Sắc ký lớp mỏng trong phân tích hóa thực vật - TLC in Phytochemistry (2008)

Sắc ký lớp mỏng trong phân tích hóa thực vật

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,

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 3edited 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 Acidsand 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 Mohrand 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 Shermaand 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: Techniquesand 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, Joseph Sherma, and Teresa Kowalska

Teresa Kowalska

University of Silesia Katowice, Poland

Thin 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.

Preface xiiiEditors xvContributors xix

Part I

and the Structure of the Book 3Monika Waksmundzka-Hajnos, Joseph Sherma,

and Teresa Kowalska

and Their Biological Activity 59Ioanna Chinou

Christian Zidorn

and Isolation of Primary and Secondary Metabolites 103Joseph Sherma

Development 119Tadeusz H Dzido and Tomasz Tuzimski

ix

Chapter 8 Derivatization, Detection (Quantification),

Bernd Spangenberg

of Natural Compounds 193

Techniques for the Separation and Isolation

of Natural Substances 215Emil Mincsovics

Svetlana Momchilova and Boryana Nikolova-Damyanova

Ravi Bhushan

Secondary Metabolites—Shickimic Acid Derivatives

of Phenolic Acids 331Magdalena Wójciak-Kosior and Anna Oniszczuk

of Coumarins 365

x

Chapter 16 Application of TLC in the Isolation and Analysis

of Flavonoids 405

Lubomír Opletal and Helena Sovová

Angelika Koch, Simla Basar, and Rita Richter

Wieslaw Oleszek, Ireneusz Kapusta, and Anna Stochmal

George Britton

Laurie Dinan, Juraj Harmatha, and Rene Lafont

Secondary Metabolites—Amino Acid Derivatives

Peter John Houghton

Monika Waksmundzka-Hajnos and Anna Petruczynik

Chapter 26 TLC of Tropane Alkaloids 685

Tomasz Mroczek

Jolanta Flieger

Secondary Metabolites—Compounds Derived

from Acetogenine (Acetylocoenzyme A)

Pharmacological Effects, and Analysis 757Lars P Christensen and Henrik B Jakobsen

xii

Part I

1 Overview of the Field

of TLC in Phytochemistry and the Structure

1.1 SURVEY OF PHYTOCHEMISTRY

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

etc., on the composition of active compounds contained in plants One part of thisdiscipline is known as allelopathy Within the framework of allelopathy, theresponses of the plant organisms to external pathological factors (e.g., environmentalpollution, the presence of pathogens, insects, etc.) are investigated

Plant biochemistry focuses on biochemical transformations that play a mental role in the biosynthesis of active compounds contained in plants, which arereferred to as primary and secondary metabolites

biochemistry and chemistry It proves to be of special importance when searching

relations are investigated between the classes of plants and the occurrence of

3

The most important application of phytochemical investigation methods is to thefield of pharmacognosy Pharmacognosy is a part of the pharmaceutical sciences and

is focused on natural products (mainly on plant materials) and the componentsthereof that show biological activity and are, therefore, used in therapy

The history of phytotherapy is almost as long as the history of civilization The

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 herbsand herbal drugs Knowledge of medicinal plants spread in West Europe and then inthe whole Western World, to a large extent through the monasteries and their schools

biologically active compounds (basically alkaloids) were isolated from the plantmaterial (e.g., morphine, strychnine, narcotine, caffeine, etc.) The golden age of

synthesized and used in therapy Then the age of chemotherapy began However, it is

in the plant world, one very often encounters strongly active substances coexistingwith the other compounds that mitigate their negative side effects Because of this, inrecent years a return to phytotherapy has been observed This return has further beenspurred by an appeal of the World Health Organization to screen plant material forthe presence of biologically active compounds contained therein and exerting, e.g., a

fully revealed, therapeutic potential exists in plants, because so far only a few percentout of 250,000 plant species have been investigated with regard to their usefulness inmedicine

Nowadays, medicines of natural origin are appreciated for their high ness and low toxicity, and they are the widely used commercial products The marketvalue of herbal preparations selling in United States alone is estimated at severaldozen million dollars per year Plant materials are often obtained from naturalsources, although many of the medicinal plants are also cultivated From these

of plant material, and further for the assessment of their identity and chemicalcomposition, in order to obtain the expected therapeutic effect

The paramount goal of pharmacognosy is comprehensive investigations of plantmaterials by use of physical, chemical, and biological methods, and also the searchfor a possibility to use these materials as natural medicines Modern pharmacognosyfocuses on the chemical components of the plant materials, including the structureand pharmacological properties that are responsible for their use in therapy Thus, itcan be concluded that the main area of interest is in the chemistry of biogeniccompounds (i.e., the chemistry of natural compounds of plant origin) This newapproach to the subject of pharmacognosy is based on the dynamic treatment of thenatural sources of drugs that takes into account their biochemical transformationsand 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 thebiologically active substances.

An interest of modern pharmacognosy, in particular compounds that occur in the

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 theplant confections, or by use of substances or substance groups isolated from the planttissues 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 fortherapy have to meet certain strictly determined standards With the most importantmaterials, these standards simply are the pharmacopoeial requirements, although avast number of herbs used in formal and popular medicine are not included in anypharmacopoeia Standardization of the plant material and of herbal preparations ismeant 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 tigate plant material, namely macroscopic (focused on botanical identity and purity

inves-of the plant material); microscopic (mostly histochemical investigations, which

and biomolecular investigations and investigations of biological activity); and ical methods Chemical investigations of the plant material have a variety of goals,such as determination of the substance groups, quantitative analysis of activecompounds, isolation of substances from the plant tissues for their further identi-fication, or physicochemical characterization, and, finally, structural analysis of theisolated unknown compounds

chem-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

they comprise an integral part of the medicinal plant analysis

The following chromatographic methods are most frequently applied in chemical analysis: one- and two-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 beused for the isolation of the individual components from the component mixtures

phyto-on a preparative and micropreparative scale

TLC is a chromatographic technique widely used for qualitative analysis oforganic compounds, isolation of the individual compounds from multicomopnent

mixtures, quantitative analysis, and preparative-scale isolation In many cases, itoutperforms the other chromatographic techniques Firstly, there is a multitude ofchromatographic systems that can be applied in TLC Many kinds of TLC and high-performance TLC (HPTLC) precoated plates are commercially available, e.g., thosewith the inorganic adsorbent layers (silica or silica gel and alumina); organic layers

gel matrix (diol, cyanopropyl, and aminopropyl); and organic, nonpolar bondedstationary phases (RP2, RP8, RP18) with different densities of coverage of the silicamatrix (starting from that denoted as W, for the lowest density of coverage and thuswettable with water) Sorbents applied in TLC have different surface characteristicsand, hence, different physicochemical properties Moreover, there is a wide choice ofmobile phases that can be used to separate mixture components; these belong tovarious selectivity groups and, thus, have different properties as proton donors,proton acceptors, and dipoles In TLC, ultraviolet (UV) absorption of the mobile

of the analytes, because the mobile phase is evaporated from the plate prior to thedetection High viscosity of a solvent can be viewed as a sole property limiting itschoice as a mobile phase component These plate and mobile phase characteristicsallow a choice from among an unparalleled abundance of TLC systems that offer abroad spectrum of separation selectivities, which is particularly important whencomplex mixtures of the plant extracts have to be separated

Another advantage of TLC is that each plate is used only once, thereby allowingsimpler sample preparation methods when compared with techniques such as GCand 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 acolumn and interfere in the analysis of subsequent samples Multiple samples can beanalyzed at the same time on a single TLC or HPTLC plate, reducing the time andsolvent volume used per sample; the processing of standards and samples on the

densitometry

Last, but not least, TLC enables usage of numerous special development

elution with stepwise variations in mobile phase composition, which is widelyapplied in HPLC, is also used in TLC Besides, there are the following specialmodes 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 anticirculardevelopment 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 ment runs IMD is performed by the stepwise increase in the development distance(the increment in the development distance is kept constant), using a steady mobilephase composition and drying the plate between the development runs It results innarrowing of the spots or zones and improved resolution In GMD, each step of therepeated chromatogram development is performed with a mobile phase of different

composition, thus enabling gradient development The development distance ofthe consecutive development runs is kept steady and it is only the mobile phasecomposition that changes, thus enabling the analysis of complex mixtures span-ning a wide polarity range When a low strength mobile phase is used, theseparation of the low polarity components is achieved on a silica layer When amedium polarity mobile phase is used, then the medium polarity components are

high polarity mobile phases, separation of the high polar components of plantextracts can be obtained BMD involves a stepwise change both of the develop-ment distance and the mobile phase composition With use of a special chamberand computer program, an improved version, known as Automated Multiple Devel-opment (AMD), can be applied, with the distance of the development increasing andthe mobile phase strength decreasing at each step AMD enables the analysis ofcomplex samples over a wide polarity range and provides focusing (tightening) ofthe zones In the circular and anticircular development modes, the mobile phasemigrates radially from the center to the periphery or from the periphery to the center,

chromatography than by means of linear chromatography, and the advantage ofthe anticircular mode is that it allows better resolution of compounds with higher

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

mobile phase is dried by evaporation, and the plate is then developed with mobilephase 2 at a right angle (perpendicular or orthogonal direction); mobile phase 2has different selectivity characteristics when compared with mobile phase 1 In thisway, complete separation can be achieved of very complex mixtures (e.g., of thecomponents of a plant extract) over the entire layer surface

Particularly valuable separation results can be achieved when using various

with cellulose one can apply a nonaqueous mobile phase to achieve the adsorptionmechanism of retention and an aqueous mobile phase to achieve the partitionmechanism In a similar way, with the polar chemically bonded stationary phasesone can use nonaqueous mobile phases to achieve the adsorption mechanism ofretention and the aqueous mobile phases to achieve the reversed-phase mechanism.Shifting from the adsorption to the partition mode causes marked differences in theseparation selectivity

After performing the separation with the optimum layer, mobile phase, anddevelopment technique combination, the zones must be detected If the zones are

indicator, a detection reagent must be applied by spraying or dipping, usuallyfollowed by heating This derivatization is mainly used in the postchromatographicmode for localization of the separated component zones on the layer Very oftenuniversal reagents are used, such as iodine vapors or sulfuric acid These reagents canlocate almost all of the existing organic compound classes Selective reagents can be

used as derivatizing reagents for individual or group identification of the analytes.

an amino group in their structure (e.g., of the amines and amino acids), and

polyphenols

TLC is coupled with densitometry to enable detection of colored, UV-absorbing,

for the standards processed with comparable chromatographic conditions, 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 canfurther be compared with the spectra of the analytes from a software library or fromstandards developed on the same plate Thus, a densitometer with a diode array

identify analytes are offered by off-line or on-line coupling of TLC with Fouriertransform infrared spectrometry, mass spectrometry, etc

Further, it is worth noting that TLC coupled with bioautographic detection ofmicrobiologically active compounds can be successfully applied in the analysis ofplant extracts Especially suitable for this purpose is direct bioautography, whichuses microorganisms (e.g., bacteria or fungi) growing directly on a TLC plate withthe previously separated mixtures of the plant extracts In this procedure, antibacter-ial or antifungal compounds appear as clear spots (i.e., without microorganismsgrowing on them) on an intensely colored background This approach can be used

as an additional analytical option in screening of biological samples, as a ization method for medicinal plant extracts, and as a selective detection method

the mobile phase Overpressured-layer chromatography (OPLC), also called mum performance laminar chromatography, makes use of a pump that feeds the

chromatog-raphy (RPC) uses centrifugal force in order to obtain an analogous effect

The advantages of TLC are particularly important with plant extracts, which arevery complex mixtures of the structurally differentiated chemical compounds Suchextracts 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 ofmain 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

phase system a nonpolar fraction moves with the mobile phase front (or it can be

prewashed with a nonpolar mobile phase prior to the development of a gram), and the polar fraction remains strongly retained near to the origin; then thefraction of interest is separated in the central part of the chromatogram.

chromato-Summing up, TLC is a principal separation technique in plant chemistryresearch It can be used in a search for the optimum extraction solvents, for

systems TLC has many advantages in plant chemistry research and development.These include single use of stationary phase (no memory effect), wide optimizationpossibilities with the chromatographic systems, special development modes anddetection methods, storage function of chromatographic plates (all zones can bedetected in every chromatogram by multiple methods), low cost in routine analysis,

1.3 ORGANIZATION OF THE BOOK

The book comprises 29 chapters, divided into two parts Part I consists of 10 chaptersand provides general information on those areas of science that are related to

chapters devoted to the technical aspects of TLC, such as the instrumentation andchromatographic systems involved

Following this chapter, Chapter 2 focuses on medicinal plants as a source ofnatural 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

having a plant origin

Chapter 3 is devoted to the medicines and the diet supplements produced from

supplements, and then they present the history of herbal drugs in the traditionalmedicines of various cultures throughout the world The botanical supplements are

botanical drugs and dietary supplements

Chapter 4 focuses on the primary and the secondary metabolites and their

second-ary is not straightforward and can be viewed differently by the different authors, weeditors will explain in the next paragraph our own ideas on this very important issue,which shape the structure of the entire volume

physiological functions (i.e., appear as the building, energetic, or the reserve 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

often happens that the secondary metabolites have a well recognized physiological

ways Most often classification is based on their chemical structure, which generallyremains in a good agreement with the biogenetic origin Sometimes, however,

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 inthe alkaloid group However, their biogenetic origin is not from amino acids but

to the presence of the tertiary nitrogen atom in the molecules of certain taxoidrepresentatives At the same time, all taxoids biogenetically belong to the class of

biogen-etic origin is sometimes impractical, and then it is recommended to refer to theirchemical structure or physicochemical properties For example, naphthoquinonesand 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 ontheir similar physicochemical properties Iridoids could formally be included in theclass of monoterpenes, but this is not done because of their differentiated physico-chemical and pharmacological properties (classical monoterpenes are the volatilecompounds present in essential oils, whereas iridoids usually are the nonvolatilespecies) For the above reasons, we decided to classify the questionable groups

of compounds according to their chemical structure Consequently, all of the

their biogenesis

Chapter 5 focuses on chemosystematics, also known as chemotaxonomy This

biochemical level, especially in the amino acid sequences of common proteins Thenthe author highlights the areas of the main interest for the chemosystematic studiesand discusses applicability of the main chromatographic modes to this area ofresearch

In Chapter 6, the sorbents and precoated layers that are particularly useful in theanalysis and preparative isolation of the primary and secondary metabolites fromplant extracts are described This chapter covers virtually all of the TLC and HPTLCanalytical and preparative layers used for separation, determination, and isolation

celluloses, and kieselguhr), and miscellaneous layers (resin, impregnated, mixed, anddual layers)

Chapter 7 starts with description of the chromatographic chambers and mobilephase compositions that can be utilized in phytochemical research Then the authorsdiscuss the development of the chromatograms in the different thin layer chromato-graphic modes This chapter covers the methods of sample application to theadsorbent layer as well

Then two chapters following deal with the detection of the analytes after theirthin layer chromatographic separation The main part of Chapter 8 is devoted toderivatization of the plant extract components by use of universal and selective

reagents The specificity of TLC—not shared with any other chromatographic

in sequence in order to identify groups or individual analytes In this chapter,

fluorescence, and mass spectrometry), as well as the methods of quantitative analysiswith use of TLC combined with densitometry

Chapter 9 deals with the methods of biodetection in TLC that enable rapid andselective determination of the biological activity (antibacterial, antifungal, and other)

of plant metabolites In this chapter the mechanisms of bioactivity of the individualcompounds are explained

separ-ability of the plant metabolites

metabolites that occur in plants Chapters 11 through 13 refer to primary metabolites.Chapter 11 deals with the chemistry of carbohydrates, with their occurrence inthe plants as mono-, oligo-, and polysaccharides, and also as glycoconjugates Itprovides an overview of the recommended analytical methods, including samplepreparation, derivatization, and the most suitable TLC systems

In Chapter 12, different classes of plant lipids are presented, and the TLCsystems applied to their separation (including normal- and reversed-phase and

fica-tion are taken into the account

Chapter 13 focuses on free amino acids, peptides, and proteins, including theiroccurrence in plants and the use of TLC to separate the individual groups of thesecompounds

The next part of the book deals with the secondary metabolites occurring in planttissues, and it is divided into sections according to the metabolic pathways in whichindividual substances are synthesized

Chapter 14 starts with the phenolic compounds that belong to the metabolicpathway of shickimic acid, i.e., phenols, phenolic acids, and tannins It describes the

preparation methods, and the various TLC systems and special techniques that areused for their separation and analysis

Chapter 15 deals with coumarins that belong to the phenol class and are alsoderived from shickimic acid Details are provided on sample preparation and isola-

chromatographic techniques Application of TLC to the measurement of biologicalactivity of coumarins is also described The chapter ends with tables of the plantfamilies in which coumarins occur

Chapter 16 is dedicated to the phenolic compounds originating from a similar

by means of TLC combined with slit-scanning and video densitometry are discussed

The section of the book on secondary metabolites ends with lignans, alsooriginating 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

prepar-ations are also reported

The next section of the book is focused on isoprenoid derivatives, which includeseveral groups of compounds It starts with Chapter 18 on the volatile compounds

importance Then the following applications of planar chromatography are

investigations, tracing of various adulterations, and analysis of cosmetics

Chapter 19 covers diterpenoids and presents their structure, physicochemicalproperties, natural occurrence, and pharmacological activity The details of samplepreparation and the analytical and preparative TLC separations of this group ofcompounds with the aid of different chromatographic systems are described, includ-

the performance of TLC with that of the other chromatographic and related niques used in diterpenoid analysis

tech-The next group of compounds that belong to the isoprenoid methabolic pathwayare triterpenes, and they are described in Chapter 20 After a short introduction onstructure and properties of this group, chromatographic systems and detectionmethods applied in the analysis of triterpenes (saponins included) are presented.The chapter emphasizes the role of planar chromatography as a technique supporting

of triterpenes

Chapter 21 focuses on tetra- and polyterpenes, and among them carotenoidsrepresent the most important group of compounds First, structure, occurrence, andproperties are presented Then the special aspects of the TLC analysis (such asdetection and instability of carotenoids) are emphasized The use of silica andalumina, 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 aresteroids, and they are presented in Chapter 22 In the introductory part of thischapter, the chromatographic systems and techniques useful for planar separation

of steroids are described Then an overview of the literature is provided, taking intothe account the classes of phytosterols, steroids (brassinosteroids, bufadienolides,cardenolides, ecdysteroids, steroidal saponins, steroidal alkaloids, vertebrate-typesteroids, and withanolides), and of the related triterpenoids (cucurbitacins) Struc-

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 structureand physicochemical properties of iridoides, the issues are emphasized related to

isolation of this group of compounds from the plant material and to sample ation Then the TLC systems and techniques applied to the analysis of iridoids are

The preparative layer chromatography of iridoids is also discussed

The next four consecutive chapters deal with alkaloids synthesized in the plantorganisms from amino acids There are several groups of alkaloids differing in theirstructure, properties, and biological activity

Chapter 24 focuses on indole alkaloids Firstly, the chemical structure, rence, and pharmacological, ecological, and chemosystematic importance of thisgroup are discussed This preliminary information is followed by a detailed descrip-tion of the TLC separations of indole alkaloids, including chromatographic systems,techniques, and detection methods Details on the separations of the particular types

occur-of indole alkaloids are also presented

Chapter 25 is devoted to the structure, properties, and biological activity ofisoquinoline 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

the separation of the isoquinoline alkaloids are presented Examples of TLC cations to quantitative analysis are shown, along with the preparative separations.Tropane alkaloids are handled in Chapter 26 Chemistry and stereochemistry of

Various methods of extraction of this entire group of compounds from plant material

extrac-tion (SPE) Then the informaextrac-tion on TLC of tropane alkaloids including their

analysis of tropane alkaloids, and a comparison is made with the results originatingfrom the other separation techniques in use

Chapter 27 focuses on the remaining groups of alkaloids, including 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

phenylethy-to each individual group are presented

The last two chapters are devoted to the secondary metabolites derived fromacetogenine (acetylocoenzym A) Chapter 28 deals with the distribution of poly-acetylenes in plants and pharmacological activity of polyacetylenes 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), theiroccurrence in plants, and pharmacological activity Applicability of the conventionalTLC 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

source of information and training on the state-of-the-art phytochemistry methodsperformed with aid of TLC It will help to considerably popularize these methods for

for many years to come A computer assisted search has found no previous book on

(1973, 1984, and 1998) by J.B Harborne (Chapman and Hall, London, UK) hadchapters organized by compound type, most of which contained some information

by B Fried and J Sherma (CRC Press, 1996), a chapter on planar chromatography in

(Springer, 2001) and a chapter on natural mixtures by M Waksmundzka-Hajnos

E Reich and A Schibli (Thieme Medical Publishers, Inc., 2007) covers the etical concepts and practical aspects of modern HPTLC as related to the analysis ofherbal drugs

now out of date Our proposed book will solve this void in information in the criticalfield of phytochemical analysis

2 Plant Materials

in Modern Pharmacy and Methods of Their Investigations

CONTENTS

Secondary Metabolites 16

Biological Activity in Different Organs of Medicinal Plants 17

in Plant Material by Various Methods(Titration, Spectrophotometric Methods) 20

and Marine Products 22

15

2.6 Chromatographic Methods and Their Role in Investigations

of Plant Material 29

References 32

Pharmacognosy is the science which treats of the history, production, commerce,

other economic materials of plant and animal origin

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 mayoccur in the crude or raw form, as dried or fresh unground or ground organs ororganisms or natural exudations of these (juice or gum), when they are termed

‘‘crude drugs.’’

These are known as herbal medicinal products (HMPs), herbal remedies, orphytomedicines and include, for example:

mild to moderate depression

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

alcohols, esters, aldehydes, or other constituents or mixtures of constituents isolatedfrom the plant or animal

Finally, also pure chemical entities exist, which are produced synthetically and

Examples include:

malaria

Also many foods are known to have beneficial effects on health:

red wine

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

follow-ing plant organs are the most important:

Numerous drugs contain also leaf material as the main component Some widely

uva-ursi), and many others

source of drugs used in phytotherapy One of the most important example is

Stem material which is often a part of those drugs is derived from all ground parts, e.g., ephedra (Ephedra sinica), hawthorn (Crataegus monogyna and

(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

procubens), tormentill (Potentilla erecta), rhubarb (Rheum palmatum), and kava (Piper methysticum) [1].

kava-2.3 METHODS OF INVESTIGATIONS OF PLANT MATERIAL

quality and purity

drug can be established by actual collection of the drug from plant or animal (among

zoological standpoint This method is rarely followed except by an investigator ofthe 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 ofpharmacognostical research

Quality of a drug refers to intrinsic value of the drug, that is, to the amount ofmedicinal principles or active constituents present in the drug A high grade ofquality in the drug is such importance that effort should be made to obtain andmaintain 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 propermanner, the preparation of the collected drug by proper cleaning, drying and garblingand proper preservation of the clean, dry, pure drug against contamination with dirt,

as follows: organoleptic, microscopic, biological, chemical, and physical [2]

organs of sense, and includes the macroscopic appearance of the drug, its odor and

the touch

For convenience of description the macroscopic characteristic of a drug may bedivided into four headings, viz.: shape and size, color and external markings, fracture

For example, description of linseed (Linum usitatissimum) is as follows: Theseed is exalbuminous, of compressed ovate or oblonglanceolate outline, pointed atone end, rounded at the other and from 4 to 6 mm in length; externally glabrous andshiny, brown to dusky red with a pale-yellow, linear raphe along one edge; the hilumand microphyle in a slight depression near the pointed end; odor slight, becomingvery characteristic in the ground or crushed drug; taste mucilaginous and oily [3]

small fragments of crude drugs and of the powdered drugs as well as in the detection

of their adulterans, for these possess few features other than color, odor, and tastewhereby clues toward their identity may be afforded Moreover, owing to the

certain entire, cellular vegetable drugs cannot be made without the examination ofmounts 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 cellcontents, when properly mounted in suitable stains, reagents or mounting media [3].Some characteristic features can be easily used to establish botanical identityand quality of the drugs The typical example is the various types of crystals formed bycalcium oxylate Several species of the family Solanaceae are used for obtainingatropine, 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 bytypical crystal structures of oxalate: sand, cluster crystals and microspheroidalcrystals, respectively These are subcellular crystal structures, which can be easilydetected using polarized light and are thus a very useful diagnostic means

Second typical example are the glandular hairs, which are characteristic for twofamilies (Lamiaceae and Asteraceae) containing many species with essential oils

View from side

FIGURE 2.1 Diagnostic features of botanical drugs, that are revealed upon microscopicexamination 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 ofPharmacognosy and Phytotherapy, Elsevier Science, Churchill Livingstone, 2004.)

including typical glandular hair (Lamiaceae and Asteraceae family)—lateral viewand view from above.

In many instances, a good idea of the quality of a drug can be ascertained byusing microchemical methods These may consist of examining mounts of sections

or powdered drug in various reagents which either form salts of contained active

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 evaporateand examining the residue There is also possible isolation of a constituents bymicrosublimation

It is often possible to detrmine whether a powdered drug has been exhausted byexamining 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 incomparison with test tube sublimation The sublimates may be directly examinedunder the microscope without mechanical alteration

2.3.3.1 Approximate Group Identification

procedure

As effect of the reaction a deep rose color is produced

reagent phenolic (the products of this reaction give purple color) All of thesereactions are also used both for qualitative and quantitative analysis (colometricreactions)

methanolic solution of KOH, and 1% methanolic solution of Naturstoffreagenz A,are used for derivatization of TLC plates It enables general evaluation of different

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 laboratorymethod of quantitative chemical analysis which can be used to determine theconcentration of known reactant Because volume measurements play a key role intitration, it is also known as volumetric analysis A reagent, called titrant, of knownconcentration (a standard solution) is used to react with a measured quantity ofreactant (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 isthe linear realationship between absorbance and concentration of an absorbingspecies It enables a quantitative determination of compounds in which solutionsabsorb light For example total concentration of pyrrolizidine alkaloids in Symohy-

2.3.3.3 Isolation of Active Compounds

When a crude extract obtained by a suitable extraction procedure shows interestingactivity (e.g., an antibacterial activity), demonstrated in bioassay, the next and one of

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 differentextraction methods Hydrophilic (polar) extracts will then usually undergoion exchange chromatography with bioassay of generated fractions A further ionexchange method of bioactive fraction would yield pure compounds, which couldnext be submitted for structure elucidation (MS, NMR)

The biological evaluation of the plant drugs is one of the most important issues ofpharmacognosy For drugs obtained from natural sources, all active compoundspresent 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 determineantiseptic value of the drugs For standardization of Digitalis spp (Foxglove) and

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

Bioassays could involve the use of in vivo systems (clinical trials, whole animalexperiments), ex vivo systems (isolated tissues and organs), or in vitro systems (e.g.,cultured cells) Collection of materials for testing in bioassays could either berandom collection of samples or directed collection, i.e., from plants known to

be used traditionally Bioassays were often linked with the processes of fractionationand isolation, known as bioassay-guided fractionation

2.4 MODERN EXTRACTION METHODS OF ACTIVE COMPOUNDS FROM PLANT MATERIAL AND MARINE PRODUCTS

Sample pretreatment is one of the most time-consuming steps of the analytical

compounds and it is important to extract all of them in the best, short and effectiveway with minimal solvent usage Proper extraction technique should also be cheapand simple [5,6] Important is high recoveries, reproducibility, low detection

solid sample pretreatment [5] Extraction and fractionation of extract is also animportant method in isolation of compound groups or individual substances fromplant material

Extraction in Soxhlet apparatus has been the leading technique mostly used for along time and still is considered to be a standard technique and the main reference towhich the other new leaching methods are compared [5]

The sample is placed in a thimble holder and during operation is continuouslyfilled with fresh portion of solvent from distillation flask When the liquid reaches the

operation is repeated until complete extraction is achieved [5,6]

Different solvents can be used in extraction process Addition of co-solvents as a

mainly depends on characteristic of matrix and of size of particles as the internaldiffusion may be a limiting step during extraction [6]

The most important advantage of conventional Soxhlet extraction technique is itscontinuous character The sample has a contact with the fresh portion of the solvent

and simple method where small experience is required The method has the bility to extract more sample mass than the other methods [5] Also wide industrial

extraction methods [6]

time of extraction, poor penetration of the matrix by the solvent, and the largeamount of solvent required, which is very expensive and causes environmentalproblems Some solvents have recently been questioned because of their toxicity.Also we cannot forget that extraction occurs for a long time at the boiling point of

the solvent so the thermal decomposition of compounds and creating artifacts ispossible [5,6] Due to all advantages and disadvantages of Soxhlet extraction method

it is the most popular one and many scientist have tried to improve it

First, the changes focused on the new design of basic units (thimble holder,siphons, condenser) what improve application and obtained results [5] There is also

Thus led to create high-pressure and focused microwave-assisted Soxhlet extraction(FMASE) High pressure was achieved by placing the extractor in autoclave or using

FMASE shows some differences comparing with other microwave-assistedextraction techniques: extraction is under normal pressure, irradiation is focused on

extraction time, capability for automation, and quantitative extraction Extraction

advantages of conventional method [8] Also there are some applications of sound-assisted Soxhlet extraction [9]

Super-critical state is achieved when the temperature and the pressure of a substance is over

both liquid and gases what makes extraction faster and more effective Many

supercritical temperature of carbon dioxide makes it attractive for the extraction ofthermolabile compounds [5,6] Application of some of them is limited because oftheir unfavorable properties with respect to safety and environmental consideration.Water in supercritical state has higher extraction ability for polar compounds, but isnot suitable for thermally labile compounds [10]

Many compounds such as phenols, alkaloids, glycosidic compounds are poorly

extractant can be increased (by addition methanol, ethanol, pentane, acetone) The

because of its lower toxicity The polarity of the analytes can also be reduced (by

lipids from the matrix, further cleanup may be necessary to remove lipids beforethe analysis

SFE is frequently used for extraction of fresh plant material The problem is

material [10]

Also very important is plant particle size Large particles can prolong extraction

speedup extraction However, powdered material may cause difficulties in keeping a

classical Soxhlet extraction are time reduction, its cleanness and safety, possibilityfor coupling on-line with detectors and chromatographs, quantitative determin-ation, the preconcentration effect, high mass transference, completeness of extrac-

than liquid solvent) Very important is that SFE offers possibility for selectiveextraction and fractionation substances from the plant material by manipulating

supercritical carbon dioxide extraction with solvent extraction of squalene and

was comparable to n-hexane Soxhlet extraction but the stigmasterol was not detectedunder these condition The method can be easily used in the laboratory for a largescale [17]

Extracts with fewer unwanted analytes may be obtained by careful manipulation

small volume of the extractor, which contains only a few grams of the material, is adisadvantage when a higher sample mass is required

Supercritical carbon dioxide is a promising solvent for the extraction of naturalcompounds, especially thermolabile ones Prevention of degradation could be

antioxi-dants would be a reasonable solution if there were no mechanical items to adsorb theoxygen [18] Also SFE eliminates time-consuming process of concentration and uses

no or minimal organic solvent what makes method environmental friendly[5,6,12,14,19]

conventional Soxhlet method (some analytes are strongly bound with matrix and notenough energy is involved in the Soxhlet process for their separation) [5]

peel of Citrus maxima [12], furanocoumarins and pentacyclic terpenoids in rhizome

of Dorstenia bryoniifolia Mart ex Miq and bark roots of Brosimum gaudichaudiiTrécul (Moraceae) [20]

Maceration under sonication gave better than SFE extraction of coumarins inMikania glomerata leaves SF extracts contain a high level of chlorophylls Also

period of time and with decreased amount of solvent [22]

Biologically active substances of rose hip seeds like unsaturated fatty acids andcarotene was extracted by SFE with carbon dioxide and propane Oil yield washigher in comparison with traditional Soxhlet extraction [23]

Effect of low, medium, and high polarity under very high pressure and with polar

Crataegus sp, and Matricaria recutita were used as models in this study Extraction

secondary metabolites present in the plant material Extractability of lipophiliccompounds increased substantially at pressure higher than 300 bar, the yields ofpolyphenolics and glycosides remained low even at pressure about 700 bar with 20%

biologically active substances are described in review article [6,7,25,26]

Pressurized liquid extraction (PLE) also known as an accelerated solvent extraction

(PFE) is a technique which uses small volume of conventional solvents at elevated

higher than 200 bar in order to keep the solvent in liquid state (the solvent is stillbelow its critical point), increasing temperature accelerates the extraction kinetics(process of the desorption of analytes from the matrix are faster compared to thecondition when solvent at room temperature are used), which gives safe and rapidmethod [6,7,27,28] ASE allows the universal use of solvents or solvent mixtureswith different polarities

In PLE, sample with sand, sodium sulfate or Hydromatrix as a dispersant are

values, then is heated in an oven to the set values During the heating cycle, solvent ispumped in and out of the cell to maintain the pressure and to perform the cycles

vial Before loading the plant materials into the extraction cell, the samples are oftenpretreated Proper size of sample enables right diffusion of analytes from the sample

to the solvent extract Drying the sample removes any moisture which may diminish

analytes of interest, minimizing the coextraction of other matrix components Itspolarity should be close to that of the target compound [27,29]

PLE can be accomplished in the static (sample and solvent are maintained for a

flows through the sample in a continuous manner) Because in most cases thedynamic mode uses water as extractant, several authors have preferred to use the

nontoxic, readily available, and an environmentally acceptable solvent PHWEwith and without the addition of a small percentage of organic solvent such

as ethanol is highly suited for the chemical standardization and quality control

of medicinal plants At the same time, it can be applied at the pilot scale as amanufacturing process for medicinal plants Further information about application

of PHWE in extraction of active compounds can be found in review papers[27,29,30]

Very important is complete automation of the whole analytical process andhighly selective extractions of compounds of different polarities [27]

Compared with classic extraction in Soxhlet apparatus, complete extraction can

be achieved in shorter time with a small volume of organic solvent and much betterpenetration of sample by the solvent in PLE Keeping the high temperature reduces

The suitability of pressurized liquid extraction (PLE) in medicinal plant analysiswas investigated PLE extracts from representative herbs containing structurallydiverse metabolites of varying polarity and solubility were compared with extractsobtained according to Pharmacopoeia monographs with respect to yield of relevantplant constituents, extraction time, and solvent consumption Experiment shows thatone to three extraction cycles of 5 to 6 min at high temperatures afforded exhaustive

or almost exhaustive extraction (instead of many hours of Soxhlet extraction) Itmarkedly reduces not only time but also solvent consumption and protect againstartifacts of extracted compounds at high temperatures Reproducibility of results wasgenerally better [31]

solvent extraction in open and close system and pressurized solvent extraction ASEgives higher yield of furanocoumarins (especially for hydrophobic) from Pastinac

the extraction condition for the analysis of rutin and isoquercitrin in Sambucus nigraflowers, leaves, and berries [34]

Ong and Len [35] have developed a method for the analysis of glycosides inmedicinal plants using PHWE The results obtained with this technique were evenbetter than those obtained with Soxhlet extraction The study showed that hydro-phobic and thermally labile components in medicinal plants could be extracted usingthe combination of surfactant and pressurized hot water at a temperature below itsboiling point and lower applied pressure

The possibility of selectivity changing for extractions of the most typicalrosemary antioxidant compounds by means of a small change of temperature hasbeen demonstrated The experiment shows that it is possible to obtain extractsenriched with different types of polyphenols [36]

PLE was examined as an alternative technology for the extraction of carotenoids inthe marine green algae Haematococcus pluvialis and Dunaliella salina and kavalac-tones in Piper methysticum The results of this study showed that PLE had comparable

amount of extracting solvent as traditional extraction, and is less time-consuming [37].PLE with ethyl alcohol of antidiabetic compound, charantin, from fruits ofMomordica charantia (bitter melon) was proposed as an alternative for conventionalSoxhlet extraction with toxic solvent such as chloroform or dichloromethane [38].PLE was more effective for the extractions of terpenes (terpenic alcohols andphytosterols), fatty acids, and vitamin E from leaves of Piper gaudichaudianum

and the manipulation of sample and solvents in comparison with Soxhlet [39]

ASE has been used in analysis of polyphenols [40], taxanoids [41], alkaloids[42] Various aspects of application of PLE of natural products were discussed inliterature [6,7,27]

2.4.4 MEDIUM-PRESSURESOLID–LIQUIDEXTRACTIONTECHNIQUE

[43], is an extraction technique based on the diffusion-dissolving processes.MPSLE can be performed in a medium-pressure liquid chromatography (MPLC)

pumped through the stationary bed The extraction distance is relatively short even

pressure Various solvents or their mixtures can be applied

exhaustive extraction method like Soxhlet extraction, but here the process of centration is not required Also all experiment is performed in close and automatedsystem with small amount of organic solvent so it is environment friendly [44]

Transposition to MPLC is straightforward and direct [45]

2.5 PURIFICATION OF CRUDE EXTRACTS AND SAMPLE

For removal of coextracted substances different cleanup procedures have

chroma-tography on polar adsorbents (Florisil or silica), and gel permeation chromachroma-tography(GPC) [27,46]

sample (cleanup, concentration, and isolation) LLE is based on the rule that when athird substance is added to a mixture of two immiscible liquids being in equilibrium,the added component will divide itself between the two liquid phases until the ratio

of its concentrations in each phase attain a certain value [47]

Method is slow, required the long time, large amount of organic solvents

very slowly and incompletely [48,49]

samples, which are to be separated by various chromatographic processes This isgenerally achieved by countercurrent methods [45]