compounds (basically alkaloids) were isolated from the plant material (e.g., morphine, strychnine, narcotine, caffeine, etc.) The golden age of
phytotherapy lasted until 1935, when thefirst sulfonamides and then antibiotics were
synthesized and used in therapy. Then the age of chemotherapy began. However, it is
a widely recognized fact that numerous synthetic drugs exert—along with a positive
therapeutic effect—also harmful and often irreversible side effects. To the contrary,
in the plant world, one very often encounters strongly active substances coexisting with the other compounds that mitigate their negative side effects. Because of this, in recent years a return to phytotherapy has been observed. This return has further been spurred by an appeal of the World Health Organization to screen plant material for the presence of biologically active compounds contained therein and exerting, e.g., a
well pronounced anticancer activity. It isfirmly believed that a great, yet still not
fully revealed, therapeutic potential exists in plants, because so far only a few percent out of 250,000 plant species have been investigated with regard to their usefulness in medicine.
Nowadays, medicines of natural origin are appreciated for their high effective-ness and low toxicity, and they are the widely used commercial products. The market value of herbal preparations selling in United States alone is estimated at several dozen million dollars per year. Plant materials are often obtained from natural sources, although many of the medicinal plants are also cultivated. From these
facts, it is clear that there is a high and increasing need for efficient purity control
of plant material, and further for the assessment of their identity and chemical composition, in order to obtain the expected therapeutic effect.
The paramount goal of pharmacognosy is comprehensive investigations of plant materials by use of physical, chemical, and biological methods, and also the search for a possibility to use these materials as natural medicines. Modern pharmacognosy focuses on the chemical components of the plant materials, including the structure and pharmacological properties that are responsible for their use in therapy. Thus, it can be concluded that the main area of interest is in the chemistry of biogenic compounds (i.e., the chemistry of natural compounds of plant origin). This new approach to the subject of pharmacognosy is based on the dynamic treatment of the natural sources of drugs that takes into account their biochemical transformations and consequently allows synthesis of the new biologically active substances. In that
way, links are being established between pharmacognosy and plant biotechnology, which involves breeding tissue cultures as a source of technological amounts of the biologically active substances.
An interest of modern pharmacognosy, in particular compounds that occur in the
plant materials, is due to their already recognized significance in therapy and also to
the importance of a steady search for new natural substances with a curing potential. In this sense, plant material has to be treated as a source of suitable medicines. The therapeutic effect can be obtained by direct use of plant materials, by use of the plant confections, or by use of substances or substance groups isolated from the plant tissues. The latter case occurs only when a given plant contains highly active sub-stances, e.g., the alkaloids in Secale cornutum, Tuber Aconiti, and Rhizoma Veratri, or cardiac glycosides in Folium Digitalis purpureae and Folium Digitalis lanatae. These materials are an important source of selected alkaloids or cardiac glycosides.
Plant materials, galenic preparations, and isolated compounds proposed for therapy have to meet certain strictly determined standards. With the most important materials, these standards simply are the pharmacopoeial requirements, although a vast number of herbs used in formal and popular medicine are not included in any pharmacopoeia. Standardization of the plant material and of herbal preparations is meant to guarantee their therapeutic value, and it is a result of the investigations on biologically active components. There are a wide number of methods to inves-tigate plant material, namely macroscopic (focused on botanical identity and purity of the plant material); microscopic (mostly histochemical investigations, which
provide the basis for identification of the material); biological (microbiological
and biomolecular investigations and investigations of biological activity); and chem-ical methods. Chemchem-ical investigations of the plant material have a variety of goals, such as determination of the substance groups, quantitative analysis of active compounds, isolation of substances from the plant tissues for their further identi-fication, or physicochemical characterization, and, finally, structural analysis of the isolated unknown compounds.
1.2 PROCEDURES OF THIN LAYER CHROMATOGRAPHY
Among the chemical methods of plant examination, chromatographic analysis plays a very important role, and it has been introduced to all the modern pharmacopoeias. Because of numerous advantages of the chromatographic methods (such as their
specificity and a possibility to use them for qualitative and quantitative analysis),
they comprise an integral part of the medicinal plant analysis.
The following chromatographic methods are most frequently applied in phyto-chemical analysis: one- and dimensional paper chromatography, one- and two-dimensional thin layer chromatography (TLC; also called planar chromatography), high-performance column liquid chromatography (HPLC), gas chromatography (GC), and counter current chromatography (CCC). These methods can also be used for the isolation of the individual components from the component mixtures on a preparative and micropreparative scale.
TLC is a chromatographic technique widely used for qualitative analysis of organic compounds, isolation of the individual compounds from multicomopnent Overview of the Field of TLC in Phytochemistry and the Structure of the Book 5
mixtures, quantitative analysis, and preparative-scale isolation. In many cases, it outperforms the other chromatographic techniques. Firstly, there is a multitude of chromatographic systems that can be applied in TLC. Many kinds of TLC and high-performance TLC (HPTLC) precoated plates are commercially available, e.g., those with the inorganic adsorbent layers (silica or silica gel and alumina); organic layers
(polyamide, cellulose); organic, polar covalently bonded modifications of the silica
gel matrix (diol, cyanopropyl, and aminopropyl); and organic, nonpolar bonded stationary phases (RP2, RP8, RP18) with different densities of coverage of the silica matrix (starting from that denoted as W, for the lowest density of coverage and thus wettable with water). Sorbents applied in TLC have different surface characteristics and, hence, different physicochemical properties. Moreover, there is a wide choice of mobile phases that can be used to separate mixture components; these belong to various selectivity groups and, thus, have different properties as proton donors, proton acceptors, and dipoles. In TLC, ultraviolet (UV) absorption of the mobile
phase solvents does not play a significant negative role in detection and quantification
of the analytes, because the mobile phase is evaporated from the plate prior to the detection. High viscosity of a solvent can be viewed as a sole property limiting its choice as a mobile phase component. These plate and mobile phase characteristics allow a choice from among an unparalleled abundance of TLC systems that offer a broad spectrum of separation selectivities, which is particularly important when complex mixtures of the plant extracts have to be separated.
Another advantage of TLC is that each plate is used only once, thereby allowing simpler sample preparation methods when compared with techniques such as GC and HPLC, in which multiple samples and standards must be applied to the column in sequence. Highly sorbed materials in plant extract samples can be left behind in a column and interfere in the analysis of subsequent samples. Multiple samples can be analyzed at the same time on a single TLC or HPTLC plate, reducing the time and solvent volume used per sample; the processing of standards and samples on the
same plate leads to advantages in the accuracy and precision of quantification by
densitometry.
Last, but not least, TLC enables usage of numerous special development
tech-niques. Most separations are carried out by a capillary flow development with a
single mobile phase (isocratic) in the ascending or horizontal configuration. Gradient
elution with stepwise variations in mobile phase composition, which is widely applied in HPLC, is also used in TLC. Besides, there are the following special modes of developing a chromatogram: unidimensional multiple development (UMD), incremental multiple development (IMD), gradient multiple development (GMD), and bivariant multiple development (BMD). Moreover, the circular and anticircular development methods can also be applied. UMD consists of repeated development of the chromatogram over the same development distance, with a given mobile phase of constant composition and with drying the plate between the individual develop-ment runs. IMD is performed by the stepwise increase in the developdevelop-ment distance (the increment in the development distance is kept constant), using a steady mobile phase composition and drying the plate between the development runs. It results in narrowing of the spots or zones and improved resolution. In GMD, each step of the repeated chromatogram development is performed with a mobile phase of different
composition, thus enabling gradient development. The development distance of the consecutive development runs is kept steady and it is only the mobile phase composition that changes, thus enabling the analysis of complex mixtures span-ning a wide polarity range. When a low strength mobile phase is used, the separation of the low polarity components is achieved on a silica layer. When a medium polarity mobile phase is used, then the medium polarity components are
separated (thefirst group is then eluted to the upper edge of the plate). With the
high polarity mobile phases, separation of the high polar components of plant extracts can be obtained. BMD involves a stepwise change both of the develop-ment distance and the mobile phase composition. With use of a special chamber and computer program, an improved version, known as Automated Multiple Devel-opment (AMD), can be applied, with the distance of the develDevel-opment increasing and the mobile phase strength decreasing at each step. AMD enables the analysis of complex samples over a wide polarity range and provides focusing (tightening) of the zones. In the circular and anticircular development modes, the mobile phase migrates radially from the center to the periphery or from the periphery to the center,
respectively. Analytes with lower RFvalues are better resolved by means of circular
chromatography than by means of linear chromatography, and the advantage of the anticircular mode is that it allows better resolution of compounds with higher
RFvalues.
TLC is also the easiest technique with which to perform multidimensional (i.e., two-dimensional) separations. A single sample is applied in the corner of a
plate, and the layer is developed in the first direction with mobile phase 1. The
mobile phase is dried by evaporation, and the plate is then developed with mobile phase 2 at a right angle (perpendicular or orthogonal direction); mobile phase 2 has different selectivity characteristics when compared with mobile phase 1. In this way, complete separation can be achieved of very complex mixtures (e.g., of the components of a plant extract) over the entire layer surface.
Particularly valuable separation results can be achieved when using various
mobile phase systems to benefit from different separation mechanisms. For example,
with cellulose one can apply a nonaqueous mobile phase to achieve the adsorption mechanism of retention and an aqueous mobile phase to achieve the partition mechanism. In a similar way, with the polar chemically bonded stationary phases one can use nonaqueous mobile phases to achieve the adsorption mechanism of retention and the aqueous mobile phases to achieve the reversed-phase mechanism. Shifting from the adsorption to the partition mode causes marked differences in the separation selectivity.
After performing the separation with the optimum layer, mobile phase, and development technique combination, the zones must be detected. If the zones are
not naturally colored orfluorescent, or do not absorb 254 nm UV light so they can be
viewed asfluorescence-quenched zones on special F-plates containing a fluorescent
indicator, a detection reagent must be applied by spraying or dipping, usually followed by heating. This derivatization is mainly used in the postchromatographic mode for localization of the separated component zones on the layer. Very often universal reagents are used, such as iodine vapors or sulfuric acid. These reagents can locate almost all of the existing organic compound classes. Selective reagents can be Overview of the Field of TLC in Phytochemistry and the Structure of the Book 7
used as derivatizing reagents for individual or group identification of the analytes.
For example, the Dragendorff’s reagent (KBiI4) is used for identification of
hetero-cyclic bases (e.g., of alkaloids), ninhydrin for identification of compounds containing
an amino group in their structure (e.g., of the amines and amino acids), and
2-(diphenylboryloxy)-ethylamineþ polyethylene glycol (PEG) for identification of
polyphenols.
TLC is coupled with densitometry to enable detection of colored, UV-absorbing,
orfluorescent zones through scanning of the chromatograms with visible or UV light
in transmission or reflectance modes. By comparison of a signal obtained with that
for the standards processed with comparable chromatographic conditions, densito-metric measurements can be used for quantitative analysis of the components con-tained in the mixtures. With multiwavelength scanning of the chromatograms, spectral data of the analytes can be directly acquired from the TLC plates and can further be compared with the spectra of the analytes from a software library or from standards developed on the same plate. Thus, a densitometer with a diode array
detector enables direct (in situ) identification of the analytes. Other possibilities to
identify analytes are offered by off-line or on-line coupling of TLC with Fourier transform infrared spectrometry, mass spectrometry, etc.
Further, it is worth noting that TLC coupled with bioautographic detection of microbiologically active compounds can be successfully applied in the analysis of plant extracts. Especially suitable for this purpose is direct bioautography, which uses microorganisms (e.g., bacteria or fungi) growing directly on a TLC plate with the previously separated mixtures of the plant extracts. In this procedure, antibacter-ial or antifungal compounds appear as clear spots (i.e., without microorganisms growing on them) on an intensely colored background. This approach can be used as an additional analytical option in screening of biological samples, as a standard-ization method for medicinal plant extracts, and as a selective detection method.
Additionally, special instruments enable the use of the forced-flow migration of
the mobile phase. Overpressured-layer chromatography (OPLC), also called opti-mum performance laminar chromatography, makes use of a pump that feeds the
sorbent bed with mobile phase at a selectedflow rate. Rotation planar
chromatog-raphy (RPC) uses centrifugal force in order to obtain an analogous effect.
Electro-osmotically driven TLC makes use of electroosmotic flow to force mobile phase
across a layer. All of these forced-flow methods provide a constant flow rate of the
mobile phase; the linear profile of the flow and elimination of vapor phase from
the system may improve system efficiency and peak resolution.
The advantages of TLC are particularly important with plant extracts, which are very complex mixtures of the structurally differentiated chemical compounds. Such extracts very often contain polar (e.g., tannins and phenols) and nonpolar (lipids, chlorophylls, and waxes) ballasts, apart from a fraction of active substances that is of main importance for phytochemistry and pharmacognosy. This latter fraction con-tains closely related compounds of a similar structure and physicochemical proper-ties. Isolation of a fraction of interest from such a mixture requires a complicated
procedure, usually liquid–liquid or solid-phase extraction. TLC enables separation
of a crude plant extract without an earlier purification. For example, in a normal
prewashed with a nonpolar mobile phase prior to the development of a chromato-gram), and the polar fraction remains strongly retained near to the origin; then the fraction of interest is separated in the central part of the chromatogram.
Summing up, TLC is a principal separation technique in plant chemistry research. It can be used in a search for the optimum extraction solvents, for
identification of known and unknown compounds, and—what is at least equally
important—for selection of biologically active compounds. TLC also plays a key
role in preparative isolation of compounds, purification of the crude extracts, and
control of the separation efficiency of the different chromatographic techniques and
systems. TLC has many advantages in plant chemistry research and development. These include single use of stationary phase (no memory effect), wide optimization possibilities with the chromatographic systems, special development modes and detection methods, storage function of chromatographic plates (all zones can be detected in every chromatogram by multiple methods), low cost in routine analysis,
and availability of purification and isolation procedures.
1.3 ORGANIZATION OF THE BOOK
The book comprises 29 chapters, divided into two parts. Part I consists of 10 chapters and provides general information on those areas of science that are related to
phytochemistry and can benefit from the use of TLC. Moreover, it also contains
chapters devoted to the technical aspects of TLC, such as the instrumentation and chromatographic systems involved.
Following this chapter, Chapter 2 focuses on medicinal plants as a source of natural drugs and on their role in modern pharmacy. It also provides a brief over-view of the methods used for the investigation of the plant material and of the
techniques used for the extraction, purification, and final assessment of the drugs
having a plant origin.
Chapter 3 is devoted to the medicines and the diet supplements produced from
plants. Firstly, the authors introduce definitions of the plant medicines and plant diet
supplements, and then they present the history of herbal drugs in the traditional medicines of various cultures throughout the world. The botanical supplements are
then discussed, and the chapter ends by discussing the tasks of TLC in thefield of
botanical drugs and dietary supplements.
Chapter 4 focuses on the primary and the secondary metabolites and their
biological activity. Because classification of the metabolites as primary and
second-ary is not straightforward and can be viewed differently by the different authors, we editors will explain in the next paragraph our own ideas on this very important issue, which shape the structure of the entire volume.
Primary metabolites are those that occur in each plant and fulfill its basic
physiological functions (i.e., appear as the building, energetic, or the reserve mater-ial). In other words, primary metabolites are indispensable for the life of a plant. Secondary metabolites are the products of metabolism and play no crucial role in the
plant’s life. This classification can be regarded as a rough and provisional only, as it
often happens that the secondary metabolites have a well recognized physiological
function in the plants as well. In practice, all metabolites can be classified in different
ways. Most often classification is based on their chemical structure, which generally remains in a good agreement with the biogenetic origin. Sometimes, however,
problems can arise with a straightforward classification of certain groups of
com-pounds that belong to the same biogenetic group and yet completely differ in terms of chemical structure. For example, steroidal alkaloids are traditionally included in the alkaloid group. However, their biogenetic origin is not from amino acids but
from steroids, and for this particular reason in this book they are classified as
steroids. In a similar way, taxoids are sometimes classified as pseudoalkaloids due
to the presence of the tertiary nitrogen atom in the molecules of certain taxoid representatives. At the same time, all taxoids biogenetically belong to the class of
diterpenes. It is noteworthy that classification of metabolites based on their
biogen-etic origin is sometimes impractical, and then it is recommended to refer to their chemical structure or physicochemical properties. For example, naphthoquinones and anthraquinones may originate both from shickimic acid and acetogenine. In fact, quinones can have a different biogenetic origin, but are joined in one group based on their similar physicochemical properties. Iridoids could formally be included in the class of monoterpenes, but this is not done because of their differentiated physico-chemical and pharmacological properties (classical monoterpenes are the volatile compounds present in essential oils, whereas iridoids usually are the nonvolatile species). For the above reasons, we decided to classify the questionable groups of compounds according to their chemical structure. Consequently, all of the
metabolites—both primary and secondary—are additionally divided according to
their biogenesis.
Chapter 5 focuses on chemosystematics, also known as chemotaxonomy. This
chapter starts from definition of this particular branch of phytochemical science,
which involves classification of the plant organisms based on the differences at the
biochemical level, especially in the amino acid sequences of common proteins. Then the author highlights the areas of the main interest for the chemosystematic studies and discusses applicability of the main chromatographic modes to this area of research.
In Chapter 6, the sorbents and precoated layers that are particularly useful in the analysis and preparative isolation of the primary and secondary metabolites from plant extracts are described. This chapter covers virtually all of the TLC and HPTLC analytical and preparative layers used for separation, determination, and isolation
within the field of phytochemistry, including silica gel, reversed phase and
hydro-philic bonded phases, nonsilica sorbents (alumina, cellulose, polyamides, modified
celluloses, and kieselguhr), and miscellaneous layers (resin, impregnated, mixed, and dual layers).
Chapter 7 starts with description of the chromatographic chambers and mobile phase compositions that can be utilized in phytochemical research. Then the authors discuss the development of the chromatograms in the different thin layer chromato-graphic modes. This chapter covers the methods of sample application to the adsorbent layer as well.
Then two chapters following deal with the detection of the analytes after their thin layer chromatographic separation. The main part of Chapter 8 is devoted to derivatization of the plant extract components by use of universal and selective
reagents. The specificity of TLC—not shared with any other chromatographic
mode—results from the possibility of applying several different detection methods
in sequence in order to identify groups or individual analytes. In this chapter,
physical methods of detection are also discussed (such as UV–Vis light absorption,
fluorescence, and mass spectrometry), as well as the methods of quantitative analysis with use of TLC combined with densitometry.
Chapter 9 deals with the methods of biodetection in TLC that enable rapid and selective determination of the biological activity (antibacterial, antifungal, and other) of plant metabolites. In this chapter the mechanisms of bioactivity of the individual compounds are explained.
Part I ends with the description of the forced-flow planar chromatographic
development techniques (Chapter 10), including their influence exerted on
separ-ability of the plant metabolites.
Part II of the book is divided into the chapters that reflect the types of the
metabolites that occur in plants. Chapters 11 through 13 refer to primary metabolites. Chapter 11 deals with the chemistry of carbohydrates, with their occurrence in the plants as mono-, oligo-, and polysaccharides, and also as glycoconjugates. It provides an overview of the recommended analytical methods, including sample preparation, derivatization, and the most suitable TLC systems.
In Chapter 12, different classes of plant lipids are presented, and the TLC systems applied to their separation (including normal- and reversed-phase and
argentation) are discussed. Class separation of lipids, their isolation, and quanti
fica-tion are taken into the account.
Chapter 13 focuses on free amino acids, peptides, and proteins, including their occurrence in plants and the use of TLC to separate the individual groups of these compounds.
The next part of the book deals with the secondary metabolites occurring in plant tissues, and it is divided into sections according to the metabolic pathways in which individual substances are synthesized.
Chapter 14 starts with the phenolic compounds that belong to the metabolic pathway of shickimic acid, i.e., phenols, phenolic acids, and tannins. It describes the
structure and classification of these compounds, their biological importance, sample
preparation methods, and the various TLC systems and special techniques that are used for their separation and analysis.
Chapter 15 deals with coumarins that belong to the phenol class and are also derived from shickimic acid. Details are provided on sample preparation and
isola-tion of coumarins with aid of classical TLC and HPTLC and the forced-flow planar
chromatographic techniques. Application of TLC to the measurement of biological activity of coumarins is also described. The chapter ends with tables of the plant families in which coumarins occur.
Chapter 16 is dedicated to the phenolic compounds originating from a similar
pathway as coumarins, i.e., flavonoids. After a short introduction on chemistry,
biochemistry, and medical significance of flavonoids, the methods for their analysis
using various TLC systems are presented, including forced-flow development
tech-niques. In this chapter, sample preparation methods and quantification of flavonoids
by means of TLC combined with slit-scanning and video densitometry are discussed. Overview of the Field of TLC in Phytochemistry and the Structure of the Book 11
The section of the book on secondary metabolites ends with lignans, also originating from shickimic acid. Chapter 17 is focused on the chemistry, occurrence in plant material, and pharmacological activity of the representatives of this group, followed by the sample preparation techniques and the TLC analysis of these
compounds. Details about quantification of lignans in herbal extracts and
prepar-ations are also reported.
The next section of the book is focused on isoprenoid derivatives, which include several groups of compounds. It starts with Chapter 18 on the volatile compounds
(mono- and sesquiterpenes), including their definition, classification, occurrence, and
importance. Then the following applications of planar chromatography are
dis-cussed: identification of the volatile fractions in pharmaceutical drugs, taxonomic
investigations, tracing of various adulterations, and analysis of cosmetics.
Chapter 19 covers diterpenoids and presents their structure, physicochemical properties, natural occurrence, and pharmacological activity. The details of sample preparation and the analytical and preparative TLC separations of this group of compounds with the aid of different chromatographic systems are described,
includ-ing derivatization and quantification methods. The chapter ends with a comparison of
the performance of TLC with that of the other chromatographic and related tech-niques used in diterpenoid analysis.
The next group of compounds that belong to the isoprenoid methabolic pathway are triterpenes, and they are described in Chapter 20. After a short introduction on structure and properties of this group, chromatographic systems and detection methods applied in the analysis of triterpenes (saponins included) are presented. The chapter emphasizes the role of planar chromatography as a technique supporting
column chromatography in identification and determination of the biological activity
of triterpenes.
Chapter 21 focuses on tetra- and polyterpenes, and among them carotenoids represent the most important group of compounds. First, structure, occurrence, and properties are presented. Then the special aspects of the TLC analysis (such as detection and instability of carotenoids) are emphasized. The use of silica and alumina, and also of the basic normal phase adsorbents, is discussed. Usefulness of TLC in screening of the plant material, in preparative separations, and in isolation of individual carotenoids is also described.
The next large group of compounds that belong to the isoprenoid pathway are steroids, and they are presented in Chapter 22. In the introductory part of this chapter, the chromatographic systems and techniques useful for planar separation of steroids are described. Then an overview of the literature is provided, taking into the account the classes of phytosterols, steroids (brassinosteroids, bufadienolides, cardenolides, ecdysteroids, steroidal saponins, steroidal alkaloids, vertebrate-type steroids, and withanolides), and of the related triterpenoids (cucurbitacins).
Struc-tural diversity, the separation systems, and the detection and quantification for each
class of compounds are presented.
Iridoides are the last group of compounds that belong to the isoprenoid pathway, and they are described in Chapter 23. After the introductory part on the structure and physicochemical properties of iridoides, the issues are emphasized related to
isolation of this group of compounds from the plant material and to sample prepar-ation. Then the TLC systems and techniques applied to the analysis of iridoids are
described, taking into account the detection methods and the forced-flow techniques.
The preparative layer chromatography of iridoids is also discussed.
The next four consecutive chapters deal with alkaloids synthesized in the plant organisms from amino acids. There are several groups of alkaloids differing in their structure, properties, and biological activity.
Chapter 24 focuses on indole alkaloids. Firstly, the chemical structure, occur-rence, and pharmacological, ecological, and chemosystematic importance of this group are discussed. This preliminary information is followed by a detailed descrip-tion of the TLC separadescrip-tions of indole alkaloids, including chromatographic systems, techniques, and detection methods. Details on the separations of the particular types of indole alkaloids are also presented.
Chapter 25 is devoted to the structure, properties, and biological activity of isoquinoline alkaloids. Information concerning problems with chromatographic sep-aration of basic compounds is also provided, and the normal phase, reversed phase, and pseudoreversed phase systems are described in detail. The use of TLC plates and
grafted plates for two-dimensional separations and forced-flow techniques applied to
the separation of the isoquinoline alkaloids are presented. Examples of TLC appli-cations to quantitative analysis are shown, along with the preparative separations.
Tropane alkaloids are handled in Chapter 26. Chemistry and stereochemistry of
tropane and the related alkaloids, and their natural occurrence, are presented first.
Various methods of extraction of this entire group of compounds from plant material
are described, followed by the pretreatment of the extracts by liquid–liquid
partition-ing (LLP), solid assisted liquid–liquid partitioning (Extrelut), and solid-phase
extrac-tion (SPE). Then the informaextrac-tion on TLC of tropane alkaloids including their
quantification is provided. The chapter gives detailed information on the OPLC
analysis of tropane alkaloids, and a comparison is made with the results originating from the other separation techniques in use.
Chapter 27 focuses on the remaining groups of alkaloids, including phenylethy-lamine derivatives, quinoline derivatives (Cinchona alkaloids), and pyrrolidine, pyrrolizidine, piridine, and piperidine derivatives (Tobacco, Lobelia, Pepper, Pelle-tierine, Sedum, Senecio alkaloids), quinolizidine alkaloids (Lupine alkaloids), xanthine, imidazole derivatives, and diterpene alkaloids. Preparation of extracts, the most frequently employed TLC systems, and the detection methods applicable to each individual group are presented.
The last two chapters are devoted to the secondary metabolites derived from acetogenine (acetylocoenzym A). Chapter 28 deals with the distribution of poly-acetylenes in plants and pharmacological activity of polypoly-acetylenes. Separation, detection, and isolation by means of TLC in various different systems are described. The results are compared with those originating from HPLC.
Chapter 29 is focused on quinones (antraquinones and naphthoquinones), their occurrence in plants, and pharmacological activity. Applicability of the conventional TLC techniques applied to the separation of quinines, and also of the special modes (e.g., gradient or two-dimensional TLC), is discussed.
The authors who agreed to contribute chapters to the book are all recognized
international experts in their respectivefields. The book will serve as a comprehensive
source of information and training on the state-of-the-art phytochemistry methods performed with aid of TLC. It will help to considerably popularize these methods for
practical separations and analyses in afield that will undoubtedly grow in importance
for many years to come. A computer assisted search has found no previous book on
TLC in phytochemistry. Three editions of the book ‘‘Phytochemical Methods’’
(1973, 1984, and 1998) by J.B. Harborne (Chapman and Hall, London, UK) had chapters organized by compound type, most of which contained some information
on TLC analysis. A chapter on‘‘Thin Layer Chromatography in Plant Sciences’’ by
J. Pothier was contained in the book‘‘Practical Thin Layer Chromatography’’ edited
by B. Fried and J. Sherma (CRC Press, 1996), a chapter on planar chromatography in
medicinal plant research in ‘‘Planar Chromatography’’ edited by Sz. Nyiredy
(Springer, 2001) and a chapter on natural mixtures by M. Waksmundzka-Hajnos
et al. in‘‘Preparative Layer Chromatography’’ edited by T. Kowalska and J. Sherma
(CRC=Taylor & Francis, 2006) included information on plant extracts. A book by
E. Reich and A. Schibli (Thieme Medical Publishers, Inc., 2007) covers the theor-etical concepts and practical aspects of modern HPTLC as related to the analysis of herbal drugs.
However, these information sources are not comprehensive, and thefirst two are
now out of date. Our proposed book will solve this void in information in the critical field of phytochemical analysis.
2
Plant Materials
in Modern Pharmacy
and Methods of Their
Investigations
Krystyna Skalicka-Wozniak, Jaros
/law Widelski,
and Kazimierz G
/lowniak
CONTENTS
2.1 Plant Material and Marine Products as Sources of Active
Secondary Metabolites ... 16
2.2 The Distribution and Concentration of Natural Compounds with
Biological Activity in Different Organs of Medicinal Plants... 17
2.3 Methods of Investigations of Plant Material ... 18
2.3.1 Macroscopic Investigations ... 18
2.3.2 Microscopic and Microchemical Methods of Investigations ... 18
2.3.3 Chemical Methods of Investigations ... 20
2.3.3.1 Approximate Group Identification... 20
2.3.3.2 Quantitative Analysis of Active Compounds
in Plant Material by Various Methods
(Titration, Spectrophotometric Methods) ... 20
2.3.3.3 Isolation of Active Compounds... 21
2.3.4 Biological Methods of Investigations... 21
2.4 Modern Extraction Methods of Active Compounds from Plant Material
and Marine Products ... 22
2.4.1 Classic Extraction Methods in Soxhlet Apparatus ... 22
2.4.2 Supercritical Fluid Extraction ... 23
2.4.3 Pressurized Liquid Extraction... 25
2.4.4 Medium-Pressure Solid–Liquid Extraction Technique ... 27
2.5 Purification of Crude Extracts and Sample Preparation ... 27
2.5.1 Liquid–Liquid Partition ... 27
2.5.2 Solid Phase Extraction ... 27
2.5.3 Gel Permeation Chromatography ... 29
2.6 Chromatographic Methods and Their Role in Investigations
of Plant Material ... 29
2.6.1 Gas Chromatography ... 29
2.6.2 High-Performance Liquid Chromatography ... 29
2.6.3 Electrophoresis and Electrochromatography ... 30
2.6.4 Coupled Methods (GC–MS, LC–MS, LC–NMR) ... 32
References ... 32
Pharmacognosy is the science which treats of the history, production, commerce,
collection, selection, identification, valuation, preservation and use of drugs and
other economic materials of plant and animal origin.
The term‘‘pharmacognosy’’ derived from the ancient Greek words pharmakon,
drug or medicine, and gnosis, knowledge, and literally means the knowledge of drugs.
2.1 PLANT MATERIAL AND MARINE PRODUCTS AS SOURCES
OF ACTIVE SECONDARY METABOLITES
Drugs are derived from the mineral, vegetable, and animal kingdoms. They may occur in the crude or raw form, as dried or fresh unground or ground organs or organisms or natural exudations of these (juice or gum), when they are termed ‘‘crude drugs.’’
These are known as herbal medicinal products (HMPs), herbal remedies, or phytomedicines and include, for example:
. Herb of St. John’s wort (Hypericum perforatum), used in the treatment of
mild to moderate depression
. Leaves of Gingko biloba, used for cognitive deficiencies (often in the
elderly), including impairment of memory and affective symptoms such as anxiety
There are also derived substances, such as alkaloids (e.g., caffeine, from the coffee
shrub—Coffea arabica—used as a stimulan), glycosides (e.g., digoxin and other
digitalis glycosides, from foxglove—Digitalis spp.—used to treat heart failure),
alcohols, esters, aldehydes, or other constituents or mixtures of constituents isolated from the plant or animal.
Finally, also pure chemical entities exist, which are produced synthetically and
referred to as ‘‘nature identical’’, but originally discovered from plant drugs.
Examples include:
. Morphine, from opium poppy (Papaver somniferum), used as an analgesic
. Quinine, from Cinchona bark (Cincoina spp.), used in the treatment of
Also many foods are known to have beneficial effects on health:
. Garlic, ginger, and many other herbs and spices
. Anthocyanin- orflavonoid-containing plants such as bilberries, cocoa, and
red wine
. Carotenoid-containing plants such as tomatoes, carrot, and many other
vegetables [1]
2.2 THE DISTRIBUTION AND CONCENTRATION OF NATURAL
COMPOUNDS WITH BIOLOGICAL ACTIVITY IN DIFFERENT
ORGANS OF MEDICINAL PLANTS
Isolated pure natural products such as numerous pharmaceuticals used in pharmacy
are thus not‘‘botanical drugs,’’ but rather chemically defined from nature. Botanical
drugs are generally derived from specific plant organs of plant species. The
follow-ing plant organs are the most important:
. Herba or aerial parts (herba)
. Leaf ( folium) . Flower (flos) . Fruit ( fructus) . Bark (cortex) . Root (radix) . Rhizome (rhizoma) . Bulb (bulbus)
Fruits and seeds have yielded important phytotherapeutic products, e.g., caraway (Carum carvi), fennel (Foeniculum vulgare), saw palmetto (Serenoa repens), horse chestnut seeds (Aesculus hippocastanum), or ispaghula (Plantago ovata), which are used often in phytotherapy.
Numerous drugs contain also leaf material as the main component. Some widely
used ones include balm (Melissa officinalis), deadly nightshade (Atropa belladonna),
ginkgo (Ginkgo biloba) peppermint (Mentha3 piperita), bearberry (Arctostaphylos
uva-ursi), and many others.
Although theflowers are of great botanical importance, they are only a minor
source of drugs used in phytotherapy. One of the most important example is
chamomile (Chamomilla recutita (Matricariase flos)). Other examples include
calendula (Calendula officinalis) and arnica (Arnica montana).
Stem material which is often a part of those drugs is derived from all above-ground parts, e.g., ephedra (Ephedra sinica), hawthorn (Crataegus monogyna and
Crataegus oxyacantha), passion flower (Passiflora incarnata), or wormwood
(Arthemisia absynthium). Also parts of the stem are used in phytotherapy like bark of Rhamnus frangula (frangula) or bark of Salix alba (willow).
Finally, underground organs (rhizome and root) of many species have yielded
pharmaceutically important drugs. Examples include: Devil’s claw (Harpagophytum
procubens), tormentill (Potentilla erecta), rhubarb (Rheum palmatum), and kava-kava (Piper methysticum) [1].
2.3 METHODS OF INVESTIGATIONS OF PLANT MATERIAL
The analytical side of pharmacognosy is embraced in the expression‘‘the evaluation
of the drug,’’ for this includes the identification of a drug and determination of its
quality and purity.
The identification of the drug is of first importance, for little consideration can be
given to an unknown drug as regards its quality and purity. The identification of a
drug can be established by actual collection of the drug from plant or animal (among
them marine organism) which can be positively identified from the botanical or
zoological standpoint. This method is rarely followed except by an investigator of the drug, who must be absolutely sure of the origin of his samples. For this reason ‘‘drug gardens’’ are frequently established in the connection with institution of pharmacognostical research.
Quality of a drug refers to intrinsic value of the drug, that is, to the amount of medicinal principles or active constituents present in the drug. A high grade of quality in the drug is such importance that effort should be made to obtain and maintain this high quality. The most important factors to accomplish this include: collection of the drug from the correct natural source at proper time and in the proper manner, the preparation of the collected drug by proper cleaning, drying and garbling and proper preservation of the clean, dry, pure drug against contamination with dirt,
moisture, fungi,filth, and insects.
The evaluation of the drug involves a number of methods, which may be classified
as follows: organoleptic, microscopic, biological, chemical, and physical [2].
2.3.1 M
ACROSCOPICI
NVESTIGATIONSOrganoleptic (lit.‘‘impression on the organs’’) refers to evaluation by means of the
organs of sense, and includes the macroscopic appearance of the drug, its odor and
taste, occasionally the sound or‘‘snap’’ of its facture, and the ‘‘feel’’ of the drug to
the touch.
For convenience of description the macroscopic characteristic of a drug may be divided into four headings, viz.: shape and size, color and external markings, fracture
and internal color, andfinally odor and taste.
For example, description of linseed (Linum usitatissimum) is as follows: The seed is exalbuminous, of compressed ovate or oblonglanceolate outline, pointed at one end, rounded at the other and from 4 to 6 mm in length; externally glabrous and shiny, brown to dusky red with a pale-yellow, linear raphe along one edge; the hilum and microphyle in a slight depression near the pointed end; odor slight, becoming very characteristic in the ground or crushed drug; taste mucilaginous and oily [3].
2.3.2 M
ICROSCOPIC ANDM
ICROCHEMICALM
ETHODS OFI
NVESTIGATIONSMicroscopical methods of valuing drugs are indispensable in the identification of
of their adulterans, for these possess few features other than color, odor, and taste whereby clues toward their identity may be afforded. Moreover, owing to the
similarity of some plant organs of allied species, definite identification even of
certain entire, cellular vegetable drugs cannot be made without the examination of mounts of thin sections of them under a microscope. Every plant possess a charac-teristic histology in respect to its organs and diagnostic features of these are ascer-tained though the study of the tissues and their arrangement, cell walls and cell contents, when properly mounted in suitable stains, reagents or mounting media [3]. Some characteristic features can be easily used to establish botanical identity and quality of the drugs. The typical example is the various types of crystals formed by calcium oxylate. Several species of the family Solanaceae are used for obtaining atropine, alkaloid used as spasmolytic in cases of gastrointestinal cramps and asthma. Species containing high amount of atropine like Atropa belladonna (deadly nightshade), Datura stramonium (thorn apple), or Hyoscyamus niger (henbane) are characterized by typical crystal structures of oxalate: sand, cluster crystals and microspheroidal crystals, respectively. These are subcellular crystal structures, which can be easily detected using polarized light and are thus a very useful diagnostic means.
Second typical example are the glandular hairs, which are characteristic for two families (Lamiaceae and Asteraceae) containing many species with essential oils.
Figure 2.1 shows diagnostic features of botanical drugs—microscopic examination
View from side
FIGURE 2.1 Diagnostic features of botanical drugs, that are revealed upon microscopic examination include typical glandular hair as found in the Lamiaceae (a) and Asteraceae (b). Top: lateral view; bottom: view from above. (From Heinrich, M. et al., Fundamentals of Pharmacognosy and Phytotherapy, Elsevier Science, Churchill Livingstone, 2004.)
including typical glandular hair (Lamiaceae and Asteraceae family)—lateral view and view from above.
In many instances, a good idea of the quality of a drug can be ascertained by using microchemical methods. These may consist of examining mounts of sections or powdered drug in various reagents which either form salts of contained active
principles, that have constant characters (microcrystallization) or show definite color
reactions, or of isolation of constituents of the powdered drug with a suitable solvent, filtering 2 or 3 drops of the extract on to a slide, permitting the solvent to evaporate and examining the residue. There is also possible isolation of a constituents by microsublimation.
It is often possible to detrmine whether a powdered drug has been exhausted by examining the crystals found in its sublimate. These have been found to be charac-teristic for many drugs. Microsublimation upon a slide is a superior technique in comparison with test tube sublimation. The sublimates may be directly examined under the microscope without mechanical alteration.
2.3.3 C
HEMICALM
ETHODS OFI
NVESTIGATIONS2.3.3.1 Approximate Group Identification
Identification of the characteristic group or groups of active constituents is one of
the basic methods of the evaluation of the drug and the first step in isolation
procedure.
For example, Borntrager’s test is commonly applied to all anthraquinone drugs.
As effect of the reaction a deep rose color is produced.
Another example is a reaction that give acids and flavonoids with Arnov’s
reagent phenolic (the products of this reaction give purple color). All of these reactions are also used both for qualitative and quantitative analysis (colometric reactions).
Characteristic reaction forflavonoids, like 1% methanolic solution of AlCl3, 5%
methanolic solution of KOH, and 1% methanolic solution of Naturstoffreagenz A, are used for derivatization of TLC plates. It enables general evaluation of different
groups of active compounds, in this caseflavonoids.
2.3.3.2 Quantitative Analysis of Active Compounds in Plant Material by Various Methods (Titration, Spectrophotometric Methods) Evaluation of plant drugs uses all of the methods known in chemical analysis. Among them we can single out the titration. Titration is a common laboratory method of quantitative chemical analysis which can be used to determine the concentration of known reactant. Because volume measurements play a key role in titration, it is also known as volumetric analysis. A reagent, called titrant, of known concentration (a standard solution) is used to react with a measured quantity of reactant (the analyte). Titration is used in quantitative analysis of tropan alkaloids, where KOH is used as a titrant and methyl red as the indicator.
Spectrophotometric techniques are used to measure the concentration of solutes in solution by measuring the amount of light that is absorbed by the solution in a
cuvette placed in the spectrophotometer. According the Beer–Lambert Law there is the linear realationship between absorbance and concentration of an absorbing species. It enables a quantitative determination of compounds in which solutions absorb light. For example total concentration of pyrrolizidine alkaloids in
Symohy-tum officinale root were investigated using UV-VIS spectra of adducts of
3,4-dehydroPAs and Erlich’s reagent [4].
2.3.3.3 Isolation of Active Compounds
When a crude extract obtained by a suitable extraction procedure shows interesting activity (e.g., an antibacterial activity), demonstrated in bioassay, the next and one of
the most difficult steps is to fractionate the extract using a different (sometimes
combined) separation method(s) so that a purified biologically active component can
be isolated.
Figure 2.2 gives a general isolation protocol starting with selection of biomass (e.g., plant, microbe or tissue culture), which is then extracted using different extraction methods. Hydrophilic (polar) extracts will then usually undergo ion exchange chromatography with bioassay of generated fractions. A further ion exchange method of bioactive fraction would yield pure compounds, which could next be submitted for structure elucidation (MS, NMR).
2.3.4 B
IOLOGICALM
ETHODS OFI
NVESTIGATIONSThe biological evaluation of the plant drugs is one of the most important issues of pharmacognosy. For drugs obtained from natural sources, all active compounds present in the plant are responsible for therapeutic effect.
There are plenty of methods for evaluation of biological properties of plant drug. For example, bacteria, such as Staphylococcus aureus are used to determine antiseptic value of the drugs. For standardization of Digitalis spp. (Foxglove) and
other‘‘heart tonic’’ drugs, pigeons and cats are used. Bioassay is the use of biological
system to detect properties of a mixture or a pure compound.
Active fractions Hydrophilic extract Lipophilic extract Extraction (soxhlet or hot/cold percolation) Organism selection Purified extract Gel chromatography Biotage flash chromatography TLC Biotage flash chromatography Ion exchange chromatography Partitioning Ion exchange chromatography HPLC Active fractions Active compounds Active compounds Structure elucidation
FIGURE 2.2 General isolation strategy for purification of bioactive natural products. (From Heinrich, M. et al., Fundamentals of Pharmacognosy and Phytotherapy, Elsevier Science, Churchill Livingstone, 2004.)
