Plant Drug Analysis

H. Wagner S. Bladt E.M.
Zgainski
Plant Drug
Analysis
A Thin Layer Chromatography Atlas
Translated by Th.A. Scott
With 170 Colored Photographs
Springer-Verlag Berlin Heidelberg GmbH 1984

Professor Dr. Hildebert Wagner
Dr. Sabine Bladt
Eva Maria Zgainski (Fachphotographin)
Institut für Pharmazeutische Biologie
der Universität München,
Karlstraße 29,
D-8000 München 2
Translator:
Dr. Thomas A. Scott
Department of Biochemistry
University of Leeds
GB-Leeds LS2 9JT
Translation of the German edition' Drogenanalyse '

© Springer-Verlag Berlin Heide1berg 1983
ISBN 978-3-662-02400-3 ISBN 978-3-662-02398-3 (eBook)

DOI 10.1007/978-3-662-02398-3
Library of Congress Cataloging in Publication Data

Wagner, H. (Hildebert), 1929-
Plant drug analysis.
Includes bibliographical references and index.
1. Materia medica, Vegetable-Analysis. 2. Drugs-Analysis. 3. Thin layer chromatography. I.
Bladt, S. (Sabine), 1945- . H. Zgainski, E.M. (Eva Maria), 1928- . III. Title.
RS190.P55W3313 1984 615'.32 84-5348
This work is subject to copyright. All rights are reserved, whether the whole or part of the
material is concerned specifically those of translation, reprinting, re-use of illustrations, broad-
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Under § 54 of the German Copyright Law where copies are made for other than private
use, a fee is payable to "Verwertungsgesellschaft Wort", Munich.
© by Springer-Verlag Berlin Heidelberg 1984
Originally published by Springer-Verlag Berlin Heidelberg New York in 1984.
Softcover reprint of the hardcover 1st edition 1984
The use of registered names, trademarks, etc. in this publication does not imply, even in
the absence of a specific statement, that such names are exempt from the relevant protective
laws and regulations and therefore for general use.
Product Liability: The publisher can give no guarantee for information ab out drug dosage
and application thereof contained in this book. In every individual case the respective user
must check its accuracy by consulting other pharmaceuticalliterature.
Reproduction of the figures : Gebrüder Czech, D-8000 München
2131/3130-543210
Preface
Thin layer chromatography is the most widely used of all the simple chromatographie
methods for the analysis of mixtures. On account of its extreme rapidity and ease
of visual evaluation, thin layer chromatography has become the most ideal analytical
method for plant drugs and for preparations that contain drug extracts or pure
drug constituents.
One disadvantage of the technique was the lack of a satisfactory method for
the permanent recording of results. Analyses were recorded simply by verbal descrip-
tion of the chromatogram (e.g., in the pharmacopoeias), by schematic drawings,
or by idealized colored diagrams. All these methods are c1early makeshift solutions
to the problem of documentation. In order to make the documentation as realistic
as possible, we have therefore attempted to make faithful photographic reproduc-
tions of thin layer chromatographie separations of drugs in visible and UV-light.
The chief difficulty that we encountered in the preparation of this TLC atlas was
knowing how to take into account the natural qualitative and quantitative variations
that occur in the composition of individual drugs. We believe we have solved this
problem by selecting, from more than 10 years of photo graphie recording, those
pictures that are the most representative and are photographically the best reproduc-
tions of chromatograms of standard drugs. In choosing solvent systems, we have
relied primarilyon the pharmacopoeias, but we have also made proposals for im-
provements and for future standardization. In view of the extent of the present-day
world drug market, analyses of many commercial, non-official drugs have also been
documented. Emphasis was placed primarily on commercial European drugs.
We hope that this chromatographie drug atlas will help those who are learning
the technique, and serve as an aid for the evaluation of thin layer chromatograms.
Furthermore, we believe that this thin layer chromatographie drug atlas represents
an aid to the standardization of industrial plant drug preparations. It will enable
the systematization of the identification and purity control of plant drugs.
The authors are especially grateful to Mrs. J. DUFOSSE of Agfa photographie
services, Munieh, for the painstaking and faithful reproduction of the color pictures.
We thank Miss MAHN and Miss SCHUCKER for technical assistance, and Mrs.
ANDRAE for drawing the formulae. Thanks are due to Springer-Verlag, and especially
to Mrs. DEIGMÖLLER, for their helpful co operation to our wishes with regard to
the reproduction of the chromatograms and the overall layout of the book.
HILDEBER T WAGNER
SABINE BLADT
EVA MARIA ZGAINSKI
Contents
Introduction
Essential Oil Drugs (Aetherolea) 5
Extraction and TLC . . . . . . . . 5

List of Essential Oil Drugs . . . . . 9
Formulae of Constituents of Essential Oils 19
Reference Compounds · Figs. 1, 2 22
Cinnamoni cortex
Calami rhizoma · Figs. 3, 4 24
Anisi fructus
F oeniculi fructus
Sassafras lignum
Basilici herba . . . . . . . . . . . . . Figs. 5, 6 26
Petroselini fructus
Myristicae semen
Macis
Caryophylli flos . . . . . . . . . . . . . Figs. 7, 8 28
Carvi fructus
Coriandri fructus
Juniperi fructus
Rosmarini folium · Figs. 9,10 30
Matricariae flos
Anthemidis flos . Figs. 11, 12 32
Lavandulae flos
Cinae flos . . . · Figs. 13, 14 34
Menthae piperitae folium
Menthae crispae folium . Figs. 15, 16 36
Salviae officinalis folium
Salviae trilobae folium
Eucalypti folium . . . . . . . . . . Figs. 17, 18 38
Thymi herba
Serpylli herba
Ajowani fructus
Melissae folium . Figs. 19,20 40
Curcumae rhizoma · Figs. 21, 22 42
VII
Ci tri pericarpium
Aurantii pericarpium . . . . . . . . . . . Figs. 23, 24 44
Terebinthinae aeth.
Pini aetherolea
Myrrha . . . . . . . . . . . . . . . . Figs. 25, 26 46
Benzoin
Balsamum tolutanum
Balsamum peruvianum . . . . . . . . . . Figs. 27, 28 48
Alkaloid Drugs 51
Extraction and TLC 51
List of Alkaloid Drugs 55
Formulae of Constituents of Alkaloid Drugs 61
Reference Compounds · Figs 1, 2 66
Y ohimbe cortex
Quebracho cortex · Figs. 3, 4 68
Rauwolfiae radix · Figs. 5, 6 70
Strychni semen
Ignatii semen . Fig. 7 72
Secale cornutum . Fig. 8 72
Chinae cortex · Figs. 9, 10 74
Ipecacuanhae radix .Figs. 11, 12 76
Opium · Figs. 13, 14 78
Berberidis radix
Colombo radix
Hydrastis rhizoma . Figs. 15, 16 80
Chelidonii herba · Fig. 17 82
Colchici ·semen · Fig. 18 82
Aconiti tuber (herba)
Sabadillae semen
Lobeliae herba
Jaborandi folium
Boldo folium · Figs. 19, 20 84
Cacao semen
Coffeae semen · Fig. 21 86
Nicotianae folium
Ephedrae herba
Spartii herba . . . . . . . . . . . . . . Fig. 22 . . . . . . . . . . 86
Belladonnae folium
Hyoscyami folium
Stramonii folium . . . . . . . Figs. 23, 24 88
Belladonnae folium/semen/radix
Hyoscyami mutici/nigri folium
Stramonii folium/semen
Scopoliae radix . . . . . . . . . . . . . . Figs. 25, 26 90
VIII
Drugs Containing Anthracene Derivatives 93
Extraction and TLC . . . . . . . . . . . . . 93

List of Drugs Containing Anthracene Glycosides 97
Formulae of Anthracene Derivatives Identified as Drug Constituents 100
Aloe resina . Figs. 1,2 102
Rhamni purshiani cortex . Fig. 3 104
TLC-Synopsis . Fig. 4 104
Frangulae cortex
Oreoherzogiae cortex
Frangulae fructus
Rhamni cathartici fructus · Figs. 5, 6 " 106
Rhei radix . . · Figs. 7, 8 . 108
Sennae folium
Sennae fructus
Hyperici herba . Figs. 9,10 110
Circular TLC
Sennae foliumjfructus . Figs. 11, 12 112
Rhei radix . . . . . · Figs. 13, 14 114
Arbutin Drugs 117

List of Drugs Containing Arbutin 119
Formulae ...... . 119
Uvae ursi folium
Vitis ideae folium
Myrtilli folium . · Figs. 1,2 120
Viburni prunifolii cortex
Viburni opuli cortex · Figs. 3,4 122
Bitter Principle Drugs 125
Extraction and TLC . . . 125

List of Bitter Principle Drugs 127
Formulae of Constituents
of Bitter Principle Drugs 130
Aurantii pericarpium
Harpagophythi radix
Gentianae radix
Centaurii herba
Condurango cortex
Menyanthidis folium . . . . . . . . . . . Figs. 1,2 . . . . . . . . . 132
Cnici herba
Marrubii herba
Absinthii herba
Quassiae lignum . . . . . . . . . . . . . Figs. 3, 4 . . . . . . . . . 134
IX
Gentianae radix
Plantaginis herba
Oleae folium/fructus · Figs. 5, 6 136
Bryoniae radix
Cucurbitae semen · Figs. 7, 8 138
Humuli strobuli · Figs. 9, 10 140
Salviae folium
Rosmarini folium · Fig. 11 142
Cynarae herba . · Fig. 12 142
Coumarin Drugs 145

List of Coumarin Drugs 147
Formulae of Constituents of Coumarin Drugs 150
Reference compounds . Fig. 1 152
Pimpinellae radix
Heraclei radix . Fig. 2 152
Angelicae radix
Levistici radix
Imperatoriae radix
Scopoliae radix. . . . . . . . . . . . . Figs. 3, 4 . . . . . . . . . 154
Herniariae herba
Meliloti herba
Asperulae herba
Abrotani herba
Rutae herba. . . . . . . . . . . . . . Figs. 5, 6 . . . . . . . . . 156
Mezerei cortex
Fraxini cortex
Asa foetida · Figs. 7, 8 158
Ammi majoris fructus
Ammi visnagae fructus · Figs. 9, 10 160
Flavonoid Drugs 163

List of Flavonoid Drugs 165
Formulae of Constituents of Flavonoid Drugs 170
Reference Compounds . Figs. 1,2 172
TLC Synopsis (flos) . . . . . . . . . . . . Figs. 3, 4 174
Arnicae flos
Calendulae flos
Cacti flos
Farfarae flos
Primulae flos
Herniariae herba . . . . . . . . Figs. 5, 6 . . . . . . . . . 176
Crataegi flos/folium/fructus
Sambuci flos
Stoechados flos
Verbasci flos . . . . . . . . . . . . . . . Figs. 7, 8 . . . . . . . . . 178
x
Acaciae flos
Pruni spinosae flos
Spireaae flos
Robiniae flos
Sambuci flos
Tiliae flos . . . . . . . . . . . . . . Figs. 9, 10 . . . . . . . . 180
Betulae folium
Juglandis folium
Anthemidis flos · Figs. 11, 12 182
Matricariae flos . Fig. 13 184
TLC Synopsis (Herba) · Fig. 14 184
Anserinae herba
Equiseti herba
Leonuri herba
Rutae herba
Sarothamni herba
Sophorae gemma
Veronicae herba
Violae tricoloris herba . Figs. 15, 16 186
Citri pericarpium
Aurantii pericarpium · Fig. 17 188
Eriodictyonis herba
Orthosiphonis folium · Fig. 18 188
Cardui mariae (Silybi) fructus · Figs. 19,20 190
Farfarae folium
Petasites folium · Figs. 21, 22 192
Cardiac Glycoside Drugs 195
Extraction and TLC . . . . 195

List of Cardiac Glycoside Drugs 198
Formulae of Constituents
of Cardiac Glycoside Drugs 200
Figs. 1,2 204
Reference compounds . . · Figs. 3, 4 206
Digitalis lanatae folium
Digitalis purpureae folium · Figs. 5-8 208
Nerii (oleandri) folium · Figs. 9, 10 212
TLC Comparison
Strophanthi semen
Adonidis herba
Convallariae herba · Figs. 11, 12 214
Strophanthi grati semen
Strophanthi kombe semen · Figs. 13, 14 216
Adonidis herba
Convallariae herb'it · Figs. 15, 16 218
XI
Uzarae (Xysmalobii) radix . Fig. 17 220
Helleborii radix · Fig. 18 220
Scillae bulbus
var. alba/var. rubra · Figs. 19,20 222
Saponin Drugs 225

List of Saponin Drugs 227
Formulae of Constituents of Saponin Drugs 230
TLC Synopsis · Figs. 1, 2 234
Primulae radix . Fig. 3 236
Saponariae radix . Fig. 4 236
Ginseng radix
Eleutherococci radix · Figs. 5, 6 238
Liquiritiae radix · Figs. 7, 8 240
A venae herba
Hederae folium . Fig. 9 242
Hippocastani semen · Fig. 10 242
TLC-Analysis of Saponins . Figs. 11, 12 244
Drugs Containing Pungent Principles 247
Extraction and TLC . . . . . . . . . . 247

List of Drugs Containing Pungent Principles 248
Formulae ........... . 249
Capsici fructus
Cubebae fructus
Piperis fructus . . . . Figs. 1, 2 . . . . . . . . . 250
Mustard Oil Drugs and Allium 253

List of Drugs . . . 255
Formulae 255
Sinapis albae (Erucae) semen
Sinapis nigri semen . Fig. 3 256
Thiourea derivatives
Allium sativum . . . Fig. 4 256
N arcotic Drugs . 259

List of Drugs . 259
Formulae 260
Cannabis herba
Hashish . . . . . . . . . . . . . . . . . Figs. 1, 2 260
XII
Drugs Containing Valepotriates 263

List of Drugs . 264
Forrnulae 265
Valerianae radix . . . . . . . . . . . . . Figs. 1, 2 266
Drugs Containing Pigments 269

List of Drugs 270
Forrnulae 271
Cyani flos
Hibisci flos
Malvae flos . . . . . . . . . . . . . . . Figs. 1, 2 . . . . . . . . . 272
Hibisci flos
Paeoniae flos
Croci stigma . . . . . . . . . . . . . . . Figs. 3, 4 . . . . . . . . . 274
Drugs with Miscellaneous Constituents 277
Extraction and TLC . . . 277

List of Miscellaneous Drugs 278
Forrnulae . . . . . . . 280
Salicis cortex . . . . . . . . . . . . . . . Fig. 1 282
Hamamelidis folium
Filicis rhizoma
Pyrethri flos . . . Fig. 2 282
Lichen islandicus . Fig. 3 284
Podophylli resina . Fig. 4 284
Visci albi herba . Figs. 5, 6 286
Amino acids . . . Figs. 7, 8 288
TLC Screening of an Unknown Commercial Drug 291

Extraction and TLC . . . . . . . . . 291
Scheme of Separation and Identification 294
TLC Analysis of Herbai Drug Mixtures 296

Spray Reagents . . . . . . 299
Abbreviations and Definitions 305
References . 307
Subject Index 309
XIII
Introduction
I. Thin Layer Chromatographie-Analysis (TLC) of Drugs

Of the many chromatographie methods presently available, thin layer chromatogra-
phy has become widely adopted for the rapid and positive analysis of drugs and
drug preparations.
There are several reasons for the popularity of this method:
- The time required for the demonstration of most of the characteristic constitutents
of a drug by TLC is very short.
- In addition to qualitative detection, TLC also provides semi-quantitative informa-
tion on the chief active constituents of a drug or drug preparation, thus enabling
an assessment of drug quality.
- TLC provides a chromatographie drug fingerprint. It is therefore suitable for
monitoring the identity and purity of drugs, and for detecting adulterations and
substitutions.
- With the aid of appropriate separation procedures, TLC can be used to analyse
drug combinations and phytochemical preparations.
- Thin layer chromatograms can be documented.
11. Doeumentation of Thin Layer Chromatograms

Various methods of documentation are possible:
- Description of the Rf values and colours of the characteristic main zones, with
reference to a standard substance or test mixture. This method has been adopted
in the 8th edition of the German pharmacopoeia, the European pharmacopoeia,
USP XX and others.
- Construction of ascale diagram of the thin layer separation, showing migration
distances and intensities of the characteristic zones. The zones observed in visible
light (vis.) in UV-254 nm and UV-365 nm are described.
- Colour photography in daylight or UV-light, under conditions that give the most
authentie reproduction of the colours and intensities of the separated zones.
- Densitometry or fluorometry of the chromatogram at certain wavelengths. Under
favourable conditions, this procedure also yields a drug fingerprint, and enables
the quantification of certain chief constituents. It suffers from the disadvantage
that a calibration graph constructed at one wavelength is applicable to only some
of the constituents.
111. Photo graphie Reeord of Thin Layer Separations of Drug

Extraets (A Photographie TLC Drug Atlas)
- A photographie TLC atlas fulfils the same function and purpose as a catalogue
of spectra. The identity or non-identity of an official drug can be established
by comparison with the chromatogram of the 'standard drug'.
- Unknown commercial drugs can be more easily identified by comparison with
the visual record in the TLC atlas.
- The photographic drug atlas is an aid to the routine identification and purity
testing of drugs in control laboratories, and it can be used without previous
pharmacognostic training.
- Photographic reproduction ofthin layer separations has a large didactic advantage
over mere graphic representation. The TLC photo-drug atlas has an immediate
clarity of representation that facilitates the learning of TLC drug analysis for
the student.
IV. Compilation of a TLC Drug Atlas

Compilation of a TLC drug atlas was governed by certain preconditions, related
to the source of the drugs, the TLC technique in general, and the photographic
reproduction of the thin layer chromatograms.
1. Source of the drugs

The drugs used in the compilation of a drug atlas must meet the standards of
the official pharmacopoeia, and they must originate from a clearly identified botani-
cal source.
Slight variations in the chromatographic picture, due to botanical varieties, or
differences in cultivation, climatic conditions, time of harvesting, drying and extrac-
tion methods are normal.
2. Extraction conditions
The chosen extraction procedures are the best available in the light ofpresent scientif-
ic knowledge. As far as possible they have been adopted unchanged from the phar-
macopoeias, and modified only when new substances and separation problems have
been encountered.
3. TLC
Reproducible TLC separations can be guaranteed only if standardized adsorption
layers are used. Commercially available TLC plates were therefore used: Si/ica gel
60F254 pre-coated TLC platesfrom Merck, Darmstadt. Since special chromatography
rooms are not always available, all TLC separations were performed at room temper-
ature, i.e. 18-22° C. Details ofthe TLC technique are to be found in pharmacopoeias
and books on methodology (see list on page 307).
Generally a distance of 15 cm is used for the development of a chromatogram.
The pictures shown in the book are approx: 1/3 of the original size of the TLC-plate.
4. Chromatography solvents
In choosing suitable solvent systems, preference has been given to those specified
in the pharmacopoeias. These often represent a compromise, and where necessary
they have been modified. In most cases, the resulting improvement in separation
has been documented with chromatograms in both systems. Where possible, an
attempt has been made to standardize the systems. Systems were chosen for their
minimal temperature sensitivity.
5. Concentration of substances for TLC

In order to obtain sharply resolved zones, the quantity of material applied to the
chromatogram should be as small as possible. Rather large sample volumes are,
however, often necessary for the detection (by colour reactions) of substances that
are present in low concentration. This inevitably results in broadening and overlap-
ping of zones.
2
6. Detection methods
- For the detection of the main, characteristic compounds of a drug, methods were
chosen that give the most striking colours.
- The active principles of a group of drugs may be very sirnilar (e.g. drugs from
Solanaceae or saponin drugs), so that differentiation and identification are difficult
or impossible on the basis of the active principles alone. In such cases, other
c1asses of compounds have been exploited for the purposes of differentiation.
- For drugs that contain unknown or incompletely known activeprinciples, identifi-
cation has been based on those constituents that can be regarded as "guide sub-
stances".
7. Photography
The developed chromatograms were photographed on Agfachrome 50L or 50S Pro-
fessional. To achieve authentie colour reproduction, each picture needs a specific
technic of exposure and deve1opment.
3
Essential Oil Drugs (Aetherolea),
Gums and Resins
Essential oils are mixtures of many substances, predominantly terpenes (ca. 90%)
and phenylpropane derivatives. Other components inc1ude simple phenols, sulphur-
containing compounds (mustard oils), methyl anthranilate and coumarins.
I. Extraction of Essential OUs

1. Official method
Steam distillation with a modified distillation apparatus according to Cocking and
Middleton (Ph. Eur. III, p. 62)
Principle. The quantity of drug used is sufficient to yield 0.1-0.3 ml essential oil.
The sample weight is 10 to 50 g, depending on the type of drug (DAB 8, Ph. Eur.),
and the rate of distillation is no greater than 2-3 ml per minute. When ebullition
has become steady, distillation is continued for between 1 1 / 2 and 4 hours. Xylene
(1 ml) is placed in the distillation flask, so a blank" xylene value" must be determined
in aseparate distillation in the absence of the drug.
Table 1 shows the essential oil drugs of the DAB 8 and Ph. Eur., with sample
weight and distillation conditions.
2. Abridged official method

If a quantitative determination of the oil content is not required, a sample of essential
oil suitable for investigation by TLC can be obtained by reducing the distillation
Table 1
Drug Content of Sampie Water Time Rate

essential oil weight (mI) (hr) (ml/min)
(ml/lOO g) (g)
Absinthii herba 0.3 50 300 3 2-3

Anisi fructus 2.0 25 200 2 2-3
Anthernidis flos 0.7 30 300 3 3-5.5
Aurantii percarpium 1.0 20 250 1.5 2-3
Carvi fructus 4.0 10 200 1.5 2-3
Curcumae rhizoina 3.5 10 200 3 3--4
Foeniculi fructus 4.0 10 200 2 2-3
Juniperi fructus 1.0 20 200 1.5 3--4
Matricariae flos 0.4 50 500 a 4 3--4
Melissae folium 0.05 40 400 2 2-3
Menthae folium 1.2 50 500 2 3-3.5
Salviae offic. folium 1.5 50 500 1.5 2-3
Salviae trilobae folium 1.8 50 500 1.5 2-3
Thyrni herba 1.2 20 300 2 2-3
a distilled from 1% NaCI solution.
5
period to one ho ur. With the exception of drugs containing e.g. eugenol, the distilla-
tion is then performed without xylene. The resulting oil is diluted 1: 10 with toluene.
Eugenol-containing oils, obtained by distillation in the presence of xylene (Ph.
Eur. III), can be applied directly to chromatograms. If the concentration of oil
is still too high, the xylene solution should be diluted 1: 5 with toluene.
3. Micromethods
a) Micro steam distillation after Luckner
1 g powdered drug and 10 ml water are placed in a 50 ml Erlenmeyer flask. A
glass U-tube (10-15 cm long; 0 ca. 5 mm) is placed between the distillation flask
and the receiver (test tube). The contents of the flask are heated to boiling (boiling
stones) and distillation via the U-tube performed slowly until about 1 ml of distillate
has collected in the test tube. The distillate is extracted by shaking with 1 ml pentane,
the pentane solution removed with a pipette, and 20-100111 of this solution are
used for TLC. A range of different sam pie concentrations is used for the TLC
separation.
Remarks: This rapid method gives only a guide to the composition of the essential oil.
b) Thermomicrodistillation after Stahl (TAS-method)

With the aid of a so-called TAS-oven (Desaga), substances that volatilize at fairly
high temperatures can be distilled from the drug onto the TLC plate.
The tapered end of a T AS-glass cartridge is cIosed with a packing of quartz
wool, followed by about 50 mg powdered drug and about 50 mg starch. The cartridge
is sealed with a cIamp and placed in the oven block of the T AS-apparatus, which
is heated to about 220° C. The point of the cartridge is directed onto the surface
of the TLC plate. Substances that are volatile at the given temperature distil onto
the starting zone of the TLC plate in about 90 sec.
Remarks: All components of essential oils and other volatile compounds, e.g. coumarins,
are obtained by this method.
c) Extraction with methylene chloride (dichloromethane (DCM)-extract)

1 g powdered drug is extracted by shaking for 15 min with 10 ml methylene chloride.
The suspension is filtered and the cIear filtrate evaporated to dryness. The residue
is dissolved in 1 ml toluene, and 50-iOO 111 are applied for TLC.
Remarks: This method also extracts other, interfering lipophilic substances.
d) Extraction with methanol (MeOH-extract)

a) Curcumae rhizoma (cinnamoyl pigments). 1 g powdered drug is extracted by
shaking for 5 min with 5 ml MeOH at about 60° C. 10111 of the cIear filtrate are
applied for TLC.
ß) Gum resins (e.g. Myrrha). 0.5 g powdered drug is extracted by shaking for 5 min
with 5 ml 96% ethanol. 20 111 of the supernatant or cIear filtrate are applied for
TLC.
y) Oleo-resins (Balsamum peruvianum, B. tolutanum). 0.5 g peru balsam is dissolved
in 10 ml ethyl acetate, and 10 111 of this solution are applied for TLC.
For tolu balsam, 10111 of a 1: 10 dilution in toluene are applied for TLC.
11. Thin Layer Chromatography

1. Reference solutions
Solutions of each of the following compounds are prepared in toluene (1: 30).
Alcohols: borneol, geraniol, linalool, menthol
Phenols: thymol, carvacrol
6
Aldehydes: anisaldehyde, citral, citronellal
Ketones: carvone, fenchone, menthone/isomenthone, piperitone, thujone
Oxides: 1.8-cineole
Phenylpropane derivatives: anethole, apiol, allyltetramethoxybenzene, eugenol, myr-
isticin, safrol
2. Adsorbent
Silica gel 60F z54 pre-coated TLC plates (Merck, Darmstadt)
3. Sampie concentration
5 111 of a 1 : 10 dilution of the essential oil in toluene are applied to the TLC plate.
3 111 of each reference solution are used.
When applied in quantities of about 100 I1g/3 111, all the reference compounds
can be detected by treatment ofthe chromatogram with VS-reagent (No. 38, p. 304).
Thymol and anethole are detectable in quantities down to 5 I1g.
A-l Toluene-ethyl acetate (93: 7)
This system is suitable for the analysis and direct comparison of.all impor-
tant essential oils.
The DAB 8 and Ph. Eur. describe different solvent systems for individual
essential oils:
Solvent system Drug or essential oil
A-2 Benzene a Anisi fructus

A-3 Chloroform Curcumae xanth. rhizoma, Melissae folium
A-4 Methylene chloride Anisi aeth., Carvi fructus, Carvi aeth., Caryo-
phylli aeth., Foeniculi aeth., Juniperi fructus, La-
vandulae aeth., Rosmarini aeth., Salviae off. and
S. trilobae folium
A-5 Benzenea-ethyl acetate (90: 10) Eucalypti aeth.
A-6 Benzenea-ethyl acetate (95: 5) Menthae piperitae aeth.
A-7 Chloroform-benzene a (75: 25) Absinthii herba, Matricariae flos, Menthae
piperitae folium, Thyrni herba
A-8 Chloroform-ethanol-glacial For the separation of cinnamoyl pigments
acetic acid (94: 5: 1) from Curcumae rhizoma
a Benzene is carcinogenic and should be replaced by toluene.
III. Detection
1. Without chemical treatment
UV-254 nm
All compounds containing at least two conjugated double bonds quench fluorescence
and appear as dark zones against the light green fluorescent background of the
TLC plate. Phenylpropane derivatives have this property, e.g. anethole, safrol, apiol,
myristicin, eugenol, asarone, methyl chavicol. Other compounds that quench fluores-
cence are cinnamic aldehyde, anisaldehyde, thymol and piperitone.
UV-365 nm
An intense blue fluorescence is given by e.g. methyl anthranilate.
7
2. Spray reagents
a) Anisaldehyde-sulphuric acid (AS No. 2, p. 299)
In the visible, the components of essential oils show strong blue, green, red and
brown colouration. Some compounds also fluoresce under UV-365 nm.
b) Vanillin-sulphuric acid (VS No. 38, p. 304)

Visible colourations are very similar to those obtained with the AS-reagent.
Exception : Thujone shows weak red with the AS-reagent, and only very weak blue
with the VS-reagent when viewed in the visible.
c) Phosphomolybdic acid (PMA No. 27, p. 303)

With the exception of anisaldehyde and fenchone, the constituents of essential oils
show uniform blue on a yellow background when viewed in the visible.
rx) Anisaldehyde shows blue with PMA-reagent only when present in concentrations higher
than 100 J..tg. At lower concentrations, its colour response varies from whitish to pale green
in visible light.
When sprayed with conc. H Z S0 4 and heated at about 100 0 C for 5 min, anis aldehyde
gives a red zone in the visible.
ß) Fenchone. After treatment of the TLC plate with PMA-reagent as described above, it
is then sprayed with a solution of 0.5 g potassium permanganate in 5 ml conc. sulphuric
acid. After heating for 5 min at 1000 C, fenchone appears as a dark blue zone in the visible.
These conditions are optimal for the detection of fenchone, but great care is advisable in
the preparation and use of the reagent.
Larger quantities (>100 J..tg) of fenchone, when heated with conc. H Z S0 4 , appear as a
yellow zone in visible light.
8
IV. List of Essential on Drugs, Gums and Resins
Chromatograms (Figs. 3-28) are reproduced on pp. 24-49.
THC = Terpene hydrocarbon(s)
Fig. Drug/Plant source/Family/Phar- Content of essential oil

macopoeia Main constituents
3 Cinnamomi Cortex Ceylon cinnamon: 1-1.5% ess. oil.

Cinnamon bark Cinnamic aldehyde (67-75%), eugenol
Cinnamomum zeylanicum BLUME (4--10%) and THC (e.g. caryophyllene, oc-pin-
Ceylon cinnamon ene).
Cinnamomum aromaticum NEES. Chinese cinnamon: 1-2% ess. oil.
(c. cassia BLUME) Cinnamic aldehyde (75-90%); eugenol absent.
Chinese or cassia cinnamon In addition, the bark contains unsubst. cou-
Lauraceae marin.
NF XV, ÖAB, Helv. VI
4 Calami Rhizoma (Radix) Triploid European race: up to 3 % ess. oil with

Sweet Flag rhizome variable content of IX-, p- and y-asarones
Acorus calamus L. (1-99%, average 50--60%).
Araceae Diploid races: 2.7-5% ess. oil, asarone absent;
containing ca. 30 compounds, e.g. isoeugenol,
ÖAB, Helv. VI isoeugenol methyl ether, acaromone, asarylal-
dehyde and artefacts formed during distilla-
tion.
5,6 Anisi Fructus 2-6% ess. oil (Ph. Eur. III specifies not less
Anise than 2%).
Pimpinella anisum L. Anethole (80--90%), methyl chavicol and
Apiaceae anisaldehyde.
NF XV (oil), Ph. Eur. III, 2. AB- Adulteration : Illicium anisatum L. (poisonous
DDR (oil) shikirni fruits I), mostly Safrol; Anethol is
absent.
5,6 Anisi stellati Fructus 5-8% ess. oil (ÖAB specifies not 1ess than
Star anise 5%)
Illicium verum HOOK. Anethole (85-90%), terpineol, phellandrene.
Illiciaceae
ÖAB
5,6 Foeniculi Fructus F. vulgare var. dulce (French sweet or Roman

Fennel Fennei): 2-5% ess. oil.
Foeniculum vulgare MILL. Anethole (50--60%), methyl chavicol (=estra-
Apiaceae goi), safrol, anisaldehyde and fenchone
ÖAB, Helv. VI, DAB 8,2. AB- (0.4--0.8%).
DDR, NF XV (oil) F. vulgare var. vulgare (French bitter Fennei) :
5-7% ess. oil (DAB 8 specifies not less than
4%)
Anethole (60--80%), methyl chavicol, anisal-
dehyde andfenchone (12-22%).
9
5 Basilici Herba 0.1-0.45% ess. oi!.

Basil Methylchavicol (ca. 55%) and linaloo!.
Ocimum basilicum L.
Lamiaceae
5 Sassafras Lignum 1-2% ess. oi!.

Sassafras wood Safrol (ca. 80%) and eugenol (ca. 0.5%).
Sassafras albidum (NUTT.) NEES.
vaL molle (RAF.) FERN. (syn. S. of-
ficinale NEES et EBERM.)
Lauraceae
7A Petroselini Fructus 3-6% ess. oi!.

Parsley fruits Phenylpropane derivatives: apiol, myristicin
Petroselinum crispum (MILL.) and allyltetramethoxybenzene.
NYM. ex hort. KEW (syn. P. hor- Apiol race: 60-80% apio!.
tense HOFFM.) vaL foliosum
(ALEF.) THELL. Leafparsley Myristicin race: 55-75% myristicin.
vaL tuberosum (BERNH.) THELL. Allyltetramethoxybenzene race: 50-60% allyl-
Root parsley tetramethoxybenzene.
Apiaceae Remarks: Petroselini radix (2. AB-DDR) con-
tains 0.2-0.3% ess. oil with apiol and myristi-
2. AB-DDR (oil) cin, as weil as thefuranocoumarins, bergapten
and isoimperatorin.
8A Myristicae Semen 6-10% ess. oil (Helv. VI specifies not less than
Nutmeg 6.5%).
Myristica fragrans HOUTT. Phenylpropane derivatives: myristicin (ca.
M yristicaceae 8%), safrol, eugenol, elemicin and TBC (IX-
pinene, limonene, p-cymene) and low concen-
Helv. VI, NF XV (oil) trations of the terpene alcohols, geraniol, bor-
neol, linalool and terpineo!.
Myristicae arillus 4-12% ess. oil, with the same qualitative com-
Mace position as the seed oi!.
Myristica fragrans HOUTT.
Myristicaceae
8B/C Caryophylli Flos 14-20% ess. oil (ÖAB specifies not less than
Cloves 16%).
Syzygium aromaticum MERR. et Eugenol (4-allyl-2-methoxyphenol) (72-90%),
PERRY aceteugenol (10-15%), ß-caryophyllene
Myrtaceae (3-12%) and epoxidihydrocaryophyllene.
NF XV (oil), Helv. VI, ÖAB, Remarks: Clove stalks contain only 5-6% ess.
DAB 8,2. AB-DDR, ÖAB (oil) oi!. Mother cloves or anthophylli (an adulter-
ation) contain 2-9% ess. oi!.
9A Carvi Fructus 2.5-7% ess. oil (DAB 8 specifies not less than
Caraway fruits 4%).
Carum carvi L. D-Carvone (50-85%), with small amounts of
Apiaceae carveol, dihydrocarveol, limonene and perillyl
alcoho!.
NF XV (oil), DAB 8, Helv. VI,
ÖAB, 2. AB-DDR
10
Fig. Drug/Plant souree/Family /Phar- Content of essential oil
9B Coriandri Fructus 0.2% ess. oil (Indian eoriander) 0.8-1 % ess.

Coriander fruits oil (Russian eoriander) (ÖAB speeifies not
Coriandrum sativum L. var. less than 0.5%).
vulgare ALEF. Linalool (50-70%) with small amounts of
Large Indian coriander geraniol and geranyl acetate, borneol and
var. microcarpum DC. citronellol, ca. 20% THC (ß-pinene, cx-terpin-
Small Russian coriander ene, myrcene).
Apiaceae
NF XV (oi!), ÖAB
9C Cardamomi Fructus 3-7% ess. oil (fruits), 4-9% ess. oil (seeds),
Cardamoms 0.5-1 % ess. oil (pericarp).
Elletaria cardamomum (L.) WHITE 'X.-Terpinyl acetate and 1,8-cineole (ca. 50%)
etMAsoN are the chief constituents, with small amounts
Zingiberaceae of borneol, cx-terpineol and limonene.
USP XX (seeds)
10A/B Juniperi Fructus 0.2-2% ess. oil (DAB 8 specifies not less than
Juniper berries 1 %).
Juniperus communis L. Varying composition of terpinene-4-01, caryo-
Cupressaceae phyllene, epoxydihydrocaryophyllene, ter-
pinyl acetate, camphor and the THC, cx-, ß-
USP XX (tar), DAB 8,2. AB- pinene, myrcene and camphene.
DDR, ÖAB, Helv. VI (fruit and
oi!)
10C Rosmarini Folium 1-2% ess. oi!.

Rosemary leaves 1,8-Cineole (15-30%), borneol (10-20%), bor-
Rosmarinus officinalis L. nyl acetate, camphene (5-10%) and cx- and
Lamiaceae ß-pinene.
DAB 8,2. AB-DDR, Helv. VI,
ÖAB (oil)
11 A, 12 MatricarJae Flos 0.5-1.5% ess. oil (Ph. Eur. specifies not less
(Chamomillae flos) Chamomile than 0.4%; 2. AB-DDR 1.2-1.8%).
flowers Chamazulene (0-15%), bisabolol (10-25%),
Chamomilla recutita (L.) RAUSCH bisabolol oxide A and B (10-25%), polyines
Asteraceae (cis- and trans-ene-ine-dicycloether, 1-40%)
and farnesene (15%).
Ph. Eur. III, Helv. VI, ÖAB,
2. AB-DDR (0.1-0.16% matricin)
11 B Anthemidis Flos 0.6-2.4% ess. oil (Ph. Eur. III specifies not
Roman chamomile flowers less than 0.7%), with a high proportion of
Chamaemelum nobile (L.) ALL. esters of angelie, methacrylic, tiglie and isobu-
tyric acids with aliphatic alcohols; cis and
Asteraceae trans-dehydromatriearia acid, polyines.
Ph. Eur. III, ÖAB Flavonoids: Apigenin, A.-7-glucoside, A.-7-
apiosylglucoside, luteolin, luteolin-7 -gluco-
side, quercitrin (see chapter on Flavonoids,
p. 182, Fig. 11/12).
11
Fig. DrugjPlant sourcejFamily jPhar- Content of essential oil
13 Lavandulae Flos 1-3% ess. oil (DAB 8 and ÖAB specify not
Lavender flowers less than 35% esters).
Lavandula angustifolia MILL. Linalyl acetate (30-50%), ünalool (10-15%),
with small quantities ofnerol, borneol, geran-
Lamiaceae
iol, cineole and caryophyllene.
USP XX (oil), DAB 8,2. AB-
DDR, ÖAB (oil)
Lavandula latifolia MED. Oil 0/ spike: esters are low or absent; chief
Lavandula hybrida, e.g. L. latifolia constituents linalool and cineole.
+ L. offic. "Lavandin oils": 20-24% or 30-32% linalyl
acetate, linalool, terpene hydrocarbons and
terpene alcohols, as in L. angustifolia.
14 Cinae Flos 2-3% ess. oil.

Wormseed 1.8-Cineole (ca. 80%) with small amounts of
Artemisia cina O.C. BERG et C.F. a-terpineol, carvacrol and sesquiterpene hy-
SCHMIDT drocarbons.
Asteraceae Bitter principles: up to 6% L-rz.-santonin and
rz.-hydroxy-santonin (artemisin).
15j16 Menthae piperitae Folium 1.3-2.1 % ess. oil.

Peppermint leaves Menthol (50-78%), (- )menthone (10-30%),
Mentha piperita L. menthyl acetate (5-20%), menthofuran
Lamiaceae (2.5-5%) with small amounts ofisomenthone,
pulegone, piperitone, cineole, limonene, jas-
NF XV (oil), Ph. Eur. III, ÖAB, mone (0.1 %).
2. AB-DDR, Helv. VI
Ph. Eur. Irr specifies not less than 1.2 % ess.
oil, containing 4.5-10% esters (calculated as
menthyl acetate), at least 44% alcohols (cal-
culated as menthol) and 15-32% ketones (cal-
culated as menthonejisomenthone).
M. arvensis L. var. piperascens 0.2-0.3% ess. oil; approx. same qualitative
HOLMES ex CHRISTY composition as the oil from M. piperita, but
NF XV (oil) menthofuran and cineole are absent.
Lamiaceae Cornmint oil (DAB 8): 3-17% esters (calc. as
menthyl acetate), at least 42% alcohols (calc.
as menthol), at least 25-40% ketones (calc.
as menthone).
Mentha pulegium L. (an adultera- 1-2% ess. oil.
tion ofM. piperita and M. arvenis) Pulegone (80-95%) with small amounts of pi-
Lamiaceae peritone, menthol and THC.
Menthae crispae Folium 1-2% ess. oil.
Spearmint leaves L-Carvone (42-67%), acetates of dihydrocar-
Mentha spicata L. emend. L. var. veol and dihydrocuminyl alcohol, THC (pin-
crispa (BENTH.) DANERT ene, limonene, phellandrene).
NF XV (oil)
Lamiaceae
12
17/18 Salviae Folium 1.3-2.6% ess. oil (DAB 8 specifies not less
Sageleaves than 1.5%).
Salvia officinalis L. Composition varies, depending on origin:
S. officinalis ssp. rninor (GMEL.)
Thujone 35-50% (S. offic. ssp. minor/major),
GAMS. S. officinalis ssp. officinalis.
cineole ca. 14%, camphor 7-8%
Dalmatian sage.
S. officinalis ssp. lavandulifolia Thujone absent (S. offic. ssp. lavandulifolia),
(VAHL) GAMS, cineoie ca. 30%,camphor 30%
Spanish sage Also present are terpene alcohols (borneol
5-8%) and THC (pinene, camphor),
Rosmarinic acid (2-3%), depside of caffeic
and IX-hydroxydihydrocaffeic acids.
Diterpene bitter principles: picrosalvin ( = car-
nosol) in Dalmatian sage (ca. 0.35%).
Bitter principles (see p. 142, Fig. 11).
Salviae trilobae Folium Up to 3% ess. oil (DAB 8 specifies not less

Greeksage than 1.8%).
Salvia trilo ba L. fil. 1,8-Cineole (60-70%), thujone (ca. 5%) and
borneol (ca. 0.35%), bornyl acetate, THC
Lamiaceae (pinene, camphene).
DAB8 Bitter principle: picrosalvin (=carnosol,
0.2-0.3%) (see Bitter principle drugs, p. 142,
Fig.11).
Flavone: salvigenin (=8-hydroxy-6,7,4'-tri-
methoxyllavone).
18 Eucalypti Folium 1-3% ess. oil.

Eucalyptus leaves 1,8-Cineole (=eucalyptol, at least 70%) with
Eucalyptus globulus LABILL., small amts. of piperitone, phellandrene, alde-
E. fruticetorum F. v. MUELLER, hydes*.
E. smithii R.T. BAKER Non-official oils sometimes contain high lev-
Myrtaceae els ofpiperitone and/or phellandrene, e.g. Eu-
NF XV (oil), Ph. Eur. III, ÖAB, calyptus dives SCHAUER
2. AB-DDR, Helv. VI (oil) * Non-rectified oils contain, e.g. butyralde-
hyde and capronaldehyde, which cause
bronchial irritation.
19 ThymiHerba 0.75-6.3% ess. oil (DAB 8 specifies not less

Thyme than 1.2%).
Thymus vulgaris L. Thymollcarvacrol (20-60%) with small
Lamiaceae amounts of 1.8-cineole, borneol, geraniol, lin-
alool, bornyl and linalyl acetate, thymol
DAB 8,2. AB-DDR, Helv. VI (oil) methyl ether and IX-pinene.
Thymus zygis L. Content and composition of ess. oil corre-
Spanish thyme spond with those of Th. vulgaris, but propor-
Lamiaceae tion of carvacrol is higher than that of thy-
DAB8 mol.
13
Serpylli Herba Composition of ess. oil similar to that of

Wild thyme Thymi Herba, with lower contents of thymol
Thymus serpyllum L. and carvacrol, and higher contents ofp-cymol
and linalool, together with terpene esters.
Lamiaceae
Helv. VI
Ajowani Fructus 2.6--4.5% ess. oil.

Ajowan fruits Thymol (35-60%) with sm all amts. of carvac-
Trachyspermum am mi (L.) rol and THC.
SPRAGUE
Apiaceae
20 Melissae Folium 0.01-0.25% ess. oil (DAB 8 specifies not less

Melissa or Lemon balm leaves than 0.05%).
Melissa officinalis L. Citronellal (ca. 39%), citral (ca. 30%), citro-
Lamiaceae nellol, linalool, geraniol and THC (caryophyl-
lene).
DAB 8,2. AB-DDR, ÖAB,
Helv. VI (oil) M etissa oU substitutes:
Java Citronella oil, Cymbopogon nardus (L.)
W. WATS., Poaceae. 0.5-1.2% ess. oil, con-
taining citronellal (25-54%) and geraniol
(16-45%).
Lemon grass oil, Cymbopogon flexuosus
(NEES et STEUD) W. WATS., Poaceae.
53-83% Citral (West Indian type) with farne-
sol, geraniol and linalool.
70-85% Citral (East Indian type).
80-84% Citral (Angola type; odourless!).
21/22 Curcumae Rhizoma

Turmeric
Curcuma xanthorrhiza RoXB. 6-11 % ess. oil (DAB 8 specifies not less than
Round turmeric 3.5%).
Zingi beraceae L-Cycloisoprenmyrcene (ca. 85%), xanthor-
DAB 8,2. AB-DDR rhizol (phenolic sesquiterpene), tolylmethyl-
carbinol (ca. 5%, an artefact), camphor
(1-5%).
Curcuma longa L. (syn. C. domes- 0.3-5% ess. oil.
tica VAHL.) Sesquiterpenes ca. 65% (e.g. turmerone),
Finger or Long turmeric zingiberene (ca. 25%), phellandrene, sabin-
Zingiberaceae ene, borneol and cineole.
Curcumins: non-steam volatile diferuloyl- and
dicinnamoyl-methane compounds.
C. xanth.: 1.2-2% curcumin and monode-
methoxy-curcumin.
C. tonga: 3-4% curcumin, monodemethoxy-
curcumin and bisdemethoxycurcumin.
14
23/24 Aurantü Pericarpium 0.6-2.2% ess. oil (DAB 8 specifies not less
SeviIIe orange peeI than 1 %).
Citrus aurantium L. ssp. aurantium (+ )-Limonene (90%) with terpene alcohols
Rutaceae and aldehydes.
NF XV (oil), DAB 8,2. AB-DDR, F/avonoids: rutin, eriocitrin, naringin, neohes-
ÖAB, Helv. VI peridin (see section on Flavonoids, p. 189,
Fig. 17).
Methyl anthranilate, coumarins.
Aurantii Flos 0.1-0.6% ess. oil (ÖAB specifies not less than
Orange flowers 0.2%).
Citrus aurantium L. ssp. amara Linalyl acetate (8-25%), linalool (ca. 30%),
ENGL. (syn. C. aurantium ssp. sin- farnesol, limonene, jasmone.
ensis) (Oil of Neroli)
Rutaceae
NF XV (oil), ÖAB, Helv. VI
23/24 Citri Pericarpium 0.1-6% ess. oiI.

Lemonpeel (+ )-Limonene (90%), citral (3.5-5%) with
Citrus limon (L.) BURM. smaII amts. of terpineol, linalyl and geranyl
Rutaceae acetate.
USP XX (oil) Coumarins: geranylmethoxycoumarin, citrop-
ten, bergamottin.
Flavonoids: rutin, eriocitrin, neo hesperidin
(see section on Flavonoids, p. 189, Fig. 17) .
Citrus aurantium (L.) ssp. berga- .. Bergamot oil" (fruit peel oil): chiefly linalyl
mia (RISSO et POlT) ENGL. acetate, with a dihydrocumin alcohol and
Rutaceae linalooI.
Coumarin: hergapten (ca. 5%).
"Oil 0/ Petit Grain" (leaf oil) contains chiefly
linalyl acetate and a terpene alcohoI.
Pine Oils
These are essential oils from the needles and branch tips of Abies, Picea and Pinus spp.
(family Pinaceae).
25 Pini pumilionis Aeth. 10% ess. oiI.

Mountain pine oil 3-10% esters, calc. as hornyl acetate (DAB 7-
Pinus mugo TURRA ssp. mugo agg. DDR). Chief terpenes: IJ(- and ß-pheIIandrene
Pinus mugo ssp. pumilio (HAENKE) (ca. 60%) and IJ(- and ß-pinene (10-20%).
FRANCO
DAB 7-DDR, ÖAB, Helv. VI,
NF XV (oil)
Pini silvestris Aeth.
Scots pine needle oil
Pinus sylvestris L.
15
25 Picea spp. (parent plant not de- Oils of Pine, Spruce and Silver fir all have
fined) similar terpene compositions. Siberian spruce
USP XX (oil) needle oils have a markedly higher content
of bornyl acetate and terpineol.
Pine needle oils
Pini sibirici Aeth.

Siberian spruce oil
Abies sibirica LEDEB. (Erg. Bd. 6)
Abies pictinatae Aeth.

Silver fir oil
Abies alba MILLER (Erg. Bd. 6)
25 Terebinthinae Aeth. Distillate of turpentine (Terebinthinae Balsa-

Terebinthinae rectificatum aeth. mum) from various Pinus spp.
Turpentine oil, rectified oil of tur- 80-90% TBC (iX-, ß-pinene, limonene, phel-
pentine landrene). Autoxidation produces iX-pinene
Pinus ssp., Pinus palustris MILLER, peroxides and subsequently verbenol and pinol
Pinus pinaster AITON et al. hydrate ( = sorbenol).
Helv. VI
Gums and Resins
26 Myrrha 2-10% ess. oil.

Myrrh Cinnamic aldehyde, cuminaldehyde, eugenol,
Commiphora molmol ENGL. m-cresol, sesquiterpenes.
Commiphora spp. 25-40% Ethanol-soluble resin fraction, con-
Burseraceae taining diterpene acids, e.g. iX-, ß- and y-com-
miphoric acids and esters.
Helv. VI
Benzresins and Balsams

Main constituents are cinnamic acid, ferulic acid and coniferyl a1cohol and their esters.
27 Benzoe tonkinensis At least 25% free or combined acids, deter-

Siam benzoin mined as benzoic acid (Ph. Eur. III).
Styrax tonkinensis (PIERRE) CRAIB Coniferyl benzoate (60-80%), cinnamoyl ben-
exHARTWICH zoate (ca. 2%), benzoic acid (10-20%), vanil-
Styracaceae lin (ca. 0.3%), iX-siaresinolic acid (19-hydro-
xyoleanolic acid).
USP XX, Ph. Eur. III, ÖAB.
Helv. VI
Benzoe Sumatra Coniferyl cinnamate and coniferyl benzoate
Sumatra benzoin (70-80%), cinnamic acid esters, styracin, cin-
Styrax benzoin DRYANDER namic acid (ca. 10%), cinnamic acid phenyl-
Styracaceae propyl ester (ca. 1 %), vanillin (ca. 1 %), su-
maresinol (6-hydroxyoleanolic acid).
16
28 Tolutanum Balsamum ca. 7.5% "cinnamein ", a mixture of benzoyl

Tolu balsam benzoate and cinnamoyl benzoate (2: 1); ca.
Myroxylon balsamum (L.) HARMS 80% resin (mostly cinnamic esters of toluresi-
var. balsamum tannol), cinnamic acid, benzoic acid, vanillin,
eugenol.
Fabaceae
USP XX (tinct.), He1v. VI, ÖAB
Peruvianum Balsamum At least 50-70% "cinnamein" (DAB 8), con-
Peru balsam sisting ofbenzoyl benzoate (25-40%) and cin-
Myroxylon balsamum (L.) HARMS namoyl benzoate (10-25%) in ratio 2.8:1,
var. pereira (ROYLE) HARMS sometimes other proportions in the range 2: 1
to 4: 1.
Fabaceae
20-28% resin (mostly cinnamic esters ofpere-
DAB 8, Helv. VI, ÖAB, 2. AB- sitannol), cinnamic acid (ca. 10%), benzoic
DDR acid, dihydrobenzoic acid and 0(- and ß-neroli-
dol (3-5%).
17
v. Formulae of Constituents of Essential Oils
Cl-Pinene ß-Pinene Carene Cl-Phellandrene Limonene Cl-Caryophyllene ß-Caryophyllene
Geraniol Nerol Carveol Terpinen-4-ol Linalool Cl-Terpineol: R = H

(trans) (cis) Terpinylacetate:
R=CHaCO
tr°
~OCOCHJ
H
&OCOCHJ
cD ~OH ~OH
Borneol Bornylacetate Linalylacetate 1,8-Cineole Thymol Carvacrol
(Eucalyptol)
~90 S:90 ~O ~O
I 'H I
I
'H
I
5!0 ctr (it

Citral Citronellal Carvone Piperitone Thujone (+ )-Fenchone Camphor
O~o
Turmerone Xanthorrh izol Cl-Santonin
19
OR
~ ~C~~ COOH
Cinnamic trans- Eugenol: R = H trans-Anethole Methyl- An isaldehyde Safrol
aldehyde Cinnamic Aceteugenol: chavicol
acid R=CH 3 CO
Apiol Myristicin Allyltetramethoxybenzene Elemicin
OCH3
Benzyl benzoate Cinnamein Ö rA;O OH
~O~
~o~ o
Coniferyl benzoate
o
Benzyl cinnamate
R, R2
Isoeugenol methyl ether - CH = CH - CH 3 (trans)H
y-Asarone - CH 2 - CH = CH 2 OCH 3
cis-Asarone (ß-Asarone) - CH = CH - CH 3 (cis) OCH 3
trans-Asarone (cx-Asarone) - CH = CH - CH 3 (trans)OCH 3
Acaramone - CH 2 - CO - CH 3 OCH 3
Asarylaldehyde - CHO OCH 3
20
D(-)-Menthol (-)-Menthone Menthofuran Jasmone
eH3
HO OCOCH, ~
r
eH3 H2
eH3
Proazulene Chamazulene cis(trans)-ene-ine-Dicycloether
(Matricin)
(-)-a-Bisabolol
/V
(l---bl
O><'OH
O? OH
(-)-a-Bisabolol oxide A (-)-a-Bisabolol oxide B (-)-a-Bisabolone oxide A
21
TLC Synopsis
Terpene and Phenylpropane Reference Compounds
Fig.l Compounds applied in order of increasing Rf-value and decreasing polarity
Fig.2 Monoterpene alcohols and their esters
Track Reference compound Rf-value Colour
Fig.1 1= borneol 0.24 violet-blue

2=linalool 0.30 blue
3 = piperitone 0.35 orange
4=cineole 0.40 blue
5=citral 0.42 blue-violet
6=carvone 0.46 red-violet
7=eugenol 0.47 yellow-brown
8=thymol 0.52 red-violet
9= citronellal 0.65 blue
10=apiol 0.65 brown-red
11 = myristicin (Macis aeth.) 0.75 red-brown
12 = anethole 0.85 red-brown
13= safrol 0.87 red-brown
Fig.2 14 = geraniol 0.25 grey-blue
15 = geranyl acetate 0.73 grey-blue
16=nerol 0.26 grey-blue
17 = neryl acetate 0.74 grey-blue
18=linalool (~2/Fig. 1) 0.36 grey-blue
19=1inalyl acetate 0.75 grey-blue
20 = trans-sabinene hydrate 0.25 violet
21 = terpineol 0.25 blue-violet
22 = phytol 0.35 violet
23 = farnesol (impure ) 0.30 blue
Solvent A-1: toluene-ethyl acetate (93: 7)

system
Detection Vanillin-sulphuric acid (VS No. 38, p. 304); vis.
The reference compounds can be divided into four main groups on the basis of
their characteristic colour reactions:
brown-red/violet phenylpropane derivatives: safrol, anethole,
myristicin, apiol and eugenol.
orange to red-violet carvone, thymol, piperitone.
blue / blue-violet citral, citronellal, cineole.
grey-blue most monoterpene alcohols and their esters
(geraniol, nerol, linalool, borneol; cf. menthol,
menthyl acetate, Fig. 15, p. 37).
Remarks: Commercially available reference substances often show weak extra zones at lower
Rf-values and sometimes also at higher Rf-values. These are partly due to resinification, decom-
position products and incompletely removed impurities.
22
-FRONT
Rf
-0.5
-START
'--_ _ _ _4_ _ _7 11
Fig.l
Tl-13
-FRONT
Rf
-0.5
-START
Fig.2
T 14 - 23
23
Cinnamomi Aetheroleum, Calami Aetheroleum
Tracks 1 = Cinnamomi ceylanici aeth. (Ceylon cinnamon oil)
2 = Cinnamomi aromatici aeth. (Cassia cinnamon oil)
3,4= Cinnamomi aeth. commercial oils I, II
5 -11 = Calami aeth. (various sourees)
Tests Tl = cinnamic aldehyde
T2 = coumarin
T3=asarone
T4=eugenol
Solvent A-1 : toluene-ethyl acetate (93: 7) Fig. 3A; 4A
system toluene-ethyl acetate (97: 3) modified Fig.4B
A-4: dichloromethane Fig. 3B; 3C
Detection Vanillin-sulphuric acid (VS No. 38, p. 304) vis. Fig. 3A, C; 4A, B
Potassium hydroxide (KOH No. 21, p. 302) UV-365 nm Fig.3B
For description of drugs see p. 9. Formulae p. 20.

Chromatogram
3 Cinnamomi aetherolea. The grey-brown zone of cinnamic aldehyde at Rf 0.5, ob-
tained after treatment with VS-reagent (cf. Tl), characterizes the TLC picture of
the cinnamic oils 1-4.
3A 1 Ceylon cinnamon oil shows an additional violet-red zone directiy above the cinnamic
aldehyde. In the region of the terpene alcohols (RfO.15-0.3) are four weak zones
(linalool Rf ca. 0.3), and the THC (a-pinene, caryophyllene) run with the solvent
front.
3A 2 Cassia cinnamon oil. In the visible this differs from official oil of cinnamon only
with respect to the secondary zones. A dear differentiation is achieved (DCM-
extract, p. 6) in UV-365 nm after treatment with KOH-reagent. The strongly blue
fluorescent zone of unsubstituted coumarin (cf. T2) appears at RfO.4. Only traces
of this coumarin are detectable in Ceylon cinnamon oil.
4 5-11 Calami aeth. After treatment with VS-reagent, chromatograms of Sweet Flag oil
are characterized by aseries of violet, blue and brown-violet zones extending from
Rf 0.1 to the solvent front. Asarone appears at Rf ca. 0.4 (cf. T3) as a red-violet
zone. The concentration varies greatly, depending on the origin and chemical race
of the drug. The oils in tracks 5 and 6 are from asarone-poor or asarone-free diploid
races of American origin.
The oils in tracks 7-11 have relatively high asarone contents and are derived
from triploid or tetraploid races.
At the Rf of the reference substance, eugenol (T4), all the oils show only a
weak zone. Above this are 3-4 blue to blue-violet zones, due to various concentra-
tions of sesquiterpenes. The lower Rf region contains e.g. asaraldehyde.
24
-FRONT
Rf
-0.5
-START
Fig.3
Tl 2 T2 2 3 4 Tl
A B
-FRONT
Rf
-0.5
-START
Fig.4
T3 5 6 7 T4 8 9 10 11 T3
25
Anisi, Foeniculi, Basilici, Sassafras Aetherolea, Anisi Fructus
Essential oils with anethole, methylchavicol or safrol as the main constituents
Tracks 1 = Anisi aeth. (Offic. anise) 5 = Basilici aeth.
2=Anisi stellati aeth. (Star anise) 6 = Sassafras acth.
3 = F oeniculi aeth. (Bitter fennel) 7= Anisi stellati fructus (DCM-extract)
4 = Foeniculi aeth. (Sweet fennei) 8 = Anisi fructus (DCM -extract)
Tests T1 = anethole T3 = eugenol
T2 = safrol T 4 = fenchone
Solvent A-l : toluene-ethyl acetate (93: 7) Fig. 5; 6A, B
system A-2: toluene Fig.6C
Detection Vanillin-sulphuric acid reag. (VS No. 38, p. 304) VlS. Fig. 5A, B
Phosphomolybdic acid reag. (PMA No. 27, p. 303) vis. Fig.6C
PMA-potassium permanganate reag. (PMA-PM No. 22,
p.302) VlS Fig.6B
conc. Sulphuric acid (H 2 S0 4 No. 34C, p. 303) vis. Fig.6A
For description of drugs see p. 9-10. Formulae p. 20.

Chromatogram
5A Anisi and Foeniculi aetherolea. In oils 1-4, treatment with VS-reagent reveals the
characteristic, main red-brown zone (Rf ca. 0.95) of the anethole-methy1chavicol
mixture (cf. Tl).
1 Offic. anise oU shows practically no terpenoid zones.
2 Star anise oil shows an additional 5-6 blue-violet zones of low intensity in the Rf
range 0.2-0.6.
6C 7 A DCM-extract of Star anise fruits developed in toluene according to Ph. Eur. III.
After treatment with PMA reagent, a weak blue zone (er T2) immediately above
the anethole zone (er Tl) can be seen. The triglycerides present in the seeds and
fruits show as a blue zone at Rf ca. 0.3.
6A, B 3,4 Fennel oil also contains fenchone (er T4, bitter fennel 12-22%, sweet fennel
0.4-0.8%) in addition to anethole (cf. Tl).
Fenchone cannot be detected with VS-reagent. In quantities greater than lOOllg
it gives an ochre-yellow zone with conc. H 2 S0 4 . At lower concentrations the PMA-
PM reagent gives a more reliable detection.
In bitter fennel (3) fenchone is c1early seen as a dark blue zone; the traces
present in sweet fennel (4) are recognizable as a whitish zone. Fenchone is not
detectable in Anise oil (1).
Anise and fennel oils contain anisaldehyde, which can be detected in the Rf
region below fenchone (violet-red zone with conc. H 2 S0 4 , or a whitish zone with
PMA-PM, both in the vis.). Anisaldehyde shows a prominent fluorescence quenching
when observed directly in UV-254 nm.
5A 5 Basilici aeth. Basil oil shows methylchavicol as a red-violet zone, and three intense
blue zones in the Rf range 0.2-0.5. The zone at Rf ca. 0.3 corresponds to linalool.
5B 6 Sassafras aeth. Sassafras wood oil is characterized by the zone of safrol at Rf ca.
0.95 (er T2). There are two weaker zones in the Rf region of the standard eugenol
(er T3), and two stronger zones in the lower Rf range (terpene a1cohols).
26
A B
-FRONT
Rf
-0.5
- START
Fig.5
Tl 2 3 4 5 T2 6 T3
A c
-FRONT
Rf
-0.5
-START
Fig.6
3 T4 T4 3 4 7 8 Tl
27
Petroselini, Myristicae, Caryophylli Aetherolea
Tracks 1-5 = Petroselini aeth. (P. fructus, steam distillate)
6 = Myristicae aeth. (M. semen, steam distillate)
7 = Macis aeth.
8= Caryophylli aeth. (e. flos, steam distillate)
Tests Tl =eugenol
T2=apiol
T3 = allyltetramethoxybenzene
T4 = aceteugenol
Solvent A-l : toluene-ethyl acetate (93: 7) Fig. 7 A, B; 8A, B
system A-2: toluene Fig.8C
Detection Vanillin-sulphuric acid-reag. (VS No. 38, p. 304) vis. Fig. 7-8
Chromatogram
7A 1-5 Petroselini aeth. After treatment with VS-reagent, oils 2-5 show the two main brown-
violet zones of apiol (T2, Rf ca. 0.75) and myristicin (Rf ca. 0.8) in the upper
Rf range, together with weaker violet-brown zones of eugenol (Tl) or eugenol methyl-
ether and allyltetramethoxybenzene (cf. T3) in the intermediate Rf range. Oi11 is
apiol-free.
7B When applied in fairly small quantities (2 111), myristicin and apio! (cf. T2) show
decreased Rf values.
There are "chemical races" of Petroselini fructus, in which the predominant constituent of
the fruit oil is myristicin (= oil 1) or apiol (= oil 2) or more rarely allyltetramethoxybenzene.
Most oils (tracks 3, 4, 5) contain approximately equal concentrations of the two main constitu-
ents. Allyltetramethoxybenzene and eugenol are present in lower concentrations. Occasionally,
as in oil 4, safrol can be detected directly above myristicin.
8A 6, 7 Myristicae aeth., Macis aeth. Both oils give similar chromatograms after treatment
with VS-reagent; there are about 8 predominantly brown to brown-vio!et zones
in the Rf range 0.2 to the solvent front.
The main zone of myristicin (Rf ca. 0.8) is more prominent in mace oil. Safrol
(Rf ca. 0.9), like eugenol (cf. Tl), is present only as a weak brown zone.
At low concentrations, terpene a1cohols (linalool, geraniol and terpineol) can be detected
as weak violet-brown zones in the Rfrange 0.25--0.4. The THC, pinene, limonene and p-cymene,
mi grate as a single violet-brown, unresolved zone at the solvent front.
8B 8 Caryophylli aeth. Treatment with VS-reagent reveals (in the vis.) the main orange-
brown zone of eugenol (cf. Tl), and the strong red-violet zone ofthe THC (humulene
and caryophyllene) at the solvent front.
8C A satisfactory separation of eugenol (cf. Tl) and aceteugenol (cf. T 4) can only
be achieved with the solvent system toluene. Epoxydihydrocaryophyllene is seen
as a red-violet zone directly below aceteugenol.
28
A B
-FRONT
Rf
-0.5
~START
Fig.7
Tl 2 3 4 5 T2 5 TJ
A c
FRONT
Rf
-0.5
-START
Fig.8
6 7 Tl 8 T4 Tl 8 T4
29
Carvi, Coriandri, Cardamomi, Juniperi, Rosmarini Aetherolea
Tracks 1 = Carvi aeth. (C. fructus, steam distillate)
2 = Carvi aeth. (commercial oil)
3=Coriandri aeth. (C. fructus, steam distillate)
4 = Coriandri aeth. (e. semen, steam distillate)
5 = Cardamomi aeth. (C. fructus, steam distillate)
6 = J uniperi aeth. (J. fructus, steam distillate)
7 = Rosmarini aeth. (official rosemary oil)
8= Rosmarini aeth. (oil from R. hispidus)
Tests T1 =carvone T5 = bornyl acetate
T2 = linalool T6 = a-terpineol (Rf ca. 0.25) and
T3=cineole a-terpinyl acetate (Rf ca. 0.75)
T4=borneol
Solvent A-1: toluene-ethyl acetate (93: 7) Fig. 9A-C; lOB, C
system A-4: dichloromethane, DAB 8 Fig.lOA
Detection Vanillin-sulphuric acid (VS No. 38, p. 304) VlS. Fig.9A-C
Anisaldehyde-sulphuric acid (AS No. 2, p. 299) vis. Fig. lOA-C
F or description of drugs see p. 10-11. F ormulae p. 19.
Chromatogram
9A 1,2 Carvi aeth. Caraway oils are characterized by the intense, raspberry-red zone of
carvone (T1) at Rf ca. 0.5. The terpene a1cohols, carveol and perillyl alcohol, migrate
together and are seen as a weakly defined, blue zone at Rf ca. 0.2.
9B 3,4 Coriandri aeth. The characteristic main zone of the oil from coriander fruits or
seeds is due to linalool (T2; Rf ca. 0.35)
Seed oils have a markedly higher linalool content. In addition, geraniol (Rf ca. 0.2) and
geranyl acetate (Rf ca. 0.7) are detectable as grey-blue zones.
9C 5 Cardamomi aeth. Cardamom oils produce 5-6 strong blue zones: a.-terpinyl acetate
(cf. T6; Rf ca. 0.75), cineole (cf. T3; Rf ca. 0.5) and less intense zones of the
terpene a1cohols, linalool (Rf ca. 0.35), borneol and terpineol (cf. T6; Rf 0.2-0.25).
The THC, limonene, appears as a weak violet zone at the solvent front.
10A, B 6 Juniperi aeth. In solvent systems A-1 or A-4 (DAB 8) juniper oil exhibits about 6
red to red-violet zones in the Rf range 0.2-0.5, with terpinene-4-o1 (ab out the same
Rf as standard cineoie ; cf. T3) and a striking red zone direcdy above it, presumably
due to epoxydihydrocaryophyllene.
Some of the zones in the lower Rf range are degradation products of THC that were initially
present in the oi!.
7, 8 Rosmarini aeth. Rosemary oil yields at least 9 red-violet or blue-violet zones over
practically the whole range of Rf values. Offiäal rosemary oi! (7) has a low content
of cineole (cf. T3), and increased levels of terpene a1cohols (borneal: Rf ca. 0.25;
cf. T4); there is a litde bornyl acetate (Rf ca. 0.7; cf. T5) and the THC (a- and
ß-pinene) mi grate with the solvent front.
The commercial oil (8) shows the characteristic main zones in the intermediate Rf range
0.35-0.5, with a predominance of cineole (Rf ca. 0.45; cf. T3) and a low proportion of terpene
alcohols (Rf range 0.2-0.3), terpene esters (Rf ca. 0.7) and THC (solvent front).
Remarks: Solvent system A-1 and A-4 (DAB 8) show practically the same separation properties
for Juniperi and Rosmarini aeth.
30
A B c
-FRONT
Rf
-0.5
-START
Fig.9
Tl 2 3 T2 4 T3 5 T6
A B c
-FRONT
Rf
-0.5
-START
Fig.10
6 T3 6 T3 T4 T3 7 8 T5
31
Matricariae (Chamomillae), Anthemidis Aetherolea
Tracks 1-13 = Matricariae aeth. (Matricariae flos, various origins, steam distillate)
14= Anthemidis aeth. (steam distillate)
15 = Matricariae flos (DCM-extract)
16= Matricariae aeth. (steam distillate)
Tests Tl = bisabolol oxide A (Rf ca. 0.2); bisabolol (Rf ca. 0.35)
T2 = linalool
T3 = bisabolol oxide A
T4 = bisabolol oxide A and B (Rf ca. 0.1--0.2); bisabolol (Rf ca. 0.35);
chamazulene (Rf ca. 0.85)
T5 = borneol (Rf ca. 0.2); bornyl acetate (Rf ca. 0.55) (Ph. Eur.)
T6 = linalool (Rf ca. 0.25); linalyl acetate (Rf ca. 0.55)
Solvent A-1 : toluene-ethyl acetate (93: 7) Fig. 11 A, B; 12A, B
system A-7:chloroform-benzene (75:25) Fig.12C
Detection Vanillin-sulphuric acid-reag. (VS No. 28, p. 303) VIS. Fig. 11A, B; 12A, B
Anisaldehyde-sulphuric acid-reag. (AS No. 2, p. 299) vis. Fig. 12 C
For description of drugs see p. 11. Formulae 21.
Chromatogram
11j12 Matricariae aeth. After treatment with VS-reagent, oils obtained by distillation and
DCM-extracts are characterized by the following main zones:
Zone Rf Colour Assignment
I ca. 0.2 yellow-green bisabolol oxide AlB
II ca. 0.25 violet terpene alcohol
III ca. 0.35 violet bisabolol
IV ca. 0.5-0.6 brown cisItrans-ene-ine-dicycloether
V ca. 0.95 red-violet azulene (non present in DCM-extract)
VI ca. 0.99 blue-violet farnesene
Compounds I-IV lie in the Rf range between borneoljlinalool and bornyl acetatej
linalyl acetate (cf. T5jT6).
11 B 14 Anthemidis aeth. shows none of the compounds I-V. The oil is characterized by
the ester zone in the upper Rf range.
11 1-13 Oil distillates of commercial chamomile flowers.
1,6 Azulene-containing oils of official quality show relatively high contents of THC, azulene,
polyines and bisabolol oxides.
2-5,7,13 Azulene-poor or azulene-free oils ofEgyptian, Bulgarian or Jugoslavian origin. The characteris-
tic oil constituents are present in very low concentrations. Oil 13 shows a high polyine content.
8, 9 Polyine-poor oils with relatively high contents of azulene and bisabolol oxides.
10,11,12 Oils with medium concentrations of azulene and variable contents of bisabolol.
12AjB 15 Dichloromethane extracts of commercial chamomile flowers
The chromatogram of a DCM-extract (15) differs from that of a steam distillate (16) primarily
by the absence of azulene; DCM only extracts proazulene (=matricin 1 ), which is located
above the start.
With slight shifts of Rf values in the lower range, solvent systems A-l and A-7 (Ph. Eur.)
give the same sequence of zones. VS-reag. produces red-violet and brown, while matricin 1
in particular produces a red-violet zone with AS-reagent.
1 Matricin can be specifically detected with EP-reagent (No. 15, p. 301).
32
A B -FRONT
VI Rf
V
IV { -0.5
ttI
11
-START
Fig.11
Tl 1-13 14 T2
A B c
-FRONT
Rf
-0.5
-START
Fig.12
15 T3 T4 16 15 T5 T6 16
Ph .Eur.III
33
Lavandulae Aetherolea. Cinae Flos
Tracks 1 = Lavandulae aeth. (Lavandin oil)
2 = Lavandulae aeth. (Barreme oil)
3 = Lavandulae aeth. (Mont Blanc oil)
4 = Lavandulae aeth. (oil of spike; L. latifolia)
5 = Lavandulae aeth. (L. angustif., steam distillate)
6 = Citri aurantii aeth. (oil of petit grain)
7 = Cinae fios (DCM extract)
Tests Tl = mixture of linalool (Rf ca. 0.35) and linalyl acetate (Rf ca. 0.7)
T2=cineole
T3 = o:-santonin
Solvent A-l : toluene-ethyl acetate (93: 7) Fig. 13A, B; 14A, B
system A-2: dichloromethane Fig.14C
Detection Vanillin-sulphuric acid-reag. (VS No. 38, p. 304) VlS. Fig. 13A, B; 14A
Phosphomolybdic acid-reag. (PMA No. 27, p. 303) vis. Fig. 14B, C
Chromatogram
13A Lavandulae aeth. After treatment with VS-reagent, chromatograms of the various
lavender oils (tracks 1-5) show intense blue zones of linalyl acetate (cf. Tl; Rf
ca. 0.7) and linalool (cf. Tl; Rf ca. 0.35), the striking, red-violet zone of epoxydihydro-
caryophyllene (Rf ca. 0.5, directly above the blue zone of cineole), and weak zones
of terpene alcohols (e.g. geraniol, borneol and nerol) between the start and Rf ca.
0.3.
1 Lavandin oil (commercial) contains all the chief terpenes in approximately equal concentrations,
with a slight predominance of linalool.
2 Freneh oil (Barreme) (commercial) is very similar to lavandin oil, with high concentrations
of linalool and linalyl acetate.
3 Freneh oil (Mont Blane) (commercial) contains about the same concentrations of linalool
and linalyl acetate as oill and 2, but only traces of cineole and epoxydihydrocaryophyllene.
4 Spike oil (commercial) contains high concentrations of cineole and linalool, and is free of
linalyl acetate.
5 The steam distillate of official lavender jlowers is similar to lavandin oil with respect to the
concentrations of the terpene alcohols, linalool, geraniol and borneol, but with a somewhat
lower concentration of linalyl acetate. Only traces of cineole are present.
6 Petit grain oil contains only linalyl acetate (up to 90% of the essential oil) and a low concentra-
tion of a terpene alcohol at Rf ca. 0.2. This oil is regarded as an adulterant.
13B Remarks: The blue zones produced with VS-reagent fade or change to green-blue
or brown with time. Borneol is then seen as a prominent brown zone.
14A, B 7 Cinae jlos. After treatment with VS-reagent, the main zones of cineole (cf. T2;
Rf ca. 0.45) and et.-santonin (cf. T3; Rf ca. 0.1) are stained blue and grey (vis.),
respectively. With PMA-reagent, they appear as dark blue, main zones.
14C Dichloromethane (solvent system A-2) gives a better separation of IX-santonin (Rf
ca. 0.4).
34
A B
-FRONT
Rf
Fig.13
Tl 2 3 4 5 6 T2 2 3
NT
-START
Fig.14
7 T2 T3 7 Tl T3 T2 7 T3
35
Menthae Aetherolea
Tracks 1 = Menthae piperitae aeth. (steam distillate) Tests Tl = menthol
2 = Menthae arvensis aeth. (commercial oil) T2 = menthone/isomenthone
3= Menthae crispae aeth. (steam distillate) T3 = menthyl acetate
4 = Carvi aeth. (commercialoil) T4 = menthofuran
5 = Menthae aeth. (commercial oil, batch I)
6 = Menthae aeth. (commercial oil, batch 11)
Solvent A-l : toluene-ethyl acetate (93: 7) Fig. lSA, B, C; 16C
system A -4: dichloromethane Fig. 16A, B
Detection Vanillin-sulphuric acid-reag. (VS No. 38, p. 304) VlS. Fig. lSA, C; 16C
Anisaldehyde-sulphuric acid-reag. (AS No. 2, p. 299) vis. Fig. 16A
Phosphomolybdic acid-reag. (PMA No. 27, p. 303) VlS. Fig. lSB; 16B

Chromatogram
15 A 1 Menthae piperitae aeth. After treatment with VS-reagent, the TLC of official pepper-
mint oil is characterized by the following terpenes:
No. Rf value Terpene Colour
I 0.3 menthol blue
11 0.35 piperitone orange
III 0.4 cineole bIue
IV 0.48 not identified blue
V 0.55 isomenthone blue-green
VI 0.70 menthone blue-green
VII 0.75 menthyl acetate blue
15B,16B After treatment with PMA-reagent, all zones are blue-black on a yellow-green back-
ground. Menthyl acetate, menthone, isomenthone and THC are more prominently
stained than after treatment with VS-reagent.
16A 2 Menthae arvensis aeth. With respect to the chief constituents, Menthae arv. aeth.
and Menthae pip. aeth show only quantitative differences. In corn mint oil, menthyl
acetate and menthol are present in fairly high concentrations. In contrast to freshly
distilled peppermint oil, however, corn mint oil contains no menthofuran (cf. T4).
The zones of THC and menthofuran have similar Rf values, and they frequently
overlap.
Remarks: Separation with dichloromethane (Ph. Eur. III) and detection with AS-reagent give
a similar chromatographic picture to that obtained with solvent system A-l and detection
with VS. The Rf values of menthyl acetate, menthone/isomenthone are shifted slightly to
lower values. Menthone and isomenthone appear yellow-brown in the visible. The advantage
of the AS-reagent is that it permits the detection of some terpenes under UV -365 nm: menthol
gives a red fluorescence, cineole green-brown, menthyl acetate blue-red, menthone yellow-
brown.
15C 3 Menthae crispae aeth. This oil is characterized by the main red-violet zone due to carvone
at Rf ca. 0.5 (cf. Carvi aeth. (4)), and the dark blue zones of dihydrocumin alcohol (Rf ca.
0.25) and dehydrocarveol acetate (Rf ca. 0.7).
16C 5,6 In addition to the blue zone of menthol (Rf ca. 0.3), the two commercial oils contain little
menthone and menthyl acetate (Rf 0.65-0.75). Piperitone is c1early seen as an orange zone,
while epoxydihydrocaryophyllene or pule gone (Rf region 0.5) give distinct brown-red zones.
This type of terpene composition indicates an adulteration with the oil of Pulegii herba.
36
A c
-FRONT
Rf
VII
VI
V
IV -0.5
-START
Fig.15
T1 T2 T3 Tl T2 T3 3 4
A
• C
-FRONT
Rf
-START
Fig.16
2 T3 T4 2 T3 T4 5 6
37
Salviae Aetherolea, Eucalypti Aetheroleum
Tracks 1, 2 = Salviae aeth. (Dalmatian oil 1/11)
3 = Salviae aeth. (Greek oil, DAB 8)
4 = Salviae aeth. (commercial oil of sage)
5 = Salviae aeth. (Spanish oil)
6, 7 = Salviae aeth. (Greek oil 1/11)
8= Eucalypti aeth.
Tests Tl = rx-, ß-thujone « - )-thujone/( + )-isothujone 35%: 65%)
T2=cineole
Solvent A-1 : toluene-ethyl acetate (93: 7)
system
Detection Vanillin-sulphuric acid-reag. (VS No. 38, p. 304) VIS. Fig. 17 A, C; 18C
Phosphomolybdic acid-reag. (PMA No. 27, p. 303) VIS. Fig. 17B, D; 18A, B

Chromatogram
17 A, C 1-7 Salviae aetherolea. After treatment with VS-reagent, the TLC of sage oils show
a main blue (vis.) zone at Rf ca. 0.5 due to cineole (cf. T2), 2-3 blue to blue-violet
zones of terpene alcohols in the Rf range 0.2-0.35 (borneol Rf ca. 0.2), and a red-
violet zone of epoxydihydrocaryophyllene directly above cineoie. This is followed
by the thujone-isothujone mixt ure (= a, cf. Tl) and immediately above that hornyl
acetate (= b) at Rf 0.6-0.7. The TBC form a strong violet zone at the solvent
front.
17 B, D; 18 A After treatment with PMA reagent the thujone mixture is seen as a strong blue-black
zone. All other terpenoids give a uniform blue-black colouration.
1-7 Differentiation ofvarious sage oils
The oils can be classified according to their contents of thujone, cineole and bornyl acetate.
1,2 Dalmatian sage oil is characterized, after treatment with PMA-reagent, by a prominent thujone
zone and a relatively weak zone of cineole, and two terpene alcohols in the Rf range 0.2-0.3.
3 Greek oils show cineole as the main zone, together with a little epoxydihydrocaryophyllene,
bornyl acetate (= b) and three terpene alcohols in the Rf range 0.2-0.4. After treatment with
PMA-reagent, thujone ( = a) can only be detected in oil 6 and not in oil 7.
5 Spanish oil can be differentiated from Greek oil by its low content of cineole, and from
Dalmatian oil by the absence of thujone. The zone of bornyl acetate (= b) and four zones
in the Rf range of the terpene alcohols are prominent.
4 Many commercial sage oils or drugs cannot be unequivocally assigned to one species of sage.
Thujone and cineole are present in approximately equal concentrations.
18B, C 8 Eucalypti aeth. is characterized by one main zone due to cineole. In addition there
are only two weak blue zones in the region of the terpene alcohols (Rf ca. 0.2-0.3),
two more weak blue zones in the ester region (Rf 0.6-0.7), and THC at the solvent
front. Thujone is absent.
38
A B c D
FRONT
Rf
-START
Fig.17
Tl 2 2 3 3 Tl T2
A B c
-FRONT
Rf
-0.5
-START
Fig.18
Tl T2 4 5 6 7 8 8
39
Thymi, Serpylli, Ajowani Aetherolea Fig.19
Tracks 1, 2 = Thymi aeth. (Thymi herba, steam distillate)
4,5, 7 = Thymi aeth. (official commercial oils from various sources)
3,8= Serpylli aeth. (Serpylli herba, steam distillate)
6 = Ajowani aeth. (Ajowani fructus, steam distillate)
Tests Tl = carvacrol
T2=thymol
Melissae Aetherolea and substitutes Fig.20

Tracks 9,10,11 = Melissae aeth. (Melissae folium, steam distillate)
12 = Citronellae aeth.
Tests T3 = lemon grass oil (for citral reference)
T 4 = citronellal
Solvent A-l : toluene-ethyl acetate (93: 7) Fig. 19A, B; 20B

system A-3: chloroform (DAB 8) Fig.20A
Detection Vanillin-sulphuric acid-reag. (VS No. 38, p. 304) VIS. Fig. 19A, B; 20B
Anisaldehyde-sulphuric acid-reag. (AS No. 2, p. 299) vis. Fig.20A

Chromatogram
19 Thymi aeth. After treatment with VS-reagent, TLC of oils from the Thymus species,
Th. vulgaris, Th. zygis (1, 2, 4, 7) and Th. serpyllum (3, 8) show a characteristic
main zone in the Rf range 0.5-0.55, due to a mixture of thymol and carvacrol
(cf. Tl/T2).
There are also lower concentrations of the terpene alcohols, borneol, geraniol and linalool
(grey zones, Rf 0.1-0.3), the terpene esters, bornyl and linalyl acetate (blue zones, Rf 0.55-0.8),
and THC at the solvent front.
3, 8 Thymi serpylli aeth. shows one or two additional ester zones at Rf ca. 0.6.
5 Thymi aeth. (rectified commercial oil) from an unknown species of thyme shows additional
zones in the Rf ranges 0.3-0.4 and 0.6-0.95, which give the characteristic red reaction of
thymol derivatives.
6 Ajowani aeth. The oil from Ajowani fructus contains practically only thymol (cf. T2), with
small quantities of terpene alcohols.
Remarks: The thymol-carvacrol mixture is efficiently separated by two-dimensional TLC:
first dimension A-l, second dimension toluene-carbon tetrachloride-o-nitrotoluene (1 : 1: 1).
20A 9-11 Melissae aeth. Chromatograms of official melissa aeth., after treatment with VS-
or AS-reagent, show a characteristic main blue zone, due to citronellal (cf. T4).
Also present are the weak grey to blue zones of citral (cf. T3) and the terpene
alcohols, geraniol, linalool and citronellol in the Rf range 0.2-0.4.
12 Melissa oil substitutes:
Ceylon or Java lemon grass oil (12) (Citronellae aeth.) resembles official Melissa oil (10)
in its high content of citronellal, and its chromatographic picture is also largely similar.
20ß Rf values in A-3 (DAß 8) are somewhat lower than those in A-l for the same
substances.
40
A
-FRONT
Rf
-0.5
-START
Fig.19
Tl 2 3 4 5 T2 T2 6 7 8
A 8
-FRONT
Rf
-0.5
-START
Fig.20
T3 9 10 11 12 T4 12 10 T3
DAS8
41
Curcumae Aetherolea
Tracks 1 = Curcumae aeth. (e. longae rhizoma, steam distillate)
2-4 = Curcumae aeth. (e. xanthorrhizae rhizoma, steam distillate)
5 = Curcumae longae rhizoma (MeOH-extract 1 g/5 ml/5 min, 60° C, p. 6)
6=Curcumae xanthorrhizae rhizoma (MeOH-extract 1 g/5 ml/5 min, 60° C)
Tests Tl = thymol
T2 = curcumin
T3 = fluorescein
Solvent A-1 : toluene-ethyl acetate (93: 7) Fig. 21 A, 22A
system A-8: chloroform-ethanol-glacial acetic acid (94: 5: 1) Fig. 21 B, 22B
Detection Vanillin-sulphuric acid-reag. (VS No. 38, p. 304) VlS. Fig.21A
Fast blue salt reagent + NH 3 vapour (FBS No. 12, p. 301) vis. Fig.22A
Without chemical treatment UV-365 nm Fig. 21B, 22B
Chromatogram
21 A 1-4 Curcumae aeth. VS-reagent gives about 8 red-violet zones in the Rf range 0.3 to
the solvent front. The zones at Rf ca. 0.8 and ca. 0.95 are especially concentrated.
Xanthorrhizol and alicyc1ic or aromatic (ar) turmerone show higher Rf values than
the standard thymol, and sesquiterpene hydrocarhons (e.g. zingiberene) are located
below the solvent front.
22A After treatment with FBS re agent, xanthorrhizol, a phenolic sesquiterpene, in particu-
lar gives an intense red.
Xanthorrhizol is a characteristic constituent of C. xanthorrhiza. Traces are also visible in
C. longa (1), where, according to the literature, it should be absent. Commercial drugs are
sometimes mixtures of both types of turmeric rhizome.
21 B; 22B These two drugs are weil distinguished on the basis of their different cinnamoyl
compounds (methanol extract). The pigment zones appear yellow in the vis., and
form yellow-white fluorescent zones in UV-365 nm.
6 Curcuma xanthorrhiza. The main compound is curcumin (cf. T2) at Rf ca. 0.6. De-
methoxycurcumin is present, in lower concentration, direct1y beneath (Rf ca. 0.5)
the zone of curcumin.
5 Curcuma longa also contains hisdemethoxycurcumin in the Rf region above the stan-
dard fluorescein (cf. T3).
Remarks: The colours can be intensified with the boron-oxalic acid reagent (DAB 8) (rubrocur-
cumines).
42
A
Fig.21
2 3 4 Tl 5 6 T2
-FRONT
Rf
-0.5
-START
Fig.22
2 3 4 Tl 5 6 T3
43
Aurantii, Citri Aetherolea, Aurantii, Citri Pericarpium
Tracks 1 = Aurantii pericarpium (steam distillate)
2 = Aurantii pericarpium (pressed oil; bitter)
3 = Aurantii pericarpium (pressed oil; sweet)
4=Citri pericarpium (steam distillate)
5 = Citri aeth. (pressed oil, DAB 7)
6 = Citri aeth. (Messina oil)
7 = Aurantii flos aeth. (Neroli oil)
8 = Citri var. bergamiae aeth. (bergamot oil)
9 = Citri var. bergamiae aeth. (petit grain oil)
lO=Citri peric. (see p. 163) (MeOH-extract)
11 = Aurantii peric. (flavonoids, see p. 188)
Test Tl = ci tral
Solvent A-1 : toluene-ethyl acetate (93: 7) Fig. 23A, B; 24A
system F-7: ethyl acetate-formic acid-water (67:7:26; upper phase) Fig.24B
Detection Vanillin-sulphuric acid-reag. (VS No. 38, p. 304) vis. Fig. 23A, B
Without chemical treatment UV-365 nm Fig.24A
Natural products-polyethylene glycol reagent
(NPjPEG No. 28, p. 303) UV-365 nm Fig.24B

Chromatogram
Aurantii and eitri pericarpium oils
23A 1,4 Steam distillation oils. After treatment with VS-reagent, the chromatograms ofboth
oils show at least 10 grey to red-violet zones (terpenes) in the Rf range 0-0.75,
and a THC zone at the solvent front.
Aurantii aeth. (1) shows about 6 main zones in the Rf range 0.15-0.4, while lemon
oil (4) shows four zones at RfO.15, 0.25, 0.4 and 0.6.
Distilled oils of C. aurantium are considered to be inferior.
2,3,5,6 Pressed oils. The commercial oils obtained from Aurantii pericarpium (2, 3) and
Citri peric. (5) by pressing procedures always have a characteristically high terpene
content. Citrat (cf. Tl) is prominent in oil5.
24A 5,6 Additional coumarin compounds, e.g. bergamottin (a), geranyl methoxycoumarin (b),
citropten (c) and a psoralene derivative (d) are characteristic components of lemon
oils.
In C. aurantium oils (1-4), the weak blue zones, seen in the Rf region of geranyl
methoxycoumarin (b), at the start and at the solvent front, originate partly from
methyl anthranilate and from blue-fluorescing flavonoids, such as sinensetin.
23 B 7 Neroli oil. The oil can be obtained from fresh orange blossoms by distillation, extrac-
tion, or the process of enfleurage. It contains a high amount of linalyl acetate
(Rf ca. 0.6), linalool (Rf ca. 0.25), and other a1cohols (terpineol, d-nerolidol, geraniol)
that migrate directly below linalool.
8, 9 Petit grain oil and hergamot oil (adulterants of neroli oil). Bergamot oil contains
lower concentrations of linalyl acetate and linalool than does neroli oil. Linalool
is complete1y absent from petit grain oil.
24 B 10, 11 MeO H-extracts of eitri and Aurantii pericarpium
The two drugs can also be differentiated on the basis of the flavonoid glycosides,
rutin, eriocitrin, naringin and neohesperidin (see section on Flavonoid drugs p. 188,
Figs. 17 and 18).
44
A
• -FRONT
Rf
- START
Fig.23
2 3 4 5 6 Tl 7 8 9
Fig.24
2 3 4 5 6 Tl 10 11
45
Pini Aetherolea, Myrrha
Tracks 1-4 = Terebinthinae aeth. (commercial)
5-9= Pi ni aeth. (commercial)
5 = "Scots pine oil "
6, 7 =" Spruce oil"
8 = "Silver fir oil"
9 = "Siberian spruce oil"
10= Myrrha
Tests Tl = terpineol
T2=cineole
T3 = bornyl acetate
Solvent A-1 : toluene-ethyl acetate (93: 7)
system
Detection Phosphomolybdic acid-reag. (PMA No. 27, p. 303) vis. Fig. 25 A
Vanillin-sulphuric acid-reag. (VS No. 38, p. 304) vis. Fig. 25B, C; 26B
Anisaldehyde-sulphuric acid-reag. (AS No. 2, p. 299) VlS. Fig.26A
Vanillin-hydrochloric acid-reag. (VHA No. 37, p. 304) vis. Fig.26C
Chromatogram
25 A, B 1-4 Terebinthinae aeth. Rectified turpentine oil (1) is characterized by a high content
of THC (a-, ß-pinene, a-, ß-phellandrene, limonene). With PMA- or VS-reagent,
these appear as blue or blue-violet zones, respectively, at the solvent front. Terpene
a1cohols and other additional zones are present only in low concentrations between
the standards of terpineol and cineole (cf. T1 and T2).
25 C In stored oils (2-4) the THC content decreases due to autoxidation. The chromato-
gram then shows increasingly blue to red-violet zones in the Rf range 0.1-0.5 (e.g.
pinene oxides, pinene hydrate, verbenol).
26A 5-9 Pine oils. This term refers to the essential oils from species of Pinus, Abies and
Picea; the source plants are rarely stated. After treatment with AS-reagent, the
chief feature of chromatograms of "Scots pine oil" (5) and "Silver fir oil" (8)
is a group of zones between the start and Rf ca. 0.45, with a low content of THC
in the area of the solvent front. In "Spruce oil" (6, 7), the main zones are due
to THC, with only weak zones on the rest of the chromatogram. Oils 5-8 show
a marked red zone at Rf ca. 0.4. "Siberian spruce oil" (9) is characterized by high
contents of bornyl acetate (cf. T3) and terpineol (cf. T1), and a low content of
THC.
26B, C 10 Myrrha. The chromatogram ofthe ethanolic resin fraction is characterized by intense
blue, blue-violet and red zones distributed over the wh oIe area between the start
and the solvent front (VS- or VSL-reagent). Red zones in the Rf range 0.4 and
0.6-0.75 (commiphoric acid esters?) are typical. Sesquiterpene hydrocarbons of the
essential oil fraction are found in the region of the solvent front.
46
B c
-FRONT
Rf
-0.5
-START
Fig.25
Tl T2 Tl T2 2 3 4
A B c
-FRONT
Rf
-0.5
-START
Fig.26
Tl T3 5 6 7 8 9 10 10
47
Resins and Balsams
Tracks 1 = benzoin (Sumatra benzoin)
2 = benzoin (Siam benzoin)
3 = Balsamum tolutanum
4 = Balsamum peruvianum
Tests Tl = benzoic acid
T2=eugenol
Solvent A-l : toluene-ethyl acetate (93: 7)
system
Detection Without chemical treatment UV-254 nm Fig. 27 A, 28A
Anisaldehyde-sulphuric acid-reag. (AS No. 2, p. 299) VlS. Fig. 27B, 28B
Phosphomolybdic acid-reag. (PMA No. 27, p. 303) VlS. Fig.28C
Description of benzoins and balsams, p. 16-17. F ormulae p. 20.
Chromatogram
27 A 1,2 Benzoins. Visualization in UV-254 nm shows pronounced quenching of fluorescence
at Rf ca. 0-0.15 (benzoic or cinnamic acid, cf. Tl), Rf ca. 0.25-0.4 (coniferyl benzoate)
and RfO.7-0.75 (main zone with a small accompanying zone: cinnamoyl cinnamate,
propyl cinnamate, cinnamoyl benzoate)
27 B The fluorescence-quenching zones are stained blue to violet in the vis. by treatment
with AS-reagent.
1 Sumatl'a benzoin is characterized by about 4 equally intense zones in UV-254 nm.
These are benzoic or cinnamic acid in the lower Rf range, coniferyl benzoate at
an intermediate Rf, and a prominent ester zone (cinnamoyl cinnamate, propyl cinna-
mate) at Rf ca. 0.7.
2 Siam benzoin pro duces zones primarily at lower and intermediate Rf values. The
main zone, due to coniferyl benzoate, is at Rf ca. 0.4. Only a very weak zone
is seen at Rf ca. 0.7.
28 3,4 Bahams
3 Tolu balsam. Direct evaluation in UV-254 nm shows the fluorescence-quenching
zones of benzoyl benzoate and benzoyl cinnamate (" cinnamein " mixture). The ratio
of the two esters is ca. 1: 2. Above the start are seen the zones of benzoic acid
(cf. Tl, Fig. 27 A) and cinnamic acid. In the intermediate Rf range are weak zones,
due in part to eugenol (cf. T2) and vanillin. After treatment with PMA-reagent,
the fluorescence-quenching zones give a prominent blue in the vis. In addition,
this reagent shows a zone ofTHC at the solvent front (cf. also AS-reagent, Fig. 28 B).
4 Peru balsam contains a much higher concentration of the "cinnamein" mixture,
with different relative proportions of the esters (ratio 2.5: 1).
Nerolidol, which is especially characteristic of Peru balsam, is made visible at Rf
ca. 0.3 by treatment with PMA- or AS-reagent.
48
-FRONT
RI
-0.5
-START
Fig.27
Tl T2 2 T2 2
• FRONT
RI
-0.5
-START
Fig.28
3 4 T2 3 4 3 4
49
Alkaloid Drugs
Most plant alkaloids are derivatives oftertiary amines, while others contain primary,
secondary or quaternary nitrogen. The basicity of individual alkaloids varies greatly,
depending on which of the four types is represented. The pKB-values (dissociation
constants) lie in the range pR 10--12 for very weak bases (e.g. purines), pR 7-10
for weak bases (e.g. Cinchona alkaloids) and pR 3-7 for medium strength bases
(e.g. opium alkaloids).
I. Preparation of Drug Extracts for TLC

1. Alkaloid drugs with high and medium alkaloid contents (~ 1% )
Powdered drug (1 g) is mixed thoroughly with 1 ml of 10% ammonia soln. or 10%
Na 2 C0 3 soln., then extracted by shaking for about 5 min with 5 ml methanol at
60° C (water bath). The filtrate is cooled and concentrated so that 100111 (the maxi-
mal quantity that should be applied to the TLC plate) contains 50--100 Ilg alkaloids
(see II, 1.)
2. Alkaloid drugs with low alkaloid contents ( < 1%)

a) Enrichment using an aluminium oxide column
Powdered drug (2 g) is ground in a mortar for about 1 min with 2 ml of 10%
ammonia soln., then mixed with 7 g basic aluminium oxide (activity stage I). The
whole mixture is packed loosely into a glass column (1.5 cm diarn., 20 cm long).
Alkaloid bases are eluted with about 10 ml CRCI 3 . The first 5 ml of eluate are
collected, evaporated to 1 ml, then used for chromatography.
This method is suitable for, e.g. the Solanaceae drugs, e.g. Belladonnae or Scopo-
liae radix and Stramonii semen. Seed drugs must first be defatted by extraction
with light petroleum.
Leaf extracts contain chlorophyll, which can interfere with the TLC separation.
In such cases the official pharmacopoeia method is recommended (see b).
b) Enrichment by extraction according to DAß 8
Powdered drug (1 or 2 g) is shaken for 5 min with 10.0 ml of 0.05 M sulphuric
acid, then filtered. To the filtrate is added 1.0 ml conc. ammonia soln. The mixture
is diluted to 10 ml with water, and extracted by shaking with 10 ml peroxide-free
ether. The ether phase is dried over anhydrous sodium sulphate, filtered, evaporated
to dryness on the water bath (fume cup board), and the residue dissolved in 0.25 ml
methanol.
This is the preferred method for Belladonnae and Stramonii folium (1 gof each)
and for Ryoscyami folium (2 15).
11. Sampie Quantities for TLC

1. Drug extracts
The sampIe applied to the TLC plate should contain 50--1OOllg total alkaloids.
The size of the sampIe can be ca1culated from the average alkaloid content of the
drug.
51
Example: powdered drug (1 g) with a total alkaloid content of 0.3%, extracted
by method 1., will yield 3 mg in 5 ml methanolic soln., containing ca. 60 Ilg total
alkaloids per 100 111.
2. Reference substances from proprietary pharmaceuticals (see table)

Alkaloids can be obtained from pharmaceutical products by extraction with metha-
nol. Since pharmaceutical products contain a wide range of different alkaloid concen-
trations, the method used for the preparation of each test solution depends on
the alkaloid content.
The sample applied to the TLC plate should contain 50-100 Ilg alkaloid.
Alkaloid content 10-250 mg per tablet or dragee: One powdered tablet or dragee
is mixed with 1 ml methanol per 10 mg alkaloid and shaken for about 5 min at
60° C. After filtration or centrifugation, the extract is applied directly; 10 111 corre-
spond to 100 Ilg alkaloid.
Alkaloid content 0.075-1.0 mg per tablet or dragee: Ten powdered tablets or dragees
are mixed with 5 ml methanol, shaken for about 5 min at 60° C, and the c1ear
filtrate evaporated to dryness. The residue is dissolved in 1 ml methanol, and, if
necessary, the solution c1eared by centrifugation. This solution is applied to the
TLC plate; 10111 contain 100llg alkaloid (1.0 mgjtablet), or 100111 contain 751lg
alkaloid (0.075 mgjtablet).
Reference substances from pharmaceutical preparations for the TLC identification

of alkaloid drugs
Drug and major Pharmaceutical a Drug and chief Pharmaceutical a

alkaloids alkaloids
Aconiti tuber Opium

Aconitine Aconitysat (drops) Codeine Codein phos. Compr.
Chinae cortex Morphine
Quinine Chinin Compr. Noscapine (Narcotine) Noflu
Quinidine Chinidin Compr. Papaverine Papaverin Tab!.
Cinchonine Sedovegan Rauwolfia spp.
Cinchonidine Sedovegan Ajmaline Gilurytmal
Caffeine drugs Raubasine Triraupin
Caffeine Coffein Compr. Rescinnamine Triraupin
Theobromine Theo-Miroton Raupine Rauwopur
Theophylline Theo-Miroton Reserpine Sedaraupin
Colchici semen Secale cornutum
Co1chicine Co1chicum Dispert Ergobasine Ergotren
Ergocristine Ergotren
Ipecacuanhae radix Ergotamine Gynergen
Cephaeline
Emetine Dicton Solanaceae drugs
Atropine Atropin sulf. Compr.
Lobeliae herba Scopolamine Boro-Scopol (drops)
Lobeline Lobelin Amp.
Strychni semen
Strychnine Dysurgal
Remarks: Some pharmaceutical preparations contain a mixture of different alkaloids, e.g.

Rauwopur dragees: 1 dragee contains 0.1 mg reserpine-HCI, 0.25 mg rescinnamine, 0.01 mg
raupine, 0.19 mg ajmaline, 0.6 mg yohimbine. Thus, in reference solutions prepared for the
detection of raupine, the concentration of yohimbine will be too high.
a Pharmaceutical preparations on the German drug market. Instead of these, other prepara-
tions, available under other names, with the same constituents can be used as references.
52
3. Reference substances
a) These are usually prepared in 1% a1coholic soln., and 10111 are applied for TLC,
e.g. atropine, brucine, strychnine, morphine.
b) Rauwolfia alkaloids. Reserpine, rescinnamine, rauwolscine, ajmaline and serpentine
are each prepared in 0.5% a1colholic soln., and 10111 are applied for TLC.
c) Colchicine is prepared as a 0.5% soln. in 70% ethanol, and 10111 are applied for
TLC (DAC).
4. Test mixtures from the pharmacopoeias

a) Cinchona alkaloid test mixture, Ph. Eur., for the TLC identification of Chinae cortex.
A mixture of 17.5 mg quinine, 0.5 mg quinidine, 10 mg cinchonine and 10 mg cin-
chonidine is dissolved in 5 ml ethanol, and 5 111 of this soln. are applied for TLC.
b) Test mixture for Ipecacuanhae radix, Ph. Eur.
4.6 mg emetine and 5.7 mg cephaeline are dissolved in 20 ml methanol, and 5 111
of this soln. are applied for TLC.
c) Test mixture for Solanaceae drugs, Ph. Eur.
24 mg atropine sulphate dissolved in 9 ml methanol. 7.5 mg scopolamine hydrobro-
mide dissolved in 10 ml methanol.
For Belladonnaefolium: 1 ml scopolamine soln. is added to 9 ml atropine sulphate
soln. 20 111 are used for TLC.
For Hyoscyami folium: 3 ml atropine sulphate soln. mixed with 0.4 ml scopolamine
hydrobromide soln., and diluted to 10 ml with methanol. 20111 are used for TLC.
For Stramonii folium: 5 ml atropine sulphate soln. mixed with 3 ml scopolamine
hydro bromide soln., and diluted to 10 ml with methanol. 20111 are used for TLC.
5. Adsorbent
Silica gel 60F 2S4 pre-coated TLC plates (Merck, Darmstadt).
Most of the alkaloids can be separated on silicic acid.
Aluminium oxide (see Fig. 16B, p. 80) is more suitable for the separation ofberberine,
columbamine and jateorhizin.
53
Solvent system Drug; Alkaloids
AL-l Toluene-ethyl acetate-diethylamine Screening system, suitable for the

(70: 20: 10) major alkaloids of most drugs.
AL-2 Chloroform-diethylamine Chinae cortex; Cinchona alkaloids
(90: 10) (Ph. Eur. III)
AL-3 Toluene-acetone-ethanol-conc. ammonia Opium; opium alkaloids (DAB 8)
(40 :40: 6: 2)
AL-4 Acetone-water-conc. ammonia Solanaceae drugs (atropine,
(90: 7: 3) hyoscyamine) according to Ph. Eur. I
AN-l Ethyl acetate-methanol-water Screening system, suitable for Rau-
(100: 13.5: 10) wolfia alkaloids, xanthine derivatives
(Caffeine drugs), Colchicum alkaloids.
AL-5 Toluene-chloroform-ethanol Secale alkaloids
(28.5: 57: 14.5)
AL-6 n-Heptane-ethylmethyl ketone-methanol Rauwolfia alkaloids (DAB 8)
(58: 34: 8)
AL-7 Chloroform-methanol Isoquinoline alkaloids
(85: 15) Ipecacuanhae radix (Ph. Eur.)
AL-8 Toluene-methanol Colchici semen (DAC). Solvent run
(86: 14) twice over 15 cm.
AL-9 n-Propanol-formic acid-water Berberidis cortex, Hydrastis rhizoma,
(90: 1: 9) Colombo radix and Chelidonii herba.
AL-l0 Cyc1ohexane-chloroform-glacial Berberine-, and protoberberine-type
acetic acid (45: 45: 10) alkaloids
111. Detection
UV-254 nm
A lot of alkaloids show a pronounced quenching of fluorescence in UV -254 nm
(e.g. strychnine, brucine, purines).
UV-365 nm
Some alkaloids (Fig. 2, p. 67) fluoresce blue or yellow in UV -365 nm.
2. Spray reagents
a) Dragendorffreagent (DRG No. 11 A-11 E, p. 300-301)
Brown or orange (vis.) zones appear immediatelyon spraying. The colours are
not stable.
Remarks: The alkaloid zones can be made more distinct by spraying first with
Dragendorff re agent and then with 5% sodium nitrite soln. or 5% ethanolic sul-
phuric acid.
54
b) Iodoplatinate reagent (IP No. 19, p. 302)
Directly after spraying, alkaloid zones appear brown, blue or whitish (vis.) on the
blue-grey background of the TLC plate.
IV. List of Alkaloid Drugs

Chromatograms (Figs. 1-26) are reproduced on pp. 66--91.
Fig. DrugjPlant source Total alkaloid content (TA)

F amily jPharrnacopoeia Major alkaloids
A. Indole alkaloids
4-6 Rauvolfiae Radix TA 0.6-1.5% (R. serpentina)
Rauwolfia root 1.3-3% (R. vomitoria)
Rauvolfia serpentina (L.) BENTHAM DAB 8 specifies not less than 1 %, ca1c. as
ex KURZ reserpine; about 50 indole alkaloids, mostly
yohimbane derivatives.
Rauvolfia vomitoria AFZEL
Main alkaloids: reserpine, rescinnamine (tert.
Apocynaceae indole alkaloids); rauwolscine (R. vomitoria
DAB 8 (R. serp.) only); ajmaline (tert. indole alk.); serpentine
USPXX (quaternary base).
Minor alkaloids: raubasine (corynantheine
type), raupine (sarpagine type).
Y ohimbe Cortex TA 2.3-5.9% (not less than 1.5% ca1c. as yo-
Y ohimbehe bark himbine)
Pausinystalia yohimba PIERRE Yohimbine is the main alkaloid. (1.- and ß-yo-
Rubiaceae himbane, pseudoyohimbine and coryantheine
are the most important minor alkaloids.
DAC (0.5-1 % ca1c. as yohimbine)
Quebracho Cortex TA 0.3-1.5%
Aspidosperma bark Main alkaloids: yohimbine, pseudoyohim-
Aspidosperma quebracho blanco bine, aspidospermine, aspidosperrnatine, que-
SCHLECHT brachamine, hypoquebrachamine, quebra-
Apocynaceae chocidine.
7,8 Secale Cornutum TA 0.2-1%

Ergot Lysergic acid alkaloids
Claviceps purpurea (FRIES.) Tu- Amide alkaloids (ergometrine = ergobasine,
LASNE "water soluble") and peptide alkaloids (ergo-
Clavicipitaceae (Ascomycetes) tamine) or alkaloids of the ergotoxin group
ÖAB ("water insoluble ").
55
Fig. Drug/Plant source Total alkaloid content (TA)
Family /Pharmacopoeia Major alkaloids
Strychni Semen TA 2-3%

Nux vomica seeds ca. 1% strychnine and 1.5% brucine. Minor
Strychnos nux vomica L. alkaloids 0(- and ß-colubrine.
Loganiaceae
2. AB-DDR, Helv. VI, ÖAB
Ignatii Semen TA 2.5-3%
Ignatius beans Strychnine (45-50% oftotal) and brucine
Strychnos ignatii BERG
Loganiaceae
B. Quinoline and isoquinoline alkaloids

Alkaloids of the morphinane type
(phenanthrene type)
9, 10 Chinae Cortex TA 4-12%
Cinchonae cortex (Ph. Eur. specifies not less than 6.5%; 2. AB-
Cinchona bark DDR specifies 7-10% quinine/cinchonine)
Cinchona pubescens VAHL (syn. Main alkaloids are the diastereomeric pairs
C. succirubra PAVON) quininel quinidine and cinchoninel cinchonidine.
Ph. Eur. III, ÖAB, 2. AB-DDR, Official Cinchona bark contains ca. 20 known
Helv. VI alkaloids (== 100%), consisting of ca. 25%
Cinchona ledgeriana MOENS. quinine, ca. 45% cinchonine and ca. 5% quin-
YeIIow Cinchona bark idine. YeIIow Cinchona bark contains up to
Rubiaceae 90% quinine.
11, 12 Ipecacuanhae Radix TA 1.8-6%

Ipecacuanha root (Ph. Eur. specifies not Iess than 2%; 2. AB-
Cephaelis ipecacuanha DDR not less than 2%, with emetine as 60%
(BROT.) RICH. (Rio and Matto- of total)
Grosso drugs) Main alkaloids are emetine and cephaeline,
Cephaelis acuminata KARSTEN and the corresponding dehydro-compounds,
(Cartagena, Panama and Costa- O-methylpsychotrine and psychotrine.
Rica drugs) Rio drug contains 3: 1 - 1: 1 ratio of emetine:
Rubiaceae cephaeline.
Ph. Eur. 1,2. AB-DDR, USP XX Panama drug contains mostly 1 : 1 ratio eme-
tine: cephaeline.
Drugs contain about 0.05% minor alkaloids.
13,14 Opium TA (raw opium) 20-29% with ca. 30 alka-

Opium loids.
Papaver somniferum L. subsp. Main alkaloids of the phenanthrene type:
somniferum and varieties morphine (3-23%), codeine (0.3-3%), thebaine
Papaveraceae (0.1-3%).
DAB 8,2. AB-DDR, Helv. VI, Benzylisoquinoline type: papaverine (0.1-2%),
ÖAB, USPXX noscapine (narcotine) (2-12%), narceine
(0.1-2%)
Opium DAB 8 (dried latex containing at least
9.5% morphine)
Opium 2. AB-DDR (at least 12% morphine)
Opium titratum DAB 8 (standardized at
9.8% morphine content)
56
Family jPharmacopoeia Major alkaloids
Opii extractum DAß 8 (dried extract from

raw opium; morphine content 19.6--20.4%)
Opii tinctura DAß 8 (from raw opium; mor-
phine content 0.95-1.05%)
C. Miscellaneous classes or alkaloids

15-18 Chelidonii Herba TA 0.35-0.9% (DAß 8 specifies not less than
Greater celandine 0.6%; 2. Aß-DDR 0.4-0.8%). About 20 al-
Chelidonium majus L. kaloids are present.
Papaveraceae ßenzophenanthridine type: chelidonine, che-
DAß 8, 2. AB-DDR lerythrine and sanguinarine.
Protoberberine type: berberine.
Protopine type: protopine, OC-, ß-allocrypto-
pine.
ßerberidis Radicis Cortex TA 0.95-3%
ßarberry bark Main alkaloids berberine, jateorhizine and
ßerberis vulgaris L. palmatine (protoberberine type).
ßerberidaceae
Hydrastis Rhizoma TA 2.5--6%, comprising ca. 3 % berberine, ca.
Golden seal rhizoma 1% tetrahydroberberine (canadine) and ca.
Hydrastis canadensis L. 1.5-4% hydrastine (a phthalidisoquinoline al-
Ranunculaceae kaloid).
Colombo Radix TA 1-2%
Colombo root with the protoberberine alkaloids, palmatine,
Jateorhiza palmata (LAM.) MIERS jateorhizin and columbamine.
Menispermaceae
Colchici Semen TA 0.5-1%
Co1chicum seeds ca. 20 alkaloids. Main alkaloid colchicine; mi-
Co1chicum autumnale L. nor alkaloid demeco1cine.
Liliaceae
DAC
19,20,22 Aconiti Tuber TA 0.3-1.5% (Helv. VI specifies not less than

Aconite root 0.6% ether-soluble alkaloids, ca1culated as
Aconitum napellus L. aconitine)
Ranunculaceae Ester alkaloids (alkamine esters), the main
Helv. VI (Aconitinum: Helv. VI, one being aconitine. Hydrolytic cleavage
ÖAB) products, benzoylaconine and aconine, are
present.
Boldo Folium TA not less than 0.1% (Helv. VI)
ßoldo 1eaves Aporphine alkaloid, boldine.
Peumus boldus J.1. MOLINA
Monimiaceae
Helv. VI
Ephedrae Herba TA up to 3.3%
Ephedra (Ma-huang) ca. 75% L-ephedrine and 25%
Ephedra distachya L. and other ( + )-pseudoephedrine and nor-pseudoephe-
species drine. The pseudoephedrines are diastereo-
Ephedraceae (Ephedrine DAB 8) isomers of ephedrine.
57
F amily jPharmacopoeia Major alkaloids
Jaborandi Folium TA 0.5-7% (Helv. VI specifies not less than

Jaborandi leaves 0.5%). The imidazole alkaloids, pilocarpine
Pilocarpus jaborandi HOLMES (Per- and isopilocarpine, may represent 25-50% of
nambuco jaborandi) the total alkaloids.
Pilocarpus pennatifolius LEMAIRE
(Paraguay jaborandi) and other
species
Rutaceae
Helv. VI; (Pilocarpine-HCI,
DAB 8 and other pharmacopoeias)
Lobeliae Herba TA not less than 6.2% (2. AB-DDR)
Lobelia Main alkaloid lobeline (piperidine ring sys-
Lo belia infla ta L. tem).
Lobeliaceae Minor alkaloid isolobinine (dehydropiperi-
2. AB-DDR, ÖAB dine ring system)
Sabadillae Semen GA 1-5%
Sabadilla seeds Steroid alkaloids (" Veratrinum alkaloid mix-
Schoenocaulon officinale ASAGRA Y ture") with C-nor-C-homo-cholestane struc-
Liliaceae ture.
Veratri Rhizoma TA not less than 1 % (Helv. VI)

White hellebore rhizome Tetraesters of protoverine, mainly Protover-
Veratrum album subsp. album, and ine A and B, together with free alkaloids.
V. lobelianum BERHN.
Liliaceae
2. AB-DDR, Helv. VI
Sarothamni (Spartii) scoparii Herba TA 0.8--1.5%
Broom Main alkaloid sparteine (a tetracyclic quinoli-
Sarothamnus scoparius (L.) WIM- zidine alkaloid)
MER ex KOCH Minor alkaloid IX-isosparteine (gentisine)
Fabaceae Flavonoids (see p. 184, Fig. 14, chapter on fla-
vonoids)
Nicotinae Folium TA variable
Tobacco leaves L-Nicotine (0.05-10%), nornicotine, anaba-
Nicotiana tabacum L., N. rustica sine and nicotyrine.
L. and other varieties
Solanaceae
D. Purine alkaloids
21 CacaoSemen 0.2-0.5% caffeine
Cacao seeds 1-2% theobromine
Theobroma cacao L.
Sterculiaceae
Coffeae Semen 0.3--2.5% caffeine
Coffee seeds (traces of theophylline)
Coffeae arabica L. and other spe- Chlorogenic acid
cies
Rubiaceae
58
Fig. Drug/Plant source Total alkaloid content ( TA)
F amily/Pharmacopoeia Major alkaloids
ColaeSemen 0.6-3% caffeine

Colaseeds ca. 0.1 % theobromine
Cola nidita SCHOTT et ENDL.
Cola acuminata SCHOTT et ENDL.
Sterculiaceae
Helv. VI, ÖAB
Mate Folium 0.5-1.5% caffeine

Mate ca. 0.05% theophylline
Ilex paraguariensis St. HILAIRE ca. 0.2-0.45% theobromine
Aquifoliaceae Chlorogenic acid
Thea Folium 2.5-4.5% caffeine

Tea 0.02-0.05% theophylline
Camellia sinensis (L.) KUNTZE, and 0.05% theobromine
other varieties
Theaceae
E. Tropine alkaloids
23-26 Belladonnae Folium TA 0.2-0.5% (Ph. Eur. I specifies not less
Belladonna leaves than 0.3%)
USPXX Main alkaloids ( - )-hyoscyamine/atropine
Ph. Eur. I, ÖAB, and scopolamine in ratio ca. 3: 1.
2. AB-DDR, He1v. VI
Belladonnae Radix TA 0.3-0.8% (2. AB-DDR specifies not less

Belladonna root than 0.4%)
Atropa belladonna L. Main alkaloids ( - )-hyoscyamine and scopola-
Solanaceae mine.
2. AB-DDR, ÖAB Minor alkaloids apoatropine, belladonnine,
cuskhygrine, norhyoscyamine, noratropine
and meteloidine.
Scopoliae Radix TA 0.4-0.95%

Scopolia root Alkaloids similar to those of Belladonnae ra-
Scopolia carniolica JACQ. dix, differentiation on basis of coumarin pat-
Solanaceae tern (see p. 90, Fig. 26)
Hyoscyami Folium TA 0.04-0.17% (Ph. Eur. specifies not less

Henbane leaves than 0.05%)
Hyoscyamus niger L. (- )-Hyoscyamine/atropine and scopolamine
Ph. Eur. I, Helv. VI, 2. AB-DDR in ratio ca. 1.2: 1.
Hyoscyami mutici Folium TA not less than 0.8% (0.8-1.4%)

Hyoscyamus muticus L. Main alkaloids ( - )-hyoscyamine/atropine
Solanaceae and scopolamine.
Minor alkaloids apoatropine and belladon-
nine.
59
Fig. Drug/Plant source Total alkaloid content (TA)
F amily /Pharmacopoeia Major alkaloids
Stramonii Folium TA 0.1-0.6% (Ph. Eur. specifies not less than

Thornapple leaves 0.25%)
Datura stramonium L. Main alkaloids ( - )-hyoscyamine and scopola-
Solanaceae mine in ratio ca. 2: 1.
Ph. Eur. I, Helv. VI, 2. AB-DDR, Minor alkaloid atropamine.
ÖAB
AU Solanaceae leaf drugs also contain flavonoids, and to some extent coumarins and plant
acids (see Fig. 26, p. 90)
60
v. Fonnulae of Constituents of Alkaloid Drugs
(- )-Emetine: R = CH a _ _---=2'-'-H_~~ O-Methylpsychotrine

Cephaline: R= H - 2H ~ Psychotrine
o
~OH
HOOe JL~Jl
0 eOOH
OH
Morphine: R, = R2 = H Meconic acid Boldine
Codeine: R, = H; R2 = CH a
Thebaine: R, = R2 = CH a, with
additional double bonds
at 6/7 and 8/14
Papaverine Noscapine
(= Narcotine)
R R
( - ) Quinine: R = OCHa (+) Quinidine: R = OCHa

Cinchonidine: R = H Cinchonine: R= H
61
ß-Carboline
~
~ChinoliZidine
N~ '\ R,
: OR 2
6CH 3
Reserpine: R, = OCH 3 ; Raubasine:

R2 = 3,4,5-Trimethoxybenzoyl (= Ajmalicine)
Yohimbane: R, = R2 = H Rescinnamine: R, = OCH 3 ;
Yohimbine: R, = CH 3 00C; R2 = OH R2 = 3,4,5-Trimethoxycinnamoyl
OH
HO
Ajmaline Sarpagine: R = H Serpentine

Raupine: R = CH 3
AMIDE ALKALOIDS
ROC
Ergometrine (Ergobasine) R = NH-CH-CH 20H

I
CH 3
Strychnine: R = R = H
Brucine: R; = R: = OCH 3
cr-Colubrine: R, = H; R2 = OCH 3
ß-Colubrine: R, = OCH 3 ; R2 = H
PEPTIDE ALKALOIDS
1
R, i OH
H 1/0,/--1
:~ Proline
L YSERGYL RESIDUE -N···C I C N

1 1-1-----1----
-::/C--: N" /C~
o : /C'" 0
---------: H 'R2
I
Alkaloids R, Amino acids R2 Amino acids
1. Ergotamine group
Ergotamine CH 3 cr-Hyd roxy-
alanine
CH 2 -<Q) Phenylalanine
2. Ergotoxine group
Ergocristine CH(CH 3 )2 cr-Hyd roxy-
valine
CH 2 -<Q) Phenylalanine
62
o
\-0
Berberine: R,; R 2 = -CH 2 - Chelidonine Chelerythrine: R, = R 2 = CH 3

Palmatine: R, = R2 = CH 3 Sanguinarine: R,; R 2 = CH 2 -
Jateorh izi ne: R, = H; R2 = CH 3
L-Hyoscyamine D-Hyoscyam i ne
I I
Atropine (DL-Hyoscyamine)
6,7-Epoxidation !-H20
T
L-Scopolamine
6
I
C=O 2Mol
IQ\- b
~II
Apoatropine
CH 2
Apoatropine Belladonnine
T = Tropyl residue
Cuskhygrine
Tropine
(Tropane-3-IX-ol)
63
Colchicine: R= COCH 3
Acontine Demecolci ne: R=CH 3
~
HO-C-H
~
H-C-OH
I
OH
I
R-CH- H c.·
2
0N
2
45
I 6 " c
CH 2- 11
~
-Q
I enant.
H CHN~C-H H-C-NHCH 3 CH3 0
3 I I
CH 3 CH 3 (-) Lobeline: R= -©
L-Ephedrine D-Ephedrine Isolobinine: R = C2 H. 4,5=
L - - - - - -v
erythro
oR
CH3
I
R-C-H
I :: OH
N R H3C-~-OH 3
••••• 0'6': 7 •• OHO! -C-CH- CH2- CH 3
C-O . . I
: H: '0 11 CH
Nicotine: . R:: ~H3 11
o 6H 6 ' 0 3
Nornicotme. R-
l~etYI
.
Protoveratr!ne A"R-H
:
Protoveratnne B. R-
OH =
64
d50 H
Pilocarpine Sparteine
R, R2 Ra
Theobromine H CH 3 CH 3
Theophylline CH a CH 3 H
Caffeine CH 3 CH 3 CH 3
65
TLC Synopsis of the Most Important Alkaloids
Alkaloids I - reference compounds detected with Dragendorff reagent

Tracks 1 = colchicine 12 = scopolamine
2 = boldine 13 = strychnine
3 = morphine 14 = yohimbine
4 = pilocarpine 15 = physostigmine
5 = quinine 16 = nicotine
6 = brucine 17 = veratrine
7 = cephaeline 18 = emetine
8= Quinidine 19= papaverine
9= atropine 20= lobeline
10= co deine 21 = aconitine
11 = cinchonine 22 = noscapine (narcotine)
Solvent AL-1 : toluene-ethyl acetate-diethylamine (70: 20: 10)
system
Detection Dragendorff reagent (No. 11 C, p. 300) vis. Fig. 1 A
Dragendorff reagent with sodium nitrite (No. 11 F, p. 301) vis. Fig. 1B; 2A
Chromato- With Dragendorff reagent alkaloids give spontaneously an orange-brown, usually

gram stable, colour in the visible. With some alkaloids, e.g. boldine (2), morphine (3)
1A and nicotine (16), the colour fades rapidly.
1 B, 2A The colour can be intensified by spraying afterwards with somum nitrite reagent.
The zones then appear dark brown (e.g. morphine (3)) or violet-brown (e.g. atropine
(9)). The colours of pilocarpine (4) and nicotine (16) are unstable.
Alkaloids 11 - reference compounds that fluoresce in UV-365 nm

Tracks 23 = serpentine 29 = emetine
24=quinine 30=yohimbine
25 = cinchonine 31 = noscapine (narcotine)
26 = quinidine 32 = hydrastine
27 = cinchonidine 33 = berberine
28 = cephaeline 34 = sanguinarine
system
Detection: Sulphuric acid reagent (5%) (No. 34, p. 303) UV-365 nm Fig.2B
Chromato- Fluorescence of these alkaloids is predominantly blue, and is intensified by treatment

gram with 5% sulphuric acid.
2B In the case of the quinine alkaloids, the initial weak blue fluorescence of quinine
and quinidine becomes a radiant blue (this appears white in the illustration), and
cinchonine and cinchonidine show a deep violet fluorescence.
Berberine and sanguinarine are exceptional in showing a yellow fluorescence.
Remarks: The commercial alkaloid reference compounds (e.g. hydrastine) frequently show
zones of minor alkaloids. Some alkaloids form degradation products in solution or during
development of the TLC plate.
66
-FRONT
Rf
n
-0.5
START
Fig.l
Tl - 21 Tl3-22
A
FRONT
Rf
START
Fig.2
T12345679 T23-34
67
Alkaloids 111 "Rauwolfia alkaloids"
Quebracho and Yohimbe Cortex
Tracks 1 = Quebracho Cortex
2 = Y ohimbe Cortex
Tests Tl = serpentine
T2 = ajmaline
T3 = yohimbine
T4 = reserpine
T5 = rescinnamine
Solvent AL-l : toluene-ethyl acetate-diethylamine (70: 20: 10) Fig. 3A; 4A
system AL-6: n-heptane-ethylmethylketone-methanol (58: 34: 8) Fig. 3B; 4B
AN-1: ethyl acetate-methanol-water (100: 13.5: 10) Fig. 3C; 4C
Detection Without chemical treatment UV -365 nm Fig. 3B, C; 4A, B
Iodoplatinate re agent (IP No. 19, p. 302) UV-365 nm Fig.3A
Dragendorffreagent (No. IlF, p. 301) vis. Fig.4C
Chromato- Rauwolfia alkaloids (reference compounds). Alkaloids of the yohimbine and corynan-
gram theine type (" Rauwolfia alkaloids" Tl-5) give an intense blue fluorescence in UV-
3 365 nm. Ajmaline is characterized by strong fluorescence quenching in UV-254 nm,
and by only a weak blue fluorescence in UV-365 nm, which can, however, be intensi-
fied by treatment with iodoplatinate reagent (Fig. 3 A).
3A-C AL-I Of the three solvent systems, AL-I gives the best separation of ajmaline and serpen-
tine, which remain in the starting region in other systems.
AL-6 shows the separation of reserpine and rescinnamine.
AN-I shows the best separation of yohimbine and reserpine/rescinnamine mixtures.
Remarks: In drug extracts containing alkaloids of the yohimbine/corynantheine type, which
occur in a variety of structural variants, it is necessary to use different solvent systems for
the analysis (see Rauwolfiae radix Fig. 5/6, p. 70).
4A 1,2 Quebracho and Yohimbe cortex. In the basic solvent system, AL-I, extracts of Quebra-
cho and Yohimbe bark show many blue fluorescent zones distributed over the whole
Rf range; the main compound is yohimbine (cf. T3) in the intermediate Rf region.
4B In the neutral system, AL-6 (cf. DAB 8/Rauwolfiae radix), most compounds are
found in the lower Rf region. The strongly fluorescent zone at Rf ca. 0.7 can be
used to differentiate between Quebracho and Yohimbe cortex.
4C AN-I Treatment with Dragendorff reagent gives brown, rapidly fading zones in the visible.
Compared with Y ohimbe cortex, Quebracho cortex shows more stable and stronger
zones in the lower Rf range and in the Rf region of standard yohimbine. Y ohimbe
cortex shows an additional zone at Rf ca. 0.8.
68
Fig.3
T1 T2 T3 T4 T5 T3 T4 T5 T3 T4 T4T5
FRONT
Rf
Fig.4
T3 2 T3 2 T3 2
69
Rauwolfiae Radix
Tracks 1 = Rauwolfiae radix (R. serpentina - "Siam drug")
2 = Rauwolfiae radix (R. vomitoria - " African drug")
3 = Rauwolfiae radix (R. serpentina - "J ndian drug")
Tests Tl = serpentine T4 = reserpinejrescinnamine test mixture
T2 = ajmaline T5 = rauwolscine (with reserpine)
T3 = reserpine T6 = Rauwopur® (see p. 52 for composition)
Solvent AL-l : toluene-ethyl acetate-diethylamine (70: 20: 10) Fig. 5
system AL-6: n-heptane-ethylmethylketone-methanol (58:34:8) Fig.6
Detection Without chemical treatment UV-365 nm Fig. 5A; 6A
Dragendorff reagent (No. 11 F, p. 301) vis. Fig. 5B; 6B
Chromato- Rauwoljia root extracts, after chromatography in solvent systems AL-l and AL-6,
gram show many light blue fluorescing zones from the starting point to Rf ca. 0.8, when
viewed in UV-365 nm.
5 1 Rauwoljiae serpentinae radix (Siam drug). In solvent system AL-l the following
pattern is obtained in order of increasing Rf:
Above the dark blue fluorescing starting zone, serpentine is weakly visible (cf.
Tl) at Rf ca. 0.1. Two pronounced blue zones are seen at Rf 0.2 and 0.25, followed
by the darker blue fluorescent zone of ajmaline (cf. T2) at Rf ca. 0.35. A further
blue fluorescent zone at Rf ca. 0.4 is followed by the hardly separated zones of
reserpine and rescinnamine (cf. T3jT4). The upper Rf range is occupied chiefly by
weakly concentrated zones, and the more prominent zone of rauhasine at Rf ca.
0.75.
3 Rauwoljiae serpentinae radix (Indian drug). The concentration of reserpine/rescinna-
mine is similar to that of the Siam drug, but the Indian drug shows a higher content
of serpentine (cf. Tl). The zones up to ajmaline (cf. T2) and the zones in the upper
Rf range are more pronounced than in the Siam drug.
2 Rauwoljiae vomitoriae radix (African drug). This drug, which is not admitted by
DAB 8, shows markedly high er contents of ajmaline, reserpine and rescinnamine.
In addition the TLC reveals rauwolscine (cf. T5) at Rf ca. 0.45, and other zones
directly above it.
6 Solvent system AL-6 (DAB 8) gives a similar range of separation for the main
alkaloids, but at lower Rf values. Serpentine and ajmaline remain at the start (cf.
TljT2). Reserpine and rescinnamine (cf. T3jT4) can be assigned to the two zones
at Rf ca. 0.3-0.35. In contrast to its behaviour in the basic solvent system, rauwolscine
mi grates with a higher Rf than reserpine.
Remarks: With Dragendorff reagent all Rauwolfia alkaloids show brown colour in the visible.
Ajmaline shows a prominent fluorescence quenching in UV-254 and can be made visible by
treatment with conc. HN0 3 (red).
70
-FRONT
Rf
-0.5
-START
Fig.5
Tl T2 2 3 T3 T4 T5 T6 T2 Tl
-FRONT
Rf
-0 .5
-START
Fig.6
Tl T2 2 3 T3 T4 T5 T6
DABS
71
Strychni, Ignatii Semen
Tracks 1 = Ignatii semen Tests Tl = brucine
2 = Strychni semen T2 = strychnine
Solvent AL-l: toluene-ethyl acetate-diethylamine (70: 20: 10) Fig. 7 A, B
system AL-2: chloroform-diethylamine (90: 10) Fig.7C
Detection Dragendorff reagent (No. 11 B)
with sodium nitrite (No. 11 F, p. 301) vis. Fig. 7 A, C
Conc. nitric acid VIS. Fig.7B
Chromato-
gram
7A After TLC separation in solvent system AL-l, followed by treatment with Dragen-
dorff reagent, both drug extracts show 4-6 orange-brown zones of differing concen-
trations in the Rfrange 0.15-0.4.
1 Ignatii semen contains hrucine (cf. Tl) and strychnine (cf. T2) in the ratio ca. 1: 3.
2 Strychni semen has a lower content of total alkaloids, with approximately equal
proportions of strychnine and hrucine. Minor alkaloids, e.g. oc- and ß-colubrine and
pseudo-strychnine, tend to mi grate ahead of the strychnine zone.
Remarks: The strychnine zone is initially prominent, but fades rapidly.
7B Brucine (cf. Tl; Rf ca. 0.15) is typically red in the visible when treated with conc.
HN0 3 • Strychnine does not react with this colour.
7C In solvent system AL-2, the main alkaloids, brucine and strychnine, migrate in
the higher Rf region (cf. Tl/T2).
Secale cornutum
Track 3 = Secale cornutum Tests T3 = ergometrine
T 4 = ergotamine
T5 = ergocristine
Solvent AL-l : toluene-ethyl acetate-diethylamine (70: 20: 10) Fig.8A
system AL-5: toluene-chloroform-ethanol (28.5: 57: 14.5) Fig.8B
Detection Van URK reagent (No. 39, p. 304) VIS. Fig. 8A, B
For description of drugs see p. 55. Formulae 'po 62.
Chromato- After treatment with van Urk reagent chromatograms of Secale extracts show
gram 5-6 blue-violet alkaloid zones in the Rf range 0.05-0.25 (solvent system AL-l) or
8A, B 0.05-0.6 (solvent system AL-5).
The zone at Rf 0.05 represents ergometrine 1 , at Rf ca. 0.03 ergotamine 1 , and
at Rf ca. 0.45 ergocristine 1 (solvent system AL-5).
TLC separations of commercially available references (T3-T5) show a second, usually weak zone. This
is also seen in Secale extracts, and is due to degradation products (Iumi-compounds or similar products).
72
A B c
-FRONT
Rf
-0.5
-START
Fig.7
Tl 2 T2 Tl 2 Tl 2 T2
A B
-FRONT
Rf
-0.5
-START
Fig.8
T3 3 T4 T5 T3 3 T4 T5
73
Chinae Cortex
Tracks 1 = Chinae (e. ledgeriana) cortex
2 = Chinae (C. succirubra) cortex
Tests TG = standard mixture of quinine alkaloids, Ph. Eur. III (for composition, see p. 53)
Ti =quinine
T2 = cinchonidine
T3 = quinidine
T 4 = cinchonine
Solvent AL-2: chloroform-diethylamine (90: 10) Ph. Eur. III Fig. 9; lOA
system AL-1 : toluene-ethyl acetate-diethylamine (70: 20: 10) Fig. lOB, C
Detection Sulphuric acid (No. 34, p. 303) UV-365 nm Fig. 9; lOC
Sulphuric acid-iodoplatinate (No. 19, p. 302) VIS. Fig. lOA, B
Chromato- After treatment with sulphuric acid, chromatograms of both cinchona drugs in sol-
gram vent system AL-2 (Ph. Eur.) reveal at least 6 intense light blue and 5-6 weaker,
9 1,2 dark violet fluorescent zones in UV -365 nm, distributed from the start to the solvent
front.
After sulphuric acid treatment, the main alkaloids, quinine (cf. Ti) and quinidine
(cf. T3), show an intense blue fluorescence, and cinchonidine (cf. T2) and cinchonine
(cf. T4) show a dark violet fluorescence in UV-365 nm. In cinchona extracts cinchoni-
dine is overlapped by the strongly blue fluorescing quinidine.
10A Treatment afterwards with iodoplatinate reagent gives a violet colouration in the
visible. The grey-violet of cinchonidine is distinct from the violet-brown of quinine,
quinidine and cinchonine.
10B, C In separations with the screening system for alkaloids (solvent system AL-I), the
four main alkaloids mi grate only in the Rf range 0-0.2.
Remarks: In addition to the four main alkaloids, further cinchona alkaloids and epiquinine
bases can also be detected. The epibases (threo-compounds) of quinine and quinidine migrate
in the lower Rf range. The dihydrocompounds of quinine, quinidine, cinchonine and cinchoni-
dine show no fluorescence in UV-365 nm. Certain yellow-green fluorescing zones may represent
quinonoid structures.
The two cinchona extracts can be dijJerentiated on the basis of their quinine contents.
2 Cinchonae succirubrae cortex contains the 4 main cinchona alkaloids in approximate-
Iy the same proportions as in the standard mixture TG (Fig. 9j10A).
1 In extracts of C. ledgerianae cortex, quinine is the predominant alkaloid (Fig. 9).
74
-FRONT
Rf
0 .5
-START
Fig.9
2 TG Tl T2 T3 T4
FRONT
Rf
START
Fig.10
TG 2 2 TG 2 TG ,
75
Ipecacuanhae Radix
Tracks 1 = Ipecacuanhae radix (CephaeIis acuminata "Cartagena/Panama drug")
2 = Ipecacuanhae radix (e. ipecacuanha "Rio/Matto-Grosso drug")
3 = Ipecacuanhae radix (commercial; plant of unspecified origin)
4= Ipeeaeuanhae radix (eommercial "Jahore")
Test TG = standard mixture of cephaeline (Rf ea. 0.2) and emetine (Rf ca. 004) Ph. Eur.
(see p. 53)
T1 = eephaeline
T2=emetine
Solvent AL-1 : toluene-ethyl acetate-diethylamine (70: 20: 10) Fig. 11 A, B, C
system AL-1: with chamber saturation Fig.12A
AL-7: chloroform-methanol (85: 15) Ph. Eur. Fig.12B
Detection Iodine-chloroform-reag. UV-365 nm Fig. 11A; 12A, 12B
(I/CHCI 3 No. 17, p. 301) VIS. Fig. 11 B
Dragendorff re agent (No. 11 B, p. 300) VIS. Fig. 11 C
Chromato- Ipecacuanhae radix. Without prior chemical treatment, the main alkaloids, emetine
gram and cephaeline (cf. TG), appear in UV-365 nm as uniformly blue fluorescing zones.
11A 1-4 After treatment with iodine reagent and brief warming, they show a characteristic
12A, B yellow-white or light blue, respectively, in UV-365 nm (Fig. 11 A; 12A, B) and red-
brown or yellowish zones, respeetively, in the visible (Fig. 11 B).
1,2 Cartagena and Rio drugs both show the intense light blue zone of cephaeline (cf.
T1) and the yellow-white zone of emetine (cf. T2). In addition there are weaker
blue or yellow fluorescent zones in the starting region and direetly above and be10w
the zone of emetine (cf. T2). The minor alkaloid, O-methylpsychotrine (in the Rf
region of emetine) also fluoresees yellow. In contrast, psychotrine, whieh is found
below cephaeline, gives a blue fluorescence.
11 C Dragendorff reagent deteets the main alkaloids as red-brown zones in the visible.
The minor alkaloids are only partly detectable.
Commercial ipecacuanha drugs can be differentiated on the basis of the eephaeline:
emetine ratio.
1 The Cartagena (Panama or Costa Riea) drug from Cephaelis acuminata contains
almost equal proportions of emetine and cephaeline.
2 In the Rio (Matto-Grosso) drug from C. ipecacuanha the emetine: cephae1ine ratio
is 2-3:1.
3,4 Ipecaeuanha drugs from other sourees (e.g. Jahore drug 4) tend to have a slightly
higher eontent of emetine than cephaeline.
12 B In the solvent system of Ph. Eur. (AL-7), with chromatographie development over
15 em, emetine and cephaeline (cf. T6) remain poorly separated in the lower Rf
range; psychotrine stays ne ar the start and methylpsychotrine mi grates above emetine.
Double development gives slightly higher Rf values for emetine and cephaeline.
76
c
-FRONT
Rf
-0.5
Fig.11
TG 2 TG 2 TG 2
_&:10 '"'""T
Fig.12
Tl 3 4 T2 TG 2
Ph . Eur I
77
Opium
Tracks 1 = Opium DAB 8 Tests Ti = morphine
2=Opium T2=codeine
3=Opii tinctura DAB 8 T3 = papaverine
T4 = noscapine
Solvent AL-3: toluene-acetone-ethanol-conc. ammonia (40: 40: 6: 2) Fig. 13A, B
system AL-1 : toluene-ethyl acetate-diethylamine (70: 20: 10) Fig. 14A, B
Detection Dragendorff re agent with NaNO z reagent (No. 11 CjF, p. 300)
VIS. Fig. 13A; 14B
Marquis reagent (No. 25, p. 302) VIS. Fig. 13B
Natural products-polyethyleneglycol re agent
(NP/PEG No. 28, p. 303) UV-365 nm Fig. 14A
Chromato-
gram
BA 1,2,3
14B After treatment with Dragendorff-NaNO z , opium extracts show six orange-brown
main zones from the start to Rf ca. 0.85. Narceine remains at the origin; in order
of increasing Rf, it is followed by morphine (cf. Ti), codeine (cf. T2), thebaine and
minor alkaloids (RfO.3-0.5 in solvent system AL-3, and 0.4-0.5 in AL-1),papave";ne
(cf. T3) and noscapine (narcotine) (cf. T4).
13 B With Marquis reagent, morphine and codeine are immediately stained violet. A weak
violet zone of thebaine, and orange-brown zones of minor alkaloids are seen in
the intermediate Rf region. Papaverine and noscapine give violet and brown zones,
respectively, with Marquis reagent.
14 An analogous separation is seen in solvent system AL-1 (screening system), with
the difference that morphine and codeine migrate with slightly higher Rf values.
14A In UV-365 nm, without prior chemical treatment, the TLC of an opium extract
(1) shows pale blue fluorescent zones over the whole Rf range. After treatment
with NP/PEG reagent, the fluorescence is intensified. Morphine (cf. Ti) fluoresces
pale blue, papaverine (cf. T3) pale green, and noscapine (cf. T4) intense blue. Codeine
gives no j1uorescence.
14B Brown to brown-red zones are obtained with Dragendorffreagent.
78
A B
-FRONT
Rf
-0.5
-START
Fig.13
Tl T2 T3 T4 Tl T2 T3 T4 2
DAß B
-FRONT
Rf
-0.5
-START
Fig. 14
Tl T2 T3 T4 Tl T2 T3 T4 3
79
Berberidis, Colombo Radix Hydrastis Rhizoma
Tracks 1 = Berberidis radix
2 = Hydrastis rhizoma
3 = Colombo radix
Tests Tl = berberine T5 = jateorhizine
T2 = palmatine-jateorhizine mixture T6 = columbamine
T3 = hydrastine (Rf ca. 0.05, Fig. 15 C) T7 = palmatine
T4 = sanguinarine
Adsorbent Silica gel 60F 254 pre-coated TLC plates Fig. 15A, B, C; 16A
Aluminium oxide TLC sheets Fig.16B
Solvent AL-9: n-propanol-formic acid-water (90: 1 : 9) Fig. 15 A, B, C
system AL-l: toluene-ethyl acetate-diethylamine (70: 20: 10) Fig.16A
AL-I0: cyc1ohexane-chloroform-glacial acetic acid
(45: 45: 10) for aluminium oxide adsorbent Fig.16B
Detection Without chemical treatment visjUV-365 nm Fig. 15A, C; 16A, B
Dragendorff reagent (No. 11, p. 300) vis. Fig.15B
Chromato-
gram 1,2,3
15 A The three drug extracts characteristically show yellow fluorescent alkaloids in UV-
15B 365 nm.
15 C 1 Berberidis radix. Berberine appears as a light yellow zone (vis.) on untreated chro-
matograms, and as a lemon-yellow fluorescent zone in UV-365 nm. With Dragen-
dorff reagent it gives a strong brown-red zone (cf. Tl). Weaker concentrations of
blue fluorescent alkaloids are seen in the lower Rf range.
15 C 2 Hydrastis rhizoma. The main zone is again berberine (cf. Tl) (yellow fluorescence
in UV-365 nm). This drug extract can be differentiated from that of Berberidis
radix by the presence of the blue-white fluorescent zone of hydrastine (cf. T3) at
the start. A second, light blue fluorescent zone, also present in the standard hydras-
tine (T3) is present at Rf ca. 0.9.
15 C 3 Colombo radix. In system AL-9, the main yellow fluorescent zone (UV -365 nm)
is a mixture ofjateorhizine-palmatine-columbamine (cf. T2).
16A In screening system AL-l, the yellow fluorescent zone of berberine (cf. Tl) migrates in the
middle of the chromatogram, while the blue fluorescent hydrastine zone has a higher Rf value
(cf. T3), thus serving for the simple differentiation of Hydrastis and Berberis extracts. Colombo
radix gives a mixt ure of yellow fluorescing alkaloids in the lower Rf range.
16B On aluminium oxide in solvent system AL-10, the alkaloids (cf. T5, T6, Ti, T7) show marked
differences in Rf values.
Remarks: Sanguinarine (cf. T4, Fig. 15C), another yellow fluorescing alkaloid, can be found
in extracts of Chelidonii herba (see Fig. 17).
80
A B
FRONT
Rf
START
Fig.15
Tl Tl Tl 2 T2 3 T3 T4
- FRONT
Rf
-START
Fig. 16
T3 2 Tl 3 T5 T6 Tl T7
81
Chelidonii Herba Colchici Semen
Tracks 1 = Chelidonii herba 2 = Colchici semen
Tests Tl = berberine
T2 = sanguinarine
T3 = papaverine
T 4 = colchicine
Solvent AL-9: n-propanol-formic acid-water (90: 1 : 9) Fig. 17 A, B
system AN-1: ethyl acetate-methanol-water (100: 13.5: 10) Fig. 18A, B
AL-8: toluene-methanol (86: 14) 2 x 15 cm; DAC Fig.18C
Detection Without chemical treatment Fig. 17 A; 18A
Draggendorff-NaN0 2 reagent (No. 11 CjF, p. 300-301) Fig. 17B; 18B
10% Ethanolic HCl (DAC) Fig.18C
For description of drugs see p. 57. Formulae p. 63-64.
Chromatogram
17 A 1 Chelidonii herba. In UV -365 nm, without treatment, the chromatogram usually
shows two to three powerfully fluorescent yellow zones in the Rf range 0.1-0.4
and three weaker, blue and yellow-green fluorescent zones in the Rfrange 0.75-0.9.
There is a prominent yellow fluorescent zone at Rf ca. 0.2; directly above this
is seen the narrow yellow-green zone of ehelerythrine, followed by the tailing yellow
zone of sanguinarine (cf. T2). The pale white fluorescent zone of protropine is located
in the same position as standard berberine (cf. Tl); this is usually overlapped by
sanguinarine.
17B After treatment with Dragendorff-NaN0 2 , ehelidonine (Rf ca. 0.6) and the other
alkaloids form rapidly fading brown (vis.) zones. Papaverine (cf. T3) can be used
for the comparison of Rf values.
18 2 Colehici semen. In UV-365 nm many blue or yellow-green fluorescent zones are
18 A seen between the origin and the solvent front. There are two prominent yellow
zones in the Rf region of standard colchicine (cf. T4). Colehicine and the minor
alkaloids, colchiceine, N-acetyldemecolcine, 1-ethyl-2-demethylcolchiceine and cor-
nigerine, give a yellowish fluorescence in UV-365 nm.
Blue fluorescent zones are given by O-benzoylcolchiceine, N-formyl-deacetyl-
colchicine, colchicoside and N-methyldemecolcine.
18 B Colehieine forms a brown (vis.) zone with DragendorJJ reagent and a lemon-yellow
18 C (vis.) zone with ethanolie hydroehlorie acid. In solvent system AL-8 (DAC) colchicine
(cf. T4) migrates at Rf ca. 0.15.
Remarks: Commercial sampies of standard colchicine contain an impurity that migrates in
the higher Rf range and stains violet with Dragendorff-NaNO z ; the same substance is present
in low concentration in the extract.
82
-FRONT
Rf
-0.5
-START
Fig.17
Tl T2 Tl T2 T3
c
-FRONT
Rf
-0.5
-START
Fig.18
T4 2 T4 2 T4 2
83
Aconiti Tuber/Herba Sabadillae Semen Lobeliae Herba
J aborandi, Boldo Folium

Tracks 1 = Aconiti tuber 4 = Lobeliae herba
2 = Aconiti herba 5=Jaborandi folium
3 = Sabadillae semen 6 = Boldo folium
Tests Tl = aconitine (alkaloid mixture) T 4 = pilocarpine
T2=veratrine (alkaloid mixture) T5= boldine
T3 = 10 beline
system
Detection Dragendorffreagent (DRG No. 11 C/F, p. 300-301) vis. Fig. 19A, B; 20B
Iodoplatinate reagent (IPL No. 19, p. 302) VlS. Fig.20A
For description of drugs see p. 57-58. Forrnulae p. 61, 64, 65.
Chromato- Treatment of chromatograms of extracts of Aconiti tuber or herba, Sabadillae semen

gram and Lobeliae herba with Dragendorff reagent shows that, in each case, the main
19 A alkaloid zones migrate in the Rf range 0.6-0.75.
1,2 Aconiti tuber or herba. Both drug extracts show different concentrations of the
same two alkaloid zones at Rf ca. 0.6-0.65, which correspond to the aconitine alka-
loid mixture (cf. Tl).
3 Sabadillae semen. Chromatograms of this drug show two main alkaloid zones at
Rf 0.5-0.7 ("veratrine alkaloid mixture", cf. T2), two weaker zones at Rf ca. 0.8
and 0.4, and a more prominent zone at Rf ca. 0.1.
4 Lobeliae herba. The extract is characterized by the presence of the main alkaloid
lobeline (cf. T3), which migrates at Rf ca. 0.65.
20A With iodoplatinate reagent all the alkaloids give an immediate brown-violet colour
(vis.), which later becomes a stable white zone on a grey-blue background.
This reagent permits a better evaluation of the drug extract, because it also reveals
the minor alkaloids.
19B 5 laborandifolium. The main alkaloid ispilocarpine (cf. T4) at Rfca. 0.1.
20B 6 Boldo folium. In system AL-1, the main alkaloid, boldine (cf. TS), mi grates at Rf
ca. 0.25. On untreated chromatograms, boldine fluoresces dark blue in UV-365 nm;
it gives a pronounced brown (vis.) with Dragendorff reagent.
84
A B
-FRONT
Rf
-0.5
-START
Fig.19
Tl 2 T2 3 T3 4 T4 5
B
-FRONT
Rf
-0.5
-START
Fig.20
Tl 2 T2 3 T3 4 6 T5
85
Purine-containing and Miscellaneous Drugs
Tracks 1 = Cacao semen 3 = Nicotianae folium
2 = Coffeae semen 4 = Ephedrae herba
5 = Spartii herba
Tests T1 = trigonelline T3 = caffeine T5 = chlorogenic acid T7 = ephedrine
T2 = theobromine T4 = theophylline T6 = nicotine T8 = sparteine
Solvent AN-i: ethyl acetate-methanol-water (100: 13.5: 10) Fig. 21A, B
system AL-i: toluene-ethyl acetate-diethylamine (70: 20: 10) Fig.22A-C
Detection Iodine-potassium iodide-HCI-reag. (I/HCI No. 20, p. 302) vis. Fig.21A
Natural products-polyethyleneglycol reagent (NP/PEG no. 28, p. 303)
UV-365 nm Fig.21B
Dragendorffreagent with NaN0 2 (No. 11 C/F, p. 300-301) VIS. Fig.22A
Ninhydrin reagent (NIH No. 29, p. 303) VIS. Fig.22B
Iodoplatinate reagent (IPL No. 19, p. 302) vis. Fig. 22C, D
For description of drugs see p. 57-59. Formulae p. 64-65.
Chromatogram
21 A l , 2 Caeao semen-Coffeae semen
In solvent system AN-i, both drug extracts show the brown zone of eaffeine (cf.
T3) at Rf ca. 0.5 after treatment with I/HCI reagent.
1 Caeao semen. Theobromine (cf. T2) can also be detected below eaffeine.
2 Coffeae semen. In addition to eaffeine, the alkaloid trigonelfine (cf. T1) is also visible
at the start.
21 B After treatment with NP/pEG reagent and visualization in UV-365 nm, chromato-
grams of Coffeae semen extracts reveal chlorogenie acid at Rf ca. 0.1 and caffeic
acid at Rf ca. 0.75; this distinguishes the drug from other caffeine-containing drugs.
Remarks: Owing to the small amount of theophylline in Coffeae semen, its presence in the
drug can only be demonstrated after enrichement of the extract.
In addition to caffeine, other purine-containing drugs, e.g. Colae semen, Theae
folium and Mate folium, usually contain detectable quantities of theobromine and
theophylline (for % see Drug List on p. 58-59).
22A 3 Nieotianaefolium. The main alkaloid, nicotine (cf. T6), shows a pronounced quench-
ing of fluorescence in UV-254 nm. With Dragendorff reagent (solvent system AL-i)
it gives only an unstable colour in the visible. In non-concentrated extracts, nornico-
tine and anabasine are only seen as weak zones in the middle and lower Rf ranges.
22B/C 4 Ephedrae herba. With ninhydrin reagent ephedrine appears with a light colour against
a red-violet background of the TLC-plate; or it becomes more violet, depending
on the extent of heating. Other reagents, like Dragendorff or iodoplatinate (Fig. 22 C)
produce only faint zones in the visible.
The isomers, pseudo-ephedrine and nor-pseudo-ephedrine, are not separated
from ephedrine in these systems.
22D 5 Sarothamni (Spartii) seoparii herba. Sparteine (cf. T8) is the characteristic main
alkaloid of the drug extract. With IPL reagent it gives a deep blue-violet (vis.)
on a red-violet background (Flavonoids, cf. Fig. 14, p. 184).
86
A
FRONT
Rf
Fig.21
Tl T2 T3 2 T4 2 TS
A c
FRONT
Rf
TART
Fig.22
T6 3 T7 4 T7 4 T8 5
87
Solanaceae Drugs I
Tracks 1 = Belladonnae folium
2 = Hyoscyami folium
3 = Stramonii folium
Tests Tl = Ph. Eur. standard solution (for Belladonnae folium), see p. 53
T2 = Ph. Eur. standard solution (for Hyoscyami folium), see p. 53
T3 = Ph. Eur. standard solution (for Stramonii folium), see p. 53
T4=atropine 100 llg
T5 = atropine 50 llg
T6 = atropine 25 llg
Solvent AL-l : toluene-ethyl acetate-diethylamine (70: 20: 10) Fig. 23A, B
system AL-4: acetone-water-conc. ammonia (90: 7: 3) Fig. 24A, B
Detection Dragendorff reagent (No. 11 C) vis. Fig. 23A
Dragendorff-NaN0 2 reagent (No. 11 CjF, p. 300-301) vis. Fig. 23B; 24A, B
Chromatogram
23 A Belladonnae, Hyoscyami and Stramonii folium
(- )-Hyoscyamine (or atropine) is the main alkaloid in all three Solanaceae drugs.
The TLC differentiation of these drugs is based on the hyoscyamine: scopolamine
ratio, and to a limited extent on the contents of the minor alkaloids, belladonnine,
atropamine and cuskhygrine. Detection of the minor alkaloids, however, requires
special enrichment prior to chromatography.
For identification and determination of alkaloid content, Ph. Eur. describes a
TLC comparison with alkaloid mixtures containing defined ratios of atropine-S0 4
to scopolamine-HBr (cf. T1-T3). Identification of the drug is then based on the
similarity of colour intensity and zone size between the standard solutions and
drug extracts.
1 Belladonnae folium. The ratio hyoscyaminejatropine (Rf ca. 0.25) to scopolamine
(Rf ca. 0.4) corresponds to that of Tl.
2 Hyoscyami folium. The hyoscyaminejatropine: scopolamine ratio of standard solu-
tion T2 is about 3: 1. The total alkaloid content of the drug does not meet the
quality requirements of the pharmacopoeia, it is generally too low in the stored
commercial drugs.
3 Stramonii foiium. The typical hyoscyaminejatropine: scopolamine ratio for this drug
is ab out 2: 1. The total alkaloid content of this sampie is higher than that demanded
by the pharmacopoeia. The concentration of scopolamine in the drug extract is
higher than that in T3.
24A In solvent system AL-4 (Ph. Eur.) hyoscyaminejatropine (Rf ca. 0.1-0.15) mi grates
in the lower Rf range, while scopolamine (Rf ca. 0.8) is found in the upper region
of the chromatogram.
Remarks: Dragendorff reagent, specified by Ph. Eur. (orange zones; vis.), suffers from the
dis advantage that it gives unstable colours with tropane alkaloids. Subsequent treatment with
sodium nitrite intensifies the colour and increases the stability of the colour of hyoscyamine/
atropine, but not of scopolamine (Fig. 23 B, 24A).
88
-FRONT
Rf
-0.5
-START
Fig.23
Tl 2 T2 3 T3 T2 3 T3
•
-FRONT
Rf
-0.5
-START
Fig.24
Tl 2 T2 3 T3 T4 T5 T6
89
Solanaceae Drugs 11
Tracks 1 = Belladonnae folium 8 = Scopoliae radix
2 = Stramonii folium 9= Belladonnae radix
3 = Stramonii semen 10= Belladonnae folium
4 = Hyoscyami mutici folium 11 = Stramonii folium
5 = Hyoscyami nigri folium 12 = Hyoscyami nigri folium
6 = Belladonnae semen 13 = Hyoscyami mutici folium
7 = Belladonnae radix (8-13: methanol extract (see
(1-7: concentrated chloro- p. 163); 20 111 applied to
form extracts; 150 Ilg) chromatogram)
Tests T1 = atropine (Rf ca. 0.25) T3 = rutin (Rf ca. 0.35), chlorogenic acid
scopolamine (Rf ca. 0.35) (Rf ca. 0.45), hyperoside (Rf ca. 0.6)
T2 = atropine T4=caffeic acid
T5 = scopoletin
Solvent AL-1 : toluene-ethyl acetate-diethylamine (70: 20: 10) Fig. 25A, B
system F-1: ethyl acetate-formic acid-glacial acetic Fig.26
acid-water (100: 11 : 11 : 27)
Detection Dragendorffreagent (No. 11 C) with NaN0 2 (No. 11 F, p. 301) VIS. Fig.25
Natural products-polyethyleneglycol reagent
(NP/PEG No. 28, p. 303) UV-365 nm Fig.26
Chromato- Tropine alkaloids. The chromatogram in Fig. 25A was obtained by applying rather
gram large quantities of the extracts (ca. 150llg, cf. p. 89). It then permits the following
25A, B differentiation:
2, 3 The scopolamine content of Stramonii semen is higher than that of Stramonii folium.
4, 5 The hyoscyamine and scopolamine contents of the industrial drug, Hyoscyamus muticus, (total
ca. 1 % according to the literature) are much higher than those of the official drug.
1,6,7 Belladonnae folium contains a higher amount of hyoscyamine than Belladonnae radix or
semen.
Remarks: It is difficult to identify unequivocally Solanaceae drug extracts only on the basis of
the alkaloid content. The flavonoid or coumarin patterns are a more certain guide to identitiy.
26 8-13 Flavonoids, coumarins
8 Scopoliae radix gives five intensely blue fluorescent zones in the Rf range 0.1-0.5, with a
main zone at Rf 0.2. Scopoletin (cf. T5) with traces of caffeie acid (cf. T4) are responsible
for blue fluorescence in the region of the solvent front.
9 Belladonnae radix. The main zone is that of scopoletin (T5; blue fluorescence in the region
of the solvent front). There are also four less intense zones between the start and Rf 0.5,
which fluoresce dark blue or blue-green.
10-13 The TLC of all lea! drugs reveals chlorophyll (red fluorescence, UV-365 nm) at
the solvent front.
10,12 On chromatograms of Belladonnae and Hyoscyami nigrifolium, the main zones are rutin (Rf
ca. 0.4; orange fluorescence) and chlorogenie acid (Rf ca. 0.45; blue fluorescence). In Hyoscyami
nigri folium, these are the only two detectable zones, whereas Belladonnae folium shows addi-
tional blue, yellow-green and orange fluorescent zones in the Rf range 0.05--0.1 (7-glucosyl-3-
rhamnogalactosides of kampferol and quercetin).
11 Stramonii folium is characterized by five orange fluorescent zones (quercetin glyeosides) in
the Rf range 0.03-0.25. The absence of rutin and chi orogenie acid c1early distinguishes this
drug from Belladonnae and Hyoscyami folium.
13 Hyoscyami mutici folium has only a low total flavonoid content, and the presence of other
compounds causes adepression of Rf values.
90
A B
-FRONT
Rf
-0.5
-START
Fig.25
Tl 2 3 4 5 6 7 T2
FRONT
Rf
START
Fig.26
T3 T4 T5 8 9 10 11 12 13
91
Drugs Containing Anthracene Derivatives
The characteristic constituents of this drug group are anthraquinones and their
reduced derivatives, oxanthrones, anthranols and anthrones. The anthraquinones,
which have purgative properties, possess phenolic groups on C-1 and C-8, and
keto groups on C-9 and C-10. In the anthrones and anthranols, only C-9 carries
an oxygen function. In addition, a methyl, oxymethyl or carboxyl group may be
present on C-3, and an hydroxy or methoxy group on C-6.
Most compounds in this group are present in the plant as O-glycosides. The
glycoside linkage is usually at C-1, C-8 or C-6. C-Glycosides are also present: these
occur in the anthrone form only, with the C-C bond always at C-10.
In the 0- and C-glycosides, the only sugars found so far are glucose, rhamnose
and apiose.
I. Extraction of Drugs and Pharmaceutical Preparations for TLC

1. Drugs
Powdered drug (0.5 g) is extracted by warrning for 5 min on the water bath with
5 ml of methanol. The dear filtrate is used directly for TLC.
Exception: Sennae folium or fructus are extracted with 50% methanol.
2. Pharmaceutical preparations
Three finely powdered dragees or tablets are extracted on a water bath with 5 ml
of methanol. The dear filtrate is used directly for TLC.
3. Hydrolysis of Rhei radix, according to DAß 8

A sampie (0.5 g) of the drug is heated under reflux for 15 min with 25 ml of 7.5%
hydrochloric acid. After cooling, the mixture is extracted by shaking with 20 ml
of ether. The ether phase is concentrated by evaporation and applied to the TLC
plate (see DAB 8).

Aloin, frangulin AlB, glucofrangulin AlB and the aglycones, rhein, Aloe-emodin and
Frangula-emodin, are applied to the TLC plate as 0.1 % methanolic solutions.
The mixture of sennosides A and Bis prepared as a 0.1 % solution in n-propanol-
ethyl acetate-water (4:4:3).
The stilbene glucoside, rhaponticin (= rhaponticoside) is applied as a 0.1 % metha-
nolic solution. Commercial "rhaponticin" also contains deoxyrhaponticin.
2. Adsorbent
Chromatography is perforrned on silica gel 60F 254 pre-coated plates (Merck, Darm-
stadt) (see also Circular TLC IV, p. 95).
93
Aloe extracts, 5 111.
Rheum, Frangula, Cascara and Senna extracts, 10111.
Reference solutions, 10 111.
With the exception of Senna preparations, the solvent system suitable for the chro-
matography of all drug extracts is ethyl acetate-methanol-water (100: 17: 13; DAB 8
and Ph. Eur.).
AN-1: ethyl acetate-methanol-water (100:17:13)
AN-2: ethyl acetate-methanol-water (100: 13.5: 10)
This slightly different ratio of components prevents the "wave shaped" sepa-
ration, which is often observed with AN-i. On the other hand, AN-2 is
also suitable for other types of constitutents, such as cardiac glycosides,
bitter principles and some alkaloids.
AN-3: n-propanol-ethyl acetate-water (40: 40: 30)
Sennae folium and fructus (Ph. Eur. I)
AN-4: toluene-ethyl formiate-formic acid (50: 40: 10)
Hypericine from Hyperiei herba (DAC)
AN-5: light petroleum-ethyl aeetate-formie acid (75: 25: 1)
Anthraquinone aglyeons (Rhei radix, DAB 8)
111. Detection
UV-254 nm
All anthraquinone derivatives queneh fluoreseence
UV-365 nm
All anthraquinone derivatives give yellow or red-brown fluoreseenee
2. Spray reagents
a) Potassium hydroxide (KOH No. 21, p. 302: Bornträger reaetion)
After spraying with 5% or 10% ethanolie KOH, anthraquinones show red in the
visible, and red fluoreseence in UV-365 nm. Anthrones and anthranols show yellow
in the visible, and yellow fluoreseence in UV-365 nm.
For detection of dianthrones, see 2e.
b) Natural produets-polyethyleneglyeol reagent (NP/PEG No. 28, p. 303)
Anthrones and anthranols give an intense yellow fluoreseenee in UV -365 nm.
e) Sennoside deteetion
Nitrie acid reagent (HN0 3 No. 33, p. 303) with potassium hydroxide reagent (KOH
No. 21, p. 302) Ph. Eur.
The TLC plate is sprayed with eone. HN0 3 , then heated for 10 min at 120 0 C.
It is then sprayed with 5% ethanolic KOH. After further heating, sennosides appear
as yellow zones in UV-365 nm, and red-brown zones in the visible.
Remarks: Sennosides can also be detected with a 1% soln. of sodium metaperiodate in 10%
ethanolic KOR. After spraying and heating (ca. 5 min), yellow-brown zones are obtained
in UV-365 nm.
d) Phosphomolybdate-H2 S0 4 reagent (PMA-H 2 S0 4 No. 27, p. 303)
Rhaponticin and deoxyrhapontiein give intense blue zones in the visible.
94
e) Hypericin detection (DAC)
A 10% soln. of pyridine in acetone intensifies the red fluorescenee of hyperiein
in UV-365 nm.
IV. TLC Methods

Anthraquinone derivatives ean be separated by the usual aseending TLC method
or by eireular TLC.
Circular thin layer chromatography
1. Method. The solvent mi grates circularly from the point of application, so that the
zones form ares.
2. Adsorbent. Silica gel 60F 254 pre-eoated plates, 20 x 20 cm (Merek, Darmstadt).
3. Solvent systems. The same solvent systems ean be used as for ascending TLC.
4. Method of application. Two diagonal pencil lines are drawn from the corners of
the TLC plate. The eentre point of the plate is marked and a eirc1e is drawn around
it with a diameter of ca. 2 cm.
The circ1e is thus divided into fOUf segments by the diagonals. The perimeter
of each segment serves for the application of drug extracts or reference solutions
(Fig. 1, below).
5. Development. 100 ml of solvent are placed in a round, straight sided chamber (glass
trough, 10 cm high, 20 cm diameter). A glass funnel is loosely packed with cotton,
which extends as a wick through the tube of the funnel. The funnel is placed in
the solvent system, so that the solvent soaks into the cotton. With the loaded side
facing downwards, the TLC plate is plaeed over the top of the trough, so that
the wick from the funne1 makes contact exaetly at the marked centre.
The solvent spreads as a circ1e over the TLC plate, and the zones ofthe separating
substanees form ares, whieh inerease in length from the starting point to the peri-
phery of the spreading solvent.
6. Detection. All the same deteetion methods can be used as for ascending TLC.
7. Application. This method is especially useful for drug extracts containing a high
proportion of ballast substances, e.g. mucilages from Sennae folium. Good separa-
tions are obtained with solvent migrations of 5-6 cm.
Funne l
+ wick
"".=..=:,=-==-==-=-,
~.-
• /
~/s~~
/-Y .;: '.' ~
.. "' .
.\.--=
' ='='::-:".=;:::::=-.1
I\. • ..
Solvent
system
Fig.l Glass trough
95
v. List of Drugs Containing Anthracene Glycosides
Fig. Drug/Plant source Main constituents (anthraquinone deriva-

Farnily/Pharmacopoeia tives) % content and identity
1,2 Aloe extractum Dried extract (aqueous) of Aloe spp., contain-

Aloe extract ing
Various types of Aloes are de- Aloin (10-C-ß-D-glucopyranoside of Aloe-
scribed by the pharmacopoeias emodin-anthrone), in most cases as a mixture
(Cape Aloes and Curacao Aloes), of the cx- and ß-stereoisomers
and as commercial drugs (Uganda
Aloes, Kenya Aloes and Indian Aloinoside A and B (stereoisomers of aloin-
Aloes); 11-cx- L-rhamnoside)
see below Aloesine A and B (" Aloe resin" with no pur-
gative action)
Aloesine B (chromone-C-glucoside)
Aloesine A (p-coumaric acid ester of aloe-
sine B)
Aglycone: Aloe-emodin
Aloe barbadensis Not less than 28% hydroxyanthracene deriva-
Curacao aloes tives ca1culated as aloin (Ph. Eur.)
Aloe barbadensis MILLER Aloin
Liliaceae Aloesine A and B (" Aloe resin ")
USP XX, Ph. Eur. III, Helv. VI
Aloe capensis Not less than 18% hydroxyanthracene deriva-
Cape aloes tives ca1culated as aloin (Ph. Eur.)
Aloe ferox MILLER and hybrids Aloin, aloinosides AlB, aloesines AlB (type
Liliaceae Cape A)
USP XX, Ph. Eur. III, Helv. VI, Aloin, aloesines AlB (type Cape B)
ÖAB
2. AB-DDR
Aloe perryi ca. 14% hydroxyanthracene derivatives ca1cu-
Socotrine aloes lated as aloin
Aloe perryi BAKER Aloin, aloinosides AlB, aleosines AlB
Liliaceae
IsoharhaÜJin test (after Klunge) for the differentiation of Aloe capensis and Aloe
barbadensis.
One drop of saturated CuS0 4 soln., 1 g NaCI and 10 ml of 90% ethanol are added
to 20 ml of an aqueous soln. of Aloe barbadensis (Curacao aloes) (1: 200); a wine
red colour is produced, which is stable for at least 12 h. Solutions of Aloe capensis
fade rapidly to yellow.
3 Rhamni purshiani Cortex Not less than 8% hydroxyanthracene deriva-

Cascarae sagradae cortex tives, of which at least 60% are cascarosides
Cascara sagrada bark ca1c. as cascaroside A (Ph. Eur. II)
Sacred bark Cascarosides A and B (diastereoisomers of
Chittem bark aloin-8-0-ß-D-glucoside)
Rhamnus purshianus DE CANDOLLE Cascarosides C and D (diastereoisomers of
Rhamnaceae deoxyaloin-8-0-ß-D-glucoside)
USP XX, Ph. Eur. II, ÖAB
Aloin and deoxyaloin
97
Fig. DrugjPlant source Main constituents (anthraquinone deriva-
Family jPharmacopoeia tives) % content and identity
5,6 Frangulae Cortex Not less than 6% anthraquinone glycosides

Rhamni frangulae cortex (Ph. Eur. H)
Alder buckthorn bark The content of anthranol derivatives should
Rhamnus frangula L. not exceed 30% (Helv. VI)
Rhamnaceae Main glycosides: Glucofrangulin A and B
Ph. Eur. H, 2. AB-DDR, Helv. VI, (emodin -6-0-ex- L-rhamnosyl -8-0-ß- D-gluco-
ÖAB side and emodin-6-0-ex-L-apiosyl-8-0-ß-D-glu-
coside)
Frangulin A and B (emodin-6-0-ex-L-rhamno-
side and emodin-6-0-ex-L-apioside)
Mono- and diglucosides of emodin, glycosides
of physcione and chrysophanol, and free agly-
cones.
Frangulae Fructus Low concentrations of anthraquinone agly-

Alder buckthorn fruits cones and traces of anthraquinone glycosides
Rhamnus frangula L. are detectable.
Rhamnaceae
Oreoherzogiae Cortex 1-3% Hydroxyanthracene derivatives.

Rhamni faIIaci cortex Falax bark is an adulterant of alder buck-
FaIIax bark thorn bark.
Rhamnus faIIax (Boiss.) VENT.
Rhamnaceae
Rhamni cathartici Fructus Low contents of frangulin and glucofrangulin.

Buckthorn berries Higher concentrations of the flavonol glyco-
Rhamnus catharticus L. sides: catharticin (rhamnocitrin-3-0-ß-rham-
Rhamnaceae ninoside), xanthorhamin (= 7-methyl-querce-
tin; rhamninoside of rhamnetin) and rhamna-
zin (3,7' -dimethylquercetin), kaempferol-3-0-
ß-D-rhamninoside.
Remarks: Rhamni cathartici cortex contains naphthalide glycosides (ex-sorigenin glu-

coside and primveroside, and ß-sorigenin primveroside) and a low concentration
of anthraquinone derivatives; it may be found as an adulterant of Frangulae cortex.
7,8 Rhei Radix 3-7.5% Hydroxyanthracene derivatives

13,14 Rhubarb rhizome (DAB 8 specifies not less than 3% caIc. as
Rheum palmatum L. rhein)
Rheum officinale BAILLON (and hy- Main glycosides: mono- and di-glucosides of
brids) physcione, chrysophanol and rhein.
Polygonaceae Aglycones: rhein, physcione, chrysophanol,
DAB 8, ÖAB, Helv. VI, emodin.
2. AB-DDR Anthrones: rheidine, sennidine.
Adulterant: Rhei rhapontici radix (R. rhapon-
ticum L.), which contains the stilbene deriva-
tives, rhapontin (rhapontizin) and deoxyrha-
pontin.
98
Fig. DrugjPlant source Main constituents (anthraquinone deriva-
F arnily jPharrnacopoeia tives) % content and identity
9, 10 Sennae Folium Both drugs contain 2-3.5% dianthrone glyco-

11,12 Senna leaves sides.
Cassia senna L. (Alexandrian Sennae folium: Ph. Eur. I specifies not less
senna) than 2.5% ca1c. as sennoside B.
Cassia angustifolia VAHL (Tinne-
velly senna)
Sennae Fructus Sennae acutifoliae frnctus (Alexandrian senna
Senna pods pods): not less than 3.6% (Ph. Eur. I)
Cassia senna L. Sennae angustifoliae fructus (Tinnevelly senna
Cassia angustifolia V AHL pods): not less than 2.5% ca1c. as sennoside B
Caesalpiniaceae (Ph. Eur. I).
USP XX, Ph. Eur. I ÖAB, The chief compounds are the dianthrone gly-
He1v. VI, 2.AB-DDR cosides, sennoside A and B. Sennoside C and
D are also present,
Mono- and diglycosides of emodin and rhein.
Rhein is found especially in Sennae folium.
Of the flavonoids present in Sennae fructus
and folium, rntin can be detected only in Sen-
nae folium.
10 Hyperici Herba Main compounds:

St. Iohn's wort Dehydrodianthrone compounds (no purgative
Hypericum perforatum L. action): hypericin, isohypericin, protohyperi-
Hypericaceae cin.
DAC Flavonoids: rutin, hyperoside.
Chlorogenie acid.
99
VI. Formulae of Anthraquinone Derivatives Identitied as Drug Constituents
OH R, R2
CH 3 OH Rheum
(= Frangula)-Emodin
CH 3 OCH 3 Physcione
CH 3 H Chrysophanol
CH 2 0H H Aloe-Emodin
COOH H Rhein
RO o OH R R,
Glucosyl Rhamnosyl Glucofrangulin A
Glucosyl Apiosyl Glucofrangulin B
H Rhamnosyl Frangulin A
H Apiosyl Frangulin B
o
OH 0 OH OH 0 OH
R
CH20R H Aloin A, B
Rhamnosyl Aloinoside A, B
Aloin A Aloin B
Aloinoside A Aloinoside B
Gluc-O 0 OH
@R
R
CH 2 0H Cascaroside A, B
CH 3 Cascaroside C, 0
H Gluc
100
Gluc-O 0 OH
Sennoside A: R, R, = COOH( + )-form

R Sennoside B: R, R, = COOH meso-form
R, Sennoside C: R=COOH R, = CH 2 0H( + )-form
Sennoside D: R=COOH R, = CH 2 0H meso-form
Gluc-O 0 OH
HO
HO
HO 0 OH
Hypericin
HO OH
P-CH=CH-60CH,
R-O
R = GI ucosyl : Rhaponticoside
R= H: Rhapontigenin
101
Aloe Resina
Tracks 1 = Aloe capensis (Cape type A)
2=Aloe capensis (Cape type B)
3=Aloe barbadensis (Curacao aloes)
4 = Aloe perryi (Socotrine aloes)
5 = aloes (Kenyan drug)
6 = aloes (U gandan drug)
7 = aloes (Indian drug)
Test T = aloin (Rf 0.45-0.50), Aloe-emodin (solvent front)
Solvent AN-2: ethyl acetate-methanol-water (100:13.5:10)
system
Detection Potassium hydroxide (KOH No. 21, p. 302) UV-365 nm Fig. 1
Chromato- After treatment of chromatograms with KOH reagent, the aloe resins (1-7) are
gram seen to contain different amounts of aloin (Rf ca. 0.4). They can be distinguished
1 mainly by the presence or absence of aloinosides A and B (RfO.2-0.25) and aloesines.
Aloesine Amigrates directly above aloin, and aloesine B dose to and below the
aloinosides.
In UV -365 nm, aloin and aloinosides A and B show the typical yellow-orange
fluorescence of anthrones. The aloesines gives strong light blue fluorescent zones.
The red fluorescent zone of aloe-emodin lies at the solvent front.
The upper and lower Rf ranges contain further blue fluorescent zones, which
can be used for differentiation (e.g. RfO.7=p-Coumaric acid methylester).
2 NP/PEG reagent intensifies the yellow fluorescence of aloin and the aloinosides
in UV-365 nm. Under these conditions the aloesines fluoresce only slightly blue.
Differentiation of aloe resins
Aloe resin Aloin Aloinosides Aloesines AlB Rernarks
1 Cape aloes type A ++ ++! ++

2 Cape aloes type B ++ ++
3 Curacao aloes ++ ++ 1. 2 zone X
4 Socotrine aloes ++ ++! ++ 2 zone X
5 Kenya aloes ++ + (+)
6 Uganda aloes ++ (+)
7 Indian aloes (+ ) +++ (+)
1 Curacao and Cape aloes can be differentiated by the isoharhaloin test of Klunge (see p. 97),
in which they give a wine red or yellow colour, respectively.
2 Zone X appears as a dark zone in UV-365 nrn and as a red-brown zone in the visible,
direcdy below aloin (Curacao aloe: X = 5-Hydroxyaloin).
Aloinosides are present in Cape aloes type A, but not in Cape aloes type B.
Socotrine aloes show aloinosides and an additional dark brown zone (zone X) below
aloin.
In contrast to Socotrine aloes, Curacao aloes have no aloinosides.
Aloes 5-7 contain only traces of aloesines.
Indian aloes show only aloinosides, and no aloin.
102
FRONT
Rf
START
Fig.l
T 2 3 4 5 6 7
FRONT
Rf
START
Fig.2
T 2 3 4 5 6 7
103
Rhamni purshiani Cortex
Tracks 1-4 = Rhamni purshiani cortex (Cascarae cortex)
(commercial drugs from various sources)
Test Tl =aloin (Rf ca. 0.5), aloinoside A (Rf ca. 0.45)
Solvent AN-l: ethyl acetate-methanol-water (100: 17: 13)
system
Detection Natural products-polyethyleneglycol reagent
(NPjPEG No. 28, p. 303) UV-365 nm Fig.3

Chromato-
gram 1-4 Cascarae cortex. After treatment with NPjPEG reagent, chromatograms of Cascarae
3 cortex extract show two pairs of strikingly yellow fluorescent zones in UV-365 nm;
these are due to cascarosides A and B (Rf 0.10-0.15) and cascarosides C and D
(Rf 0.20-0.25). Weak yellow zones appear at Rf ca. 0.5 (aloin A, B), Rf ca. 0.6
(deoxyaloin). The predominant cascarosides are normally A and B.
The red fluorescent zones at the solvent front are due to the aglycones, emodin,
aloe-emodin and chrysophanol.
Additional blue and green-blue fluorescent zones are seen in the Rf range
0.35-0.75. All drug sampies show 4-5 blue fluorescent zones below the standard
aloin (presumably naphthalide derivatives).
TLC Synopsis
Tracks 5 = Aloe capensis
6 = Aloe barbadensis
7 = Rhamni purshiani cortex
8 = Frangulae cortex
Test T2 = aloin (Rf ca. 0.5), frangulin A (Rf ca. 0.85)
Solvent AN-l: ethyl acetate-methanol-water (100: 17: 13)
system
Detection Potassium hydroxide reagent (KOR No. 21, p. 302)
UV-365 nm Fig.4

Chromato-
gram 5-8 Chromatograms of the four official drugs, Cape aloes, curacao aloes, american and
4 official alder huckthorn hark, can be differentiated after treatment with KOR reagent
and inspection in UV -365 nm, by the presence of characteristic yellow or red fluores-
cent zones at defined Rf values.
Yellow fluorescent zones are due to the an throne glycosides, aloin and deoxyaloin,
and to aloinosides and cascarosides (see p. 102-104).
Red fluorescent zones are given by frangulins A and B, glucofrangulins A and B
and their aglycones (see p. 106).
Remarks: AN-l has a higher methanol and water content than AN-2, and it separates aloin
and aloinosides in a higher Rf range (cf. aloin Rf ca. 0.4, AN-2, Fig. 1, p. 102). The higher
methanol and water content often results in the formation of wavy zones (see (Fig. 3/4).
104
FRONT
Rf
START
Fig.3
Tl 2 3 4
FRONT
Rf
Fig.4
T2 5 6 7 8
105
Frangulae Cortex and aduIterant. Rhamni cath. Fructus
Tracks 1 = Frangulae cortex
2 = Oreoherzogiae cortex
3 = Frangulae fructus
4 = Rhamni cath. fructus
Tests Tl = glucofrangulins A and B (Rf ca. 0.25-0.3), aloin (Rf ca. 0.45),
frangulin A (Rf ca. 0.75), frangula-emodin (solvent front)
T2 = rutin (Rf ca. 0.4), chlorogenic acid (Rf ca. 0.45),
hyperoside (Rf ca. 0.6)
Solvent AN-2: ethyl acetate-methanol-water (100:13.5:10) Fig. 5A, B; 6A
system F-l: ethyl acetate-forrnic acid-glacial acetic
acid-water (100: 11 : 11: 27) Fig. 6B
Detection Potassium hydroxide reagent (KOH No. 21, p. 302)
vis./UV-365 nm Fig. 5A, B
(NP/PEG No. 28, p. 303) UV-365 nm Fig. 6A, B

Chromato-
gram 1 Frangulae cortex. The KOH-treated chromatogram is characterized by four red (vis.),
5A, B or red fluorescent (UV-365 nm) zones; these are Jrangulins A and B (Rf 0.75-0.85)
and glucoJrangulins A and B (Rf 0.25-0.35). The aglycones,frangula-emodin, physcion
and chrysophanol, are not separated and migrate with the solvent front.
Additional anthraquinones are detected above the glucofrangulin zones,
especially at Rf ca. 0.5 (emodin monoglycosides).
The yellow zone directly below the glucofrangulins is due to a flavonol glycoside
(see also Fig. 6A, UV-365 nm).
5A, B 2 Oreoherzogiae cortex. KOH treatment shows the red zones (vis. and UV-365 nm)
of the aglycones (solvent front), anthraquinone monoglycosides (Rf ca. 0.5) and digly-
cosides (Rf 0.2-0.35), and glucoJrangulins A and B (Rf ca. 0.25). Only traces of
frangulins are present. In contrast to official alder buckthorn bark, four characteris-
tic red zones of the glycosides of emodin and physcione are seen above the zone
of the glucofrangulins. As in Frangulae cortex, a flavonol glycoside is detectable
below the zone of the glucofrangulins.
5B 3 Frangulae Jructus. After KOH treatment the chromatogram shows only one red
zone (vis. and UV-365 nm) in the aglycone region.
6A/B After NP/PEG treatment, yellow-green fluorescent zones (UV-365 nm) are detect-
able at Rf 0.15 and 0.4-0.45, and at the solvent front. The lowest fluorescent zone
(see also solvent system F-l, Fig. 6B) is due to the flavonol glycoside, catharticin.
5B 4 Rhamni cathartici Jructus. After KOH treatment only glucoJrangulin A, Jrangulin A
and emodin are detectable (vis. and UV-365 nm) (cf. Tl).
6A NP/PEG reagent produces several yellow, and green-orange zones (UV-365 nm)
in the Rf ranges 0-0.25, and 0.7 to the solvent front, respectively (solvent system
AN-2).
6B Overlapping yellow and orange fluorescent zones are seen in the lower Rf range
(catharticin, "xanthorhamnin" and kaempJerol-3-0-rhamninoside). The yellow fluo-
rescent zone of catharticin is found in extracts of Frangulae and Rhamni cath.
fructus.
106
Fig.5
Tl 2 Tl 2 3 4
-FRONT
Rf
-0.5
-START
Fig.6
3 4 T2 4 3
107
Rhei Radix
Tracks 1 = Rhei rhapontici radix
2 = Rhei palmati radix
3 = Rhei radix hydrolysate (see p. 93)
Tests Tl = rhein
T2 = rhapontizin/deoxyrhapontizin mixture (Rf 0.45-0.55)
Solvent AN-2: ethyl acetate-methanol-water (100: 13.5: 10) Fig. 7 A, B; 8A, B
system AN-5: light petroleum-ethyl acetate-formic acid (75: 25: 1) Fig.8C
Detection Potassium hydroxide reagent (KOH No. 21, p. 302)
UV-365 nm/vis. Fig. 7 A, B; 8C
(NP/PEG No. 28, p. 303) UV-365 nm Fig.8A
Phosphomolybdic acid-H 2 S0 4 reagent (PMA No. 27, p. 303)
vis. Fig.8B
Chromato- 2 Rhei radix. After treatment with KOH, chromatograms of the official drug are
gram characterized by zones at Rf 0.45 and 0.55, which are orange-red in UV-365 nm
7 A, B a n d red in the visible. The lower zone is due to rhein (cf. Tl). The broad upper
zone represents a mixture of 8-0-monoglucosides of emodin, physcione and chryso-
phanol. The corresponding diglycosides are present in lower concentrations in the
Rf range 0.05-0.3. The aglycones mi grate as an orange-red zone at the solvent
front.
8A 1 Rhei rhapontici radix (adulterant). The pattern of anthraquinones corresponds closely
to that of the official Rheum drug.
In additon, especially after NP/PEG treatment, the intermediate Rf range con-
tains a broad light blue fluorescent zone (UV-365 nm) between rhein and the mono-
glycoside mixture; this zone contains the stilbene derivatives, rhaponticin (rhaponti-
coside) and deoxyrhaponticin.
8B With PMA-H2 S0 4 reagent, the stilbene glycosides give powerfully dark blue zones
in the visible.
8C 3 Rhei radix (aglycones). Hydrochloric acid hydrolysis (see p. 93) of Rheum extract
pro duces a mixture of aglycones. Aloe-emodin and rhein migrate at Rf 0.2-0.25,
emodin at Rf ca. 0.35, chrysophanol and physcione at Rf 0.6-0.7.
In UV 365 nm, all the aglycones fluoresce uniformly yellow, or orange-red if
the chromatogram is treated with KOH (no Fig.).
If the TLC plate is heated after KOH treatment (Fig. 8C), frangula(rheum)-
emodin appears as a dark zone in UV 365 nm.
Remarks: see also Circular TLC Fig. 13/14, p. 114.
108
-FRONT
Rf
-0.5
-START
Fig.7
Tl T2 2 Tl T2 2
-FRONT
Rf
Fig.8
Tl T2 2 2 T2 3
109
Dianthrones Dehydrodianthrones
Sennae Folium, Fructus Hyperici Herba
Tracks 1 = Sennae folium 3 = Hyperici herba
2 = Sennae fructus
Tests T1 = rhein TA = rutin (Rf ca. 0.4), chlorogenie acid
T2 = sennoside A (Rf ca. 0.45), hyperoside (Rf ca. 0.6)
T3 = sennoside B
Solvent AN-3: n-propanol-ethyl acetate-water (40:40:30) Fig. 9A, B
system AN-4: toluene-ethyl formate-formic acid (50:40:10) Fig.10C
F-l: ethyl acetate-formic acid-glacial acetic acid-water Fig. lOA, B
(100: 11: 11 :27)
Detection Nitric acid-KOH reagent (No. 33, p. 303) vis./UV-365 nm Fig. 9A, B
(NP/PEG No. 28, p. 303) UV-365 nm Fig. lOA, B
Pyridine (10% in ethanol) UV-365 nm Fig. lOC
Chromato- 1 Sennaefolium. After treatment with HN0 3 /KOH reagent, 7-9 red-brown (vis.) zones
gram are seen between the start and the solvent front. In UV-365 nm the same zones
9A, B fluoresce lemon yellow or light blue. The main zones are due to sennoside A (Rf
0.4, cf. T2) and sennoside B (Rf 0.2, cf. T3). Sennoside Dappears at Rf 0.5--0.55,
and directly above the rhein-8-0-glueoside is found. Sennoside Cis seen as a weakly
coloured zone at Rf ca. 0.7, and rhein as an orange-red zone (cf. Tl) at Rf ca.
0.8. The other aglycones migrate together at the solvent front.
10A NP/PEG reagent (solvent system Fl) reveals at least 8 yellow to yellow-green fluores-
cent (UV-365 nm) jlavonoid zones. The main zone corresponds to rutin (cf. TA).
Two further zones are found above and below rutin, two weak zones above the
hyperoside standard (cf. TA, Rf ca. 0.6), and a zone of flavonoid aglycones that
mi grates with the solvent front.
9A, B 2 Sennae fruetus. Compared with Sennae folium, the concentrations of sennosides A,
B, C, D and rhein are somewhat lower.
10A The jlavonoid pattern shows two main zones below rutin, two zones above hyperoside
and a zone at the solvent front. Rutin is not present.
10C 3 Hyperici herba. In solvent system AN-4, the hypericins mi grate in the Rf range
0.45-0.5. In UV 365 nm they appear as reddish zones (untreated chromatograms)
or orange-red zones (after pyridine treatment).
lOB Chromatography in solvent system Fl, followed by NP/PEG treatment, shows the
flavonoids, rutin and hyperoside, at Rf 0.35 and 0.6 (cf. TA), other weaker flavonoid
zones at Rf ca. 0.7-0.8, and aglycones on the solvent front. Chlorogenie and neo-
ehlorogenie acid (Rf 0.45-0.55, cf. TA) give a light blue fluorescence, and the hyperi-
eins (Rf 0.9) show a red fluorescence.
110
FRONT
Rf
START
Fig.9
Tl T2 TJ 2 Tl T2 TJ 2
FRONT
Rf
START
Fig. 10
TA 2 TA 3 3
111
Sennae Folium, Fructus Circular TLC
Segment Sennae folium (upper segment)
Sennae fructus (lower segment)
sennoside-A=Test 1 +rhein=Test 4
sennoside B = Test 2 + aloin = Test 5
Solvent n-propanol-ethyl acetate-water (40: 40: 30)
system
Detection HN0 3 -KOH reagent (No. 33, p. 303) vis. Fig.11
UV -365 nm Fig. 12

For description of circular TLC, see p. 95.
Chromato- Sennae folium. The two yellow (vis.) zones of sennoside B (Test 2) and sennoside A
gram (Test 1) have intermediate Rf values. Above these lie the weak red-violet zone of
11 rhein-8-0-monoglucoside and the yellow zones of sennosides D and C on either side
of the monoglucoside zone (cf. 3).
Rhein is overlapped by yellow zones (Test 4). Weak zones of anthraquinone agly-
cones and flavonoids are found at the solvent front, just ahead of the aloin standard
(cf. 5).
Sennae fructus. As in Sennae folium, sennosides Band A (cf. Tests 2 and 1) are
followed by the red zone of rhein-8-0-glucoside and rhein.
12 In UV-365 nm sennoside B appears in both drug preparations in approximately the
same concentration [yellow fluorescent zone (cf. standard 2)]. Sennoside A is more
concentrated in Sennae folium than in Sennae fructus.
Rhein-8-0-monoglucoside and rhein are more distinct in the visible than in UV-
365 nm, where they show only a weak orange-red fluorescence above Test 4.
Sennae folium can be clearly distinguished from Sennae fructus by the presence
of a pink fluorescent zone just above the aloin standard (not present in the extract).
112
.... SENNAE FOLIUM
ENNAE FRUCTUS
Fig.11
.... SENNAE FOLIUM
.... SENNAE FRUCTUS
Fig.12
113
Rhei Radix Comparison of Circular TLC and Ascending TLC
Tracks 1= Rhei rhapontici radix
2 = Rhei palmati radix
Tests 3 = rhaponticin/deoxyrhaponticin mixture
4= rhein
Solvent
system AN-2: ethyl acetate-methanol-water (100: 13.5: 10)
Detection Potassium hydroxide reagent (KOR No. 21, p. 302) vis. Fig. 13A, B
UV-365 nm Fig. 14A, B
Chromato- Rhei radix

gram Ascending TLC
1,2 The two red (vis.) or red-orange fluorescent (UV-365 nm) anthraquinone zones are
due to rhein (Rf ca. 0.5) and to a mixture of 8-0-monoglucosides of emodin, physcione
and chrysophanol (Rf ca. 0.6).
(For further data, see Fig. 7/8, p. 108)
Circular TLC
With a development distance of 5-6 cm, the following zones can be identified in
the visible:
- aglycone mixture (rheum-emodin, physcione, chrysophanol) as a red zone on the
periphery of the chromatogram. Then moving inwards towards the centre:
- a weak orange zone
- the main orange zone (mixture of the monoglucosides of the three aglycones)
- a weak zone of rhein
In UV-365 nm:
- a pronounced orange fluorescent zone on the periphery (aglycone mixture). Then
moving inwards towards the centre:
- a dark zone between two blue fluorescent zones
- the orange zone of the monoglucoside mixture
- rhein, partly overlapped by blue fluorescent zones.
In Rhei rhapontici radix, the strikingly light blue fluorescent zone of rhaponticin
(rhaponticoside) is easily recognisable (cf. T3).
114
2 FRONT
Rf
3 4
Fig.13 START
c-oc 2
FRONT
Rf
START
Fig.14
c- OC 2
115
Arbutin Drugs
The main constituents ofthe arbutin drugs are arbutin (hydroquinone-ß-O-glucoside)

and methylarbutin, with small amounts of 2-0-galloyl-arbutin, 6-0-acetyl-arbutin
and free hydroquinone. The presence of gallo- and ellagotannins is also characteristic
of these drugs.
I. Preparation of Extracts of Drugs and Pharmaceutical

Preparations for TLC
1. Drug extracts (for the detection of arbutin)

The powdered drug (2 g) and calcium carbonate (0.2 g) are mixed, then extracted
for 15 min under reflux (water bath) with 20 ml of 50% methanol. The mixture
is filtered and the residue washed with 50% methanol to give a total filtrate volume
of 20 ml. To remove tannins, 10 ml of this solution are treated with 0.5 g basic
lead acetate, shaken, then filtered. The pale yellow filtrate (30 ~l) is applied direct1y
to the chromatogram.
2. Extraction of bearberry leaves according to DAß 8

The powdered drug (5 g), is extracted with 75% methanol (50 ml) under reflux
for 30 min, then filtered. The filtrate is evaporated to about 12 ml and transferred
to a separating funnel together with 50 ml water. This solution is extracted twice
with 30 ml ether and the ether extracts are discarded. It is then extracted three
times with 50 ml ethyl acetate, the combined ethyl acetate extracts evaporated to
dryness and the residue dissolved in 10 ml methanol: 20 ~l are applied to the chro-
matogram. This extract is suitable for the TLC investigation of phenol glucosides
and flavonoids.
3. Extract of Viburni prunifolii and V. opuli cortex

To remove interfering resins and lipids, the powdered drug (2 g) is extracted under
reflux for about 15 min with 50 ml light petroleum. The drug residue is extracted
by boiling for about 20 min with 20 ml methanol, then filtered. The filtrate is evapor-
ated to about 1 ml, and 50 ~l are applied to the chromatogram.
Remarks: Turbidity or sediment which may appear in the concentrated filtrate, can be ignored.
4. Extracts of pharmaceutical preparations

Five powdered tablets or dragees are mixed with calcium carbonate (0.1 g) and
extracted with 25 ml of 50% methanol for about 10 min on a water bath. To the
first 10 ml of filtrate are added 200 mg basic lead acetate. This is again filtered
and the filtrate is used direct1y for TLC.
117
Tl: 25 mg arbutin and 25 mg hydroquinone are dissolved in 10 ml of 50% methanol,
and 10 111 of this soln. are applied to the chromatogram.
T2: 25 mg hydroquinone, 25 mg gallic acid and 25 mg arbutin are dissolved in
10 ml methanol, and 10 111 of this soln. are applied to the chromatogram (DAB 8).
Scopoletin, amentoflavone and catechin/epicatechin are prepared separately as 0.1 %
methanolic solns., and 10111 of each are applied for chromatography.
2. Adsorbent
Silica gel 60F 254 pre-coated TLC plates (Merck, Darmstadt).
AN 2: ethyl acetate-methanol-water (100: 13.5: 10)
DAß 8: ethyl acetate-formic acid-water (88:6:6)
F-3: chloroform-acetone-formic acid (75: 16.5: 8.5)
111. Detection

Arbutin and other phenolic compounds show a pronounced quenching of fluores-
cence in UV -254 nm.
2. Spray reagents
a) Berlin blue reaction (BB No. 5, p. 299)
Arbutin, methylarbutin, hydroquinone and methylhydroquinone appear as blue
zones (vis.).
b) Millons reagent (ML No. 26, p. 303)
Arbutin, methylarbutin, hydroquinone and methylhydroquinone appear as yellow
zones (vis.).
c) ReagentsJrom DAB 8
a) Jor arbutin and hydroquinones: 1% 2,6-dichloroquinonechloroimide in methanol
(DCC No. 8, p. 300). The chromatogram is sprayed with the reagent then exposed
to ammonia vapour. Arbutin appears as a violet (vis.) zone.
b) flavonoids: saturated methanolic aluminium chloride shows flavonoids as orange
or blue fluorescent zones in UV-365 nm.
d) Natural products-polyethyleneglycol reagent (NP/PEG No. 28, p. 303)
Intense orange or blue fluorescent zones are seen in UV-365 nm. Amentoflavone
gives a yellow-brown fluorescence.
e) Potassium hydroxide reagent (KOH No. 21, p. 302)
After spraying with 5% ethanolic KOH, arnentoflavone gives a yellow-green fluorescence,
and cournarins and phenol carboxylic acids give a light blue fluorescence in UV-365 nrn.
J) Fast blue salt reagent (FBS/KOH No. 12, p. 301)
Phenolic compounds appear as red-brown zones (vis.).
118
IV. Important Arbutin Drugs
Drug Plant of origin Total Pharmaco-
Family hydroquinone poeia
content
Figs. 1,2 Uvae ursi folium Arctostaphylos 4-12% DAB 8 (not

Bearberry leaves uva-ursi (L.) SPRENGEL less than
Ericaceae 6%), ÖAB,
Helv. VI.
Vitis idaeae folium Vaccinium vitis idaea L. 5.5-7%
Cowberry leaves Ericaceae
Myrtilli folium Vaccinium myrtillus L. 0.4-1.5%
Bilberry leaves Ericaceae
Ericae (Callunae) herba Calluna vulgaris (L.) HULL. 0.6-0.68%
Heather Ericaceae
Pyri communis folium Pyrus communis L. up to 4.7%
Pear leaves Rosaceae (young leaves)
Figs. 3, 4 Viburni prunifolii cortex Viburnum prunifolium L. 0.5%
Black haw bark Caprifoliaceae
Bergeniae crassifoliae folium Bergenia crassifolia ca. 12%
Bergenia (L.) FRITSCH (tannins
Saxifragaceae 15-25% !)
Remarks: Other constituents such as flavonoids or coumarins can also be used

for the identification of arbutin drugs:
Uvae ursi folium contains the flavonoids, quercitrin, isoquercitrin, myricitrin and
quercetin-3-ß-D-6-0-galloyl-galactoside.
Viburni prunifoli cortex is characterized by the biflavone, amentoflavone (see p. 170)
and the coumarins, scopoletin and scopoline.
~ OH
Hydroquinone
~
Arbutin
O-Gluc
R=H
Methylarbutin: R = eH a
119
Arbutin Drugs
Tracks 1 = Uvae ursi folium
2 = Vitis ideae folium
3 = Myrtilli folium
Tests Tl = arbutin (Rf ca. 0.4), hydroquinone (Rf ca. 0.95)
T2 = arbutin (Rf ca. 0.25), gallic acid + hydroquinone (Rf 0.9-0.95)
Solvent AN-2: ethyl acetate-methanol-water (100: 13.5: 10) Fig. 1
system DAB 8: ethyl acetate-forrnic acid-water (88: 6: 6) Fig. 2
Detection Berlin blue reagent (BB No. 5, p. 299) VIS. Fig.1A
Millons reagent (ML No. 26, p. 303) vis. Fig. 1B
DichlorpquinonechioroimidejNH 3 reagent (DCC No. 8, p. 300) VIS. Fig.2A
(NPjPEG No. 28, p. 303) UV-365 nm Fig.2B

Chromato-
gram 1-3 Chromatograms of Uvae ursi folium and Vitis ideae folium show a distinct blue
1A, B (BB) or yellow (ML) zone (Rf ca. 0.4) of arbutin. Myrtilli folium gives onIy a very
weak zone of arbutin; above and below this zone are several weak blue (BB) or
yellow (ML) zones. All three extracts contain only traces of free hydroquinone (Rf
ca. 0.95). BIue zones (BB) at and directly above the start are due to tannins.
2A 1 After treatment with DCC reagent, chromatograms of Uvae ursi folium show a
strong vioiet (vis.) zone (Rf ca. 0.25; cf. T2) of arbutin. Six further zones (flavones
and acids) are seen between arbutin and Rf ca. 0.75, and three brown-violet zones
(gallic acid and hydroquinone; cf. T2) are present below the solvent front.
2B Uvae ursi folium shows four orange fluorescent (NP/PEG reagent; UV-365 nm)
zones of flavone glycosides in the Rf range 0.2-0.4: quercetin-3-ß-D-6-0-galloyl-
galactoside, isoquercitrin, quercitrin and myricitrin. Pale blue fluorescent zones in
the Rf range 0.5-0.8 are typical, e.g. of cinnamic acid derivatives. Gallic acid gives
an intense blue fluorescence at Rf ca. 0.9 (cf. T3).
Remarks: Arbutin does not fluoresce in UV-365 nm.
120
Fig.l
Tl 2 3 Tl 2 3
FRONT
Rf
START
Fig.2
T2 T2
DAS 8
121
Vihurni Cortex
Tracks 1 = Viburni prunifolii cortex
2 = Viburni opuli cortex
Tests Tl = scopoletin
T2 = amentoflavone
T3 = catechin/epicatechin mixture
Solvent F-3: chloroform-acetone-formic acid (75: 16.5: 8.5)
system
Detection Potassium hydroxide reagent (KOH No. 21, p. 302) UV-365 nm Fig.3A
Fast blue salt reagent (FBS No. 12; followed by KOH, p. 301) VlS. Fig.3B
For description of drugs see p. 119. Formulae p. 150 and 170.
Chromato- Viburni prunifolii cortex and Viburni opuli cortex. After KOH treatment both extracts
gram show at least seven blue fluorescent zones (UV -365 nm) between the start and Rf
3A ca. 0.7 and a blue or orange fluorescent zone at the solvent front.
1 Viburni prunifolii cortex is characterized by the prominent main zones (light blue
fluorescence) of the coumarins, scopoletin (cf. Tl) and scopoline (near the start),
and by the presence of the biflavone, amentojlavone (cf. T2; green fluorescence
4 with KOH, yellow-green fluorescence with NP/PEG). Other blue fluorescent zones
are due to plant acids, e.g. caffeic acid.
2 Viburni opuli cortex shows blue fluorescent zones in more or less the same Rf regions
as in V. prunifolium; scopoletin is less pronounced and amentoflavone is completely
absent.
3B The two extracts can also be differentiated by treatment of chromatograms with
FBS-KOH. This reveals the catechin/epicatechin mixture, which is present only in
V. opulus, as a brown-red zone (vis.) at RfO.l-0.2 (cfT3).
Remarks: Arbutin (ca. 0.5%) is not detectable under the above conditions. Extracts must
be specially concentrated prior to chromatography for the detection of arbutin.
122
Fig.3
Tl T2 2 T3 T2 2
RI
5
Fig.4
Tl T2 2
123
Bitter Principle Drugs
Most ofthe bitter principles in important official drugs possess a terpenoid structure,
representing derivatives of mono terpenes (secoiridoids), sesquiterpenes, diterpenes
and triterpenes.
Powdered drug (1 g) is extracted for 10 min with 10 ml methanol at 60° C on the

water bath. The mixture is filtered and the filtrate is evaporated to a volume of
ca. 2 ml.
Exceptions:
1. Humuli lupuli strobulus
a) The drug (1 g) is extracted for 24 h with 15 ml of cold ether. The filtrate is
allowed to stand for 12 hin the refrigerator, precipitated waxy materials are removed
by filtration, and the filtrate is evaporated to dryness at room temperature. The
residue is dissolved in 1 ml methanol, and this soln. is used for chromatography.
b) The drug (1 g) is extracted for 3 h at room temperature with 10 ml dichlorometh-
ane. The filtrate is evaporated to about 3 ml, and used for chromatography.
2. Salviae folium and Rosmarini folium

Coarsely powdered leaves (3 g) are extracted under reflux (water bath) for 1 h with
100 ml ether. The filtrate is evaporated to about 3 ml, and this soln. is used for
chromatography.
All standard substances are prepared as 1% methanolic solutions.
2. Adsorbent
Drug extracts 40 111
Reference solutions 20 111
125
Solvent system For detection of:
B-1 ethyl acetate-methanol-water (77: 15: 8) "Screening system"

B-2 acetone-chloroform-water (70: 30: 2) amarogentin/Gentianae radix
B-3 chloroform-methanol (95: 5) absinthin/ Absinthii herba
quassin/Quassiae lignum
B-4 chloroform-methanol (97: 3) carnosolic acid, carnosol/
Rosmarini and Salviae folium
B-5 acetone-chloroform (30: 40) cnicin/Cardui benedicti herba
B-6 n-propanol-toluene-glacial acetic acid-water aucubine/Plantaginis herba
(25 :20: 10: 10)
B-7 ethyl acetae-dioxan-water (30: 10: 0.3) oleuropein/Oleae folium
B-8 iso-octane-iso-propanol-formic acid }
(83.5: 16.5 :0.5) humulone and lupulone (bitter
B-9 n-heptane-iso-propanol-formic acid acids)/Humulus lupulus
(90: 15:0.5)
B-10 chloroform-ethanol (95: 5) cucurbitacins/Bryoniae radix
111. Detection

UV-254 nm
Substances with conjugated double bond systems show fluorescence quenching (e.g.
quassin).
UV-365 nm
Mostly unspecific fluorescence, with the exception of e.g. the flavonoid compounds
in Aurantii pericarpium.
2. Spray reagents
a) Vanillin-sulphuric acid (VS No. 38, p. 304)
Visualization after ca. 10 min at 100 0 C:
neo hesperidin, naringin, harpagoside red-violet
gentiopicroside, swertiamarin brown-red
condurangins blue-green
foliamenthin, menthiafolin, quassin blue
marrubiin, absinthin, cnicin blue
b) Anisaldehyde-sulphuric acid (AS No. 2, p. 299)
Similar colours (vis.) to those obtained with VS re agent.
c) Liebermann-Burchard reagent (LB No. 16, p. 301)
The TLC plate is sprayed with freshly prepared soln., heated for 10 min at 100 0 C,
and inspected in UV-365 nm or vis.
Absinthin: sand colour in UV-365 nm; dark brown in vis.
Cnicin: light grey in UV-365 nm; weak grey in vis.
126
d) Fast red salt B (FRS No. 13, p. 301)
For reducing substances and phenolic compounds, chromatograms are inspected
in visible light
Amarogentin: orange
Gentiopicroside: red
e) Benzidine reagent (BZ No. 4, p. 299)
Aucubine: brown-black (vis.)
f) FeCl 3 (10% FeCl 3 soin. No. 14, p. 301)
The TLC plate is inspected immediately after spraying. Oleuropein, carnosolic acid
(carnosol) and hop bitter principles show yellow-brown to yellow-green (vis.).
g) Vanillin-phosphoric acid reagent (VPA No. 36B, p. 304)
Cucurbitacins: blue or red-violet (vis.); bIue, pink, yellow and green fluorescence
in UV-365 nm.
h) Natural products-polyethyleneglycol-reagent (NPjPEG No. 28, p. 303)
Dicaffeoylquinic acids (e.g. cynarinjCynarae herba) show intense blue to bIue-green
fluorescence in UV-365 nm.
IV. List of Bitter Principle Drugs
Chromatograms (Figs. 1-12) are reproduced on pp. 13i-143.
Fig. DrugjPlant source Main constituents

Family jPharmacopoeia Bitterness index (BI)
Drugs containing terpenoid bitter principles

1. Monoterpenes (C-10)
1,2 Centaurii Herba Secoiridoid bitter principles:
Centaury swertiamarin, gentiopicroside (= gentiopic-
Centaurium minus MOENCH rin); traces of amarogentin.
Gentianaceae BI plant: at least 2000 (10,000 Helv. VI)
DAB 8, Helv. VI, ÖAB BI flowers: ca. 12,000
Gentianae Radix Secoiridoid bitter principles:
Gentian root gentiopicrin (2-3.5%; BI 12,000)
Gentiana lutea L. amarogentin (0.05%; BI 58 x 10 6 ).
Gentianaceae Trisaccharide bitter principle:
Ph. Eur. 1,2. AB-DDR, gentianose (BI 120).
Helv. VI, ÖAB BI ofthe drug 10,000-30,000.
Harpagophyti Radix Iridoid bitter principles:
Grapple plant root harpagoside, isoharpagoside, procumbid.
Grapple plant BI of the drug 600-2,000.
Harpagophytum procumbens
(BRUCH) DC ex MEISSEN
Pedaliaceae
Scrophulariae Herba Substitute drug for Harpagophyti radix with
Figwort the same qualitative composition of bitter
Scrophularia nodosa L. principles, but about half the content of har-
Scrophulariaceae pagoside.
127
Fig. Drug/Plant source Main constituents
F amily /Pharmacopoeia Bitterness index (BI)
Menyanthidis Folium Secoiridoid bitter principles:

Trifolii fibrini folium foliamenthin, menthiafolin, T,8'-dihydrofo-
Buckbean leaf liamenthin and sweroside.
Menyanthes trifoliata L. Verbenalin type bitter principle:
Menyanthaceae loganin.
Helv. VI, ÖAB Monoterpene alkaloids:
gentianin, gentianidine.
BI of the drug 4,000-10,000
6 Oleae Folium Iridoid bitter principles:

Olive leaf gentianin type: ca. 0.75% oleuropein in fresh
Olea europea L. ssp. leaves. On storage, this is c1eaved to 3,4-di-
silvestris hydroxyphenylethyl a1cohol and a dicarb-
oxylic acid methyl ester.
Oleaceae
Olives contain up to 2% oleuropein.
5 Plantaginis Folium Iridoid bitter principle:

Ribwort leaf aucubine (1.9-2.4%)
Plantago lanceolata L. Remarks: aucubin is also present in Aucuba
Plantaginaceae japonica and Vitex agnus castus.
Helv. VI, ÖAB (Folium),
2. AB-DDR (Herba)
2. Sesquiterpenes (C-15)
3 Cardui benedicti Herba Germacran type bitter principles (ca. 0.25%),
Cnici herba with cnicin, an unsaturated sesquiterpene di-
Blessed thistle hydrolactone, and artemisiifolin (a sesquiter-
Cnicus benedictus L. pene lactone).
Asteraceae BI ofthe drug 800-1,800.
DAC, Helv. VI, ÖAB
Absinthii Herba Bitter principle content ca. 0.3% in leaves,
Wormwood 0.15% in flowers.
Artemisia absinthium L. Sesquiterpene lactones: absinthin (ca. 0.2%)
Asteraceae and its isomer, anabsinthin.
DAB 8,2. AB-DDR, Helv. VI, Artabsin can be detected only in freshly har-
ÖAB vested plants.
BI of the drug 10,000-25,000
BI of absinthin ca. 12,700,000.
3. Diterpenes (C-20)
4 Marrubii Herba Bitter principle content 0.3-1 %. Marrubiin
White Horehound (BI 65,000) is the main constituent. Marru-
Marrubium vulgare L. biol, marrubenol and its half acetal are not
Lamiaceae bitter.
ÖAB
11 Salviae Folium The bitter principle of both drugs is picrosal-
Sage leaves vin (=carnosol). During extraction picrosal-
Rosmarini Folium vin is converted into the non-bitter, opened
Rosemary leaves ring form, carnosolic acid.
For further details see section on
Essential oil drugs, pp. 11 and 13.
128
Family/Pharmacopoeia Bitterness index (BI)
4. Triterpenes (C-30)
7 Bryoniae Radix Tetracyclic triterpene bitter principles: Cucur-
Bryonyroot bitacins B, D, E, I, J, K, Land dihydrocucurbi-
Bryonia alba L. and Bryonia cre- tacins E and B possess the same ring system,
tica ssp. dioica PLANCH. but differ with respect to substitution in
ring A.
Cucurbitaceae
Bryonia alba and dioica show qualitatively
similar contents of bitter principles, but the
content of B. dioica is about 10 times higher.
8 Cucurbitae pepo Semen Main compounds are sterols besides amines

Melon pumpkin seeds e.g. cucurbitine.
Cucurbita pepo L.
Cucurbitaceae
3 Quassiae Lignum Secotriterpenes ca. 0.25%:

Quassia wood quassin (0.1-0.15%), neoquassin and 18-hy-
Quassia amara L. droxy-quassin.
Picrasma excelsa PLANCH. BI of the drug 40,000-50,000
Simarubaceae BI of quassin/neoquassin 17 x 10 6 •
5. Pregnane type (steroids)

Condurango Cortex Bitter principles ca. 1-2%.
Condurango bark Condurangins are ester glycosides of a pre-
Marsdenia condurango gnane derivative of the digitanol group. Con-
REICHB. duragenin A is linked to a pentasaccharide,
Asc1epidiaceae and esterified with cinnamic and acetic acid.
Helv. VI, ÖAB BI of the drug ca. 15,000.
Drugs containing non-terpenoid bitter principles

Aurantü Pericarpium Bitter principles:
Seville orange peel FIavanone glycosides, neo hesperidin and nar-
Citrus aurantium L. ssp. aurantium ingin.
Rutaceae The triterpene bitter principle, limonin (BI
DAB 8, Helv. VI, ÖAB 10 6 ) is found chiefly in the seeds. In the fresh
(see sections on flavonoids, p. 169 plant it is present in a non-bitter form.
and essential oils, p. 15) BI of the flavanone glycosides ca. 500,000
BI ofthe drug 600-1,500.
9, 10 HlUßuli lupuli Strobuli Bitter principles:

Hops Chief compounds are the prenylated phloro-
Humulus lupulus L. glucinol derivatives, iso-humulone and lupu-
Moraceae lone. These are very unstable, and are trans-
formed into the so-called bitter acids.
ÖAB
12 Cynarae Herba Sesquiterpene lactones:

Artichoke e.g. cynaropicrin, grossheimin
Cynara scolymus L. Phenol carboxylic acids:
Asteraceae e.g. 1,3-dicaffeoylquinic acid (cynarin) and
chlorogenic acid.
129
v. Formulae of Constituents of Bitter Principle Drugs
MONOTERPENES
~o
o
~ -....;::
IH o OH
O-Gluc
Gentiopicrin Amarogenti n: R, = H, R2 = OH Gentianin

(= Gentiopicroside) Amaroswerin: R, = OH, R2 = OH
Amaropanin: R,=H, R2 =H
R R R
c R-0;i5
CO
~CH~ ~
f;l
:"
0
: CH,OH
OH IA :
O-Gluc
Foliamenthin Dihydrofolia- Menthiafolin
menthin
HO
HO
~oy- _
0
15
~
0
-. .;:
0
OC H3
O-Gluc
R = OH Procumbid Oleuropein (= Oleuropaeoside) Aucubin

R= H Harpagid
130
SESQUITERPENES
Cnicin
~ o
Artabsin Absinthin
DITERPENES
OH
Carnosol Marrubiin
(Picrosalvin)
TRITERPENES
OR
HO
o
Quassin Cucurbitacin E: R=COCH 3
Cucurbitacin I: R = H
OTHER BITTER PRINCIPLES
Humulone
Condurangin A R, = Cinnamoyl
Neohesperidose R2 = Pentaglycosyl
Naringin: R, R, = H Limonin
Neohesperidin: R=CH 3 ; R,=OH
131
Drugs with Bitter Principles
TLC Synopsis
Tracks 1 = Aurantii pericarpium
2= Harpagophyti radix
3= Gentianae radix
4= Centaurii herba
5= Condurango cortex
6= Menyanthidis folium
Test T = neo hesperidin
Solvent B-l: ethyl acetate-methanol-water (77: 15: 8) Fig.l
system F-l: ethyl acetate-formic acid-glacial acetic acid-water (100: 11: 11 :26) Fig.2
Detection Vanillin-sulphuric acid reagent (VS No. 38, p. 304) vis. Fig. 1
Anisaldehyde-sulphuric acid re agent (AS No. 2, p. 299) vis. Fig.2
For description of drugs see p. 127. Formulae p. 130-131
Chromato-
gram 1 Aurantii pericarpium. The TLC shows two characteristic intense zones, which are
1,2 red-orange (VS) or brown-violet (AS), in the Rfrange 0045-0.5 (naringin and neohe-
speridin).
The non-bitter flavonoids, rutin, eriocitrin and chlorogenie acid, can be located
with the NP/PEG re agent (see Flavonoids, p. 189, Fig. 17 AlB), which produces fluo-
rescent zones (orange, violet-red and blue-green, respective1y) in UV-365 nm.
2 Harpagophyti radix is characterized by two prominent violet zones at Rf ca. 0.5
and 0.1-0.2 (harpagoside and procumbid or harpagid).
Remarks: Scrophulariae herba, which contains about 50% less harpagoside, counts as an
acceptable substitute.
3 Gentianae radix shows gentiopicrin (= gentiopicroside) as a brown-violet main zone
at Rf ca. 0045 (B-l) or 0.35 (F-l). Immediately below there is a weak brown zone
due to swertiamarin, the main constituent of Centaurii herba (cf. 4).
Amarogentin, which quenches fluorescence in UV-254 nm (Rf 0.8), can be better
located in the visible with the fast red reagent (see p. 136 Fig. 5 A).
4 Centaurii herba is characterized by 4 yellow or orange-brown main zones in the
Rf range 0.25-0.5 (B-1) or 0.2-004 (F-l). The main zone at Rf ca. 004 (B-1) or
0.3 (F-1) is due to swertiamarin. The weak brown zone directly above is due to
gentiopicrin (cf. 3, Gentianae radix).
5 Condurango cortex shows several intense, black-green zones in the Rfrange 0045-0.5,
due to condurangins.
6 Menyanthidis folium. Treatment with VS re agent produces one typical dark blue
zone at Rf ca. 0.6, and two in the Rf range 0.8-0.85 (B-1). These correspond to
foliamenthin, menthiafolin and dehydrofoliamenthin. With AS reagent and solvent
system F -1, these zones are not c1early defined.
Remarks: Chromatograms 1-6 show dark blue zones of essential oils and aromatic acids
below the solvent front. The black or brown zones at and above the start originate partly
from sugars.
132
FRONT
Rf
-0.5
-START
Fig.l
T 2 3 4 5 6
FRONT
Rf
-0.5
-START
Fig.2
T 2 3 4 5 6
133
Cnici, Absinthi, Marrubii Herba Quassiae Lignum
Tracks 1 = Gnici herba
2 = Absinthii herba
3 = Quassiae lignum
4 = Marrubii herba
Tests Tl =cnicin
T2 = absinthin (anabsinthin)
T3=quassin
T4 = marrubiin
Solvent B-3: chloroform-methanol (95: 5) Fig. 3A, B; 4C
system B-5: acetone-chloroform (30: 40) Fig. 4A, B
Detection Liebermann-Burchard reagent (LB No. 16, p. 301) UV 365 nm Fig. 3A; 4A
Vanillin-sulphuric acid reagent (VS No. 38, p. 304) vis. Fig. 3B; 4B, C
For description of drugs see p. 128-129·. Formulae p. 131
Chromato-
gram 1 Cnici herba. Treatment with LB reagent, followed by observation in UV-365 nm,
3A reveals at least 14 fluorescent zones (mainly light blue, red-brown or yellow green)
between the start and the solvent front. In UV -365 nm cnicin shows yellow-green
fluorescence (Rf ca. 0.05).
4A, B In solvent system B-5 cnicin migrates at Rf ca. 0.4 (cf. Tl). LB and VS reagents
give only weak, grey-violet colours in the visible.
3A 2 Absinthii herba. LB reagent reveals two main zones with intense ochre fluorescence
in UV-365 nm (absinthinfanabsinthin, Rf 0.3-0.4), together with at least ten other
mainly blue or green fluorescent zones. After VS reagent, absinthin and anabsinthin
appear as dark blue and violet zones (vis.), respectively (cf. T2-3, Fig. 4B).
Remarks: Treatment with 50% sulphuric acid also produces ochre fluorescent zones (in UV-
365 nm) of absinthin and anabsinthin.
3A, B 3 Quassiae lignum. After treatment with LB reagent, about 11 weak blue or blue-green
fluorescent zones (in UV-365 nm) are seen between Rf 0.1 and the solvent front
(partly alkaloids and coumarins).
The bitter principle, quassin, shows a distinct quenching of fluorescence in UV-
254 nm, but does not itself fluoresce in UV -365 nm. The weak blue and green fluores-
cent zones (UV-365 nm) accompanying the quassin standard (T3) are due to impuri-
ties of alkaloids and coumarins.
3B After treatment with VS reagent, the quassin (cf. T3) appears as a violet (vis.)
zone. In solvent system B-5 quassin migrates at Rfca. 0.9 (cf. Fig. 4B).
4C 4 Marrubii herba. Treatment with VS reagent pro duces violet zones, in particular
three pronounced violet (vis.) zones in the Rf range 0.5-0.9. The zone at Rf ca.
0.8 is due to marrubiin (cf. T4); the zone at Rfca. 0.5 is presumably due to premarru-
biin.
Remarks: Visualization in UV-365 nm without chemical treatment reveals only non-specific
fluorescent zones.
134
-FRONT
Rf
-0.5
START
Fig.3
Tl T2 2 T3 3 T3 3
c
-FRONT
- Rf
=0 .5
-START
Fig.4
Tl T4 4
135
Gentianae Radix/amarogentin
Track 1 = Gentianae radix (G.lutea) Test T1 = amarogentin
Solvent B-1: ethyl acetate-methanol-water (77: 15: 8) Fig.5A
system B-2: acetone-chloroform-water (70: 30: 2) Fig.5B
Detection Fast red salt reagent (FRS No. 13, p. 301)+NaOH VlS. Fig.5A
or+25% NH 3 vis. Fig.5B
Gentianae radix contains 0.01-0.04% amarogentin; only traces are present in Centaurii herba.
Chromato- With fast red salt reagent, amarogentin shows orange ( + KOlI) or red-violet (+ NH 3 )
gram colour (vis.). It migrates at Rf ca. 0.8 (solvent system B-1) or ca. 0.45 (B-2).
5A, B Gentianae radix also shows a strong zone of the xanthone, gentisin, at the solvent
front.
Chromatograms of root extracts of G. pannonica, purpurea and punctata differ from
those of G. lutea by the presence of the additional bitter principles, amaropanin and amaro-
swerin. These compounds migrate direct1y above and below amarogentin, respectively, and
they also give various reds with FRS reagent.
Plantaginis Herba/aucubine
Track 2 = Plantaginis herba (Plantago lanceolata L.) Test T2 = aucubine
Solvent B-6: n-propanol-toluene-glacial acetic acid-water (25: 20: 10: 10)
system
Detection Benzidine reagent (BZ No. 4, p. 299) VlS. Fig.5C
Chromato- After treatment with benzidine reagent, aucubine becomes visible as a brown zone
gram at Rf ca. 0.35. Plantaginis herba shows additional yellow zones between the start
5C and Rf 0.3, and another brown zone at the solvent front.
Oleae Folium, Fructus/oleuropein

Tracks 3 = Oleae folium Test T3 = oleuropein (Rf ca. 0.25)
4 = Oleae fructus (fresh)
5 = Oleae fructus (stored)
Solvent B-7: ethyl acetate-dioxan-water (30: 10: 0.3)
system
Detection Without chemical treatment vis. Fig.6A
10% FeCl 3 soln. vis. Fig.6B
Oleuropein is easily detectable in freshly harvested olive leaves and fruits. During storage it is
cleaved into 3,4-dihydroxyphenylethyl alcohol and an iridoid carboxylic acid.
Chromato- Without treatment of the chromatogram, oleuropein is visible as a weak brown

gram zone at Rf 0.25-0.35. Treatment with FeCl 3 produces a stronger brown (vis.). A
6A,B zone below oleuropein becomes dark brown. Breakdown products appear as grey-
green zones near the solvent front and at the start.
136
A c
-FRONT
Rf
-0.5
-START
Fig.5
Tl Tl T2 2
A
• -FRONT
Rf
-0.5
-START
Fig.6
T3 3 4 T3 3 4 5
137
Bryoniae Radix Cucurbitae Semen
Tracks 1 = Bryoniae dioicae radix} . Id
. d··
2 = Bryomae lOIcae ra d·IX commerCIa rugs
3 = Bryoniae albae radix
4 = Cucurbitae semen
Tests T1 = 25-0-acetylbryomarid
T2 = cucurbitacin D
T3 = dihydrocucurbitacin D
T4 = cucur bitacin I
T5 = cucurbitacin L
Solvent B-l0: chloroform-ethanol (95:5)
system
Detection Vanillin-phosphoric acid reag. (VPA No. 36, p. 304)
UV-365 nm Fig. 7 A, 8B
vis. Fig. 7B, 8A, C
For description of drugs see p. 129. Formulae p. 131
Chromato-
gram 1,2 Bryoniae dioicae radix. After treatment with VPA reagent, chromatograms of Bryo-
7A, B niae dioicae radix extracts show at least 12 blue, bIue-green or orange fluorescent
zones (UV-365 nm) between the start and the solvent front. The same zones appear
violet or brown violet in vis.
Cucurbitacins B, D, E, I, K, Land the corresponding dihydrocucurbitacins migrate
in the Rf range 0.2-0.4. Bryomarid (at the start) and acetylbryomarid (directly above
the start) (cf. T1) appear as pale blue fluorescent zones in UV-365 nm, and as
violet zones in vis.
3 Bryoniae albae radix has alm ost the same qualitative pattern of cucurbitacins as
B. dioicae radix, but shows normally a lower content of bitter principles.
The specific source plant of any commercial preparation of Bryoniae radix cannot
be unequivocally identified. Moreover, the cucurbitacin content depends on the
time of harvesting and extraction method.
8B, C 4 Cucurbitae semen. In UV-365 nm the chromatogram shows four characteristic,
strong orange-brown fluorescent zones at Rf ca. 0.4, 0.6, 0.85 and at the solvent
front. After 5-10 min the fluorescence changes to blue. The same zones appear
violet in vis. Most of these zones are due to sterols, Cucurbitacins are not present.
4,1 After treatment with VPA reagent and visualization in UV-365 nm, chromatograms
of Cucurbita and Bryonia extracts appear somewhat similar; but a five-fold greater
quantity of Cucurbitae semen extract must be applied for the detection of the zones
in the Rf range 0.1-0.3.
Remarks: Solvent system B-I0 is temperature-dependent, resulting in shifts in the Rf values
ofthe zones (cf. Fig. SA/SC). Shorter solvent migrations are therefore recommended, as shown
in Fig. SC.
138
-0.5
-5
Fig.7
Tl T2 T3 2 3 T4 T5 2 3 T4 T5
Rf
Fig.8
Tl 2 3 T2 T3 T4 T5 4 4
139
Humuli Strobuli (Glandulae)
Tracks 1 = Humuli strobuli DCM extract (commercial pattern I)
2 = Humuli strobuli ether extract (commercial pattern II)
3 = H umuli stro buli methanol extract (commercial pattern I)
4 = Humuli strobuli (fresh hops pattern I)
5 = Humuli strobuli (fresh hops pattern II)
Tests Tl = lupulone with degradation products

T2 = humulone with degradation products
TG = flavonoid mixture : rutin (Rf ca. 0.35), chlorogenic acid (Rf ca. 0.5), hyperoside
(Rf ca. 0.6).
Solvent B-8: iso-octane-isopropanol-formic acid
system (83.5:16.5:0.5) developed over 15 cm Fig. 9A, B
B-9: n-heptane-isopropanol-formic acid
(90: 15: 0.5) developed twice over 15 cm Fig. 10A, B, C
F-1: ethyl acetate-formic acid-glacial acetic
acid-water (100: 11 : 11 : 27) Fig.9C
Detection Fast blue salt-KOH (FBSjKOH No. 12, p. 301) vis. Fig.9A
Without chemical treatment UV-365 nm Fig. 9B; lOC
FeCl 3 reagent (No. 14, p. 301) VlS. Fig. 10A, B
(NPjPEG No. 28, p. 303) UV-365 nm Fig.9C
Chromato-
gram 1,2 Commercial drugs. The phloroglucinol derivatives, lupulone and humulone, are unsta-
9 A, B ble. The commercial drugs are therefore found to contain mostly breakdown or
decomposition products, known as bitter acids.
After treatment with FBS re agent, chromatograms of DCM or ether extracts
show a broad, red-brown (vis.) zone of bitter acids at Rf 0.4-0.45. In the same
Rfrange, visualization in UV-365 nm reveals weak blue or reddish fluorescent zones.
10 A 1 Double development of the TLC plate over 15 cm in solvent system B-9 gives better
resolution of the individual zones between Rf 0.3-0.6.
lOB, C 4,5 Freshly harvested, Jreeze-dried drugs. Ether extracts of hops (patterns land II) still
contain lupulone (Rf ca. 0.5) andjor humulone (Rf ca. 0.75). Track 4, however, shows
that an extensive breakdown to bitter acids has already occurred (Rf ca. 0.4 and
0.4-0.6).
9C 3 Flavonoids and ehlorogenie aeid:
After treatment with NP/PEG reagent and visualization in UV-365 nm, chromato-
grams of methanolic drug extracts mainly show yellow-green fluorescent zones of
rutin (Rf ca. 0.35), hyperoside (Rf ca. 0.55), ehlorogenie acid (Rf ca. 0.45) (cf. TG),
and an additional flavonoid monoglycoside (Rf ca. 0.7).
140
A
FRONT
Rf
START
Fig.9
Tl 2 Tl 2 TG 3
A B
-FRONT
Rf
0 .5
START
Fig. 10
Tl Tl 4 5 T2 Tl 4 5 T2
141
Salviae, Rosmarini FoliumjCarnosolic acid (Carnosol) Cynarae Herba
Tracks 1 = Salviae folium 3 = Cynarae herba (fresh plant)
2 = Rosmarini folium 4 = Cynarae herba (dried drug)
Tests T = carnosol (picrosalvin)
Tl = rutin
T2 = chlorogenie acid
T3 = luteolin-7-0-glucoside
T4=cynarin
T5 = caffeic acid (Rf ca. 0.9), isochlorogenie acid (Rf ca. 0.8),
chlorogenie acid (Rf ca. 0.4)
Solvent B-4: chloroform-methanol (97: 3) Fig.11
system F-1: ethyl acetate-formic acid-glacial
acetic acid-water (100: 11: 11 :27) Fig.12
Detection Without chemical treatment VlS. Fig. 11 A
FeCl 3 reagent (FeCI 3 No. 14, p. 301) vis. Fig. 11 B
Vanillin-sulphuric acid reagent (VS No. 38, p. 304) VlS. Fig. 11 C
Natural products-polyethyleneglycol-reagent
For description ofdrugs see p. 128-129. Formulae p.131 and 171
Chromato- 1 Salviae folium

gram 2 Rosmarini folium
11A, B The bitter principle, earnosol, is present in both drugs, but it undergoes ring opening
to earnosolie acid during extraction, and is detected as such on the chromatogram.
Without chemical treatment, carnosolic acid is seen as a yellow-brown zone at Rf
ca. 0.2. Treatment with FeCl 3 reagent converts this to a green-brown (vis.) zone.
In Rosmarini folium a second strong zone is seen at Rf ca. 0.4.
11C VS-reagent pro duces violet (vis.) zones of the terpenoid compounds of the essential
oil fraction (see p. 30, Fig. 10; p. 38, Fig. 17).
Remarks: Rosmarini folium also shows blue fluorescent zones (UV-365 nm) of rosmarinic
acid and the jlavonoid, sinensetin (Rf 0.8-0.85).
12 3,4 Cynarae herba. The detection ofthe bitter principles, eynaropierin, dehydroeynaropie-
rin and grossheimin is only possible after enrichment of the extract. Identification
is therefore more conveniently based on the presence of eynarin 1 (an hepatoactive
compound), a 1,3-dicaffeoylquinic acid, other phenol carboxylic acids and flavon-
oids.
3 In fresh plant extracts 1 , treatment of chromatograms with NPjPEG reagent shows
eynarin as a blue fluorescent zone (UV-365 nm) at Rf 0.6-0.65 (cf. T4). The other
acids appear at RfO.4-0.55 (cf. T2; ehlorogenie and neoehlorogenie acid) and between
Rf 0.7 and the solvent front (isoehlorogenie aeid and eajJeie acid, cf. T5).
Luteolin-7-0-glueoside (cf. T3) is seen as a yellow fluorescent, main zone at
Rf ca. 0.6. Luteolin-3-0-rutinoside is located in the same region as the rutin standard
(cf. Tl), with the weak zone of luteolin-7-0-rutinosyl-4'-O-glueoside below it.
1 Remarks: Cynarin easily isomerizes to 1,5-dicaffeoylquinic acid during extraction. Extracts
of stored drugs (= 4) show chiefly caffeic acid (Rf ca. 0.9) and isomerization products
(Rf 0.75-0.85), and little cynarin (T4).
142
A c
-FRONT
Rf
-0.5
-START
Fig.11
2 T 2
Fig.12
Tl T2 3 4 T3 T4 T5
143
Coumarin Drugs
The active principles of coumarin drugs are benzo-a.-pyrones. They can be further
c1assified as folIows:
1. Simple coumarins
Most of the compounds in this series are substituted with OH or OCH 3 at positions
C-6 and C-7. Substitution at C-5 and C-8 is less common.
Drugs:
Angelicae, Imperatoriae, Levisticae, Pimpinellae and Herac1ei radix,
e.g. umbelliferone.
Scopoliae radix, e.g. scopoletin.
Abrotani herba, e.g. scopoletin and umbelliferone.
Fraxini cortex, e.g. fraxidin, isofraxidin and fraxetin.
Herniariae herba, e.g. herniarin.
2. C-prenylated coumarins
Drugs:
Rutae herba, e.g. rutamarin.
Angelicae radix, e.g. umbelliprenin.
Imperatoriae radix, e.g. ostruthin.
3. "Condensed coumarins"
a) Furanocoumarins
A furan ring is fused at C-6 and C-7 (psoralen-type) or C-7 and C-8 (angelicin-type)
of the coumarin ring system.
Drugs:
Angelicae, Imperatoriae, Levisticae, Pimpinellae and Herac1ei radix, e.g. bergapten,
angelicin, imperatorin.
Ammi majoris fructus, e.g. bergapten, xanthotoxin.
Rutae herba, e.g. bergapten, psoralene.
b) Pyranocoumarins
A pyran ring is fused at C-7 and C-8 ofthe coumarin ring system (seselin-type).
Drug:
Amrni majoris fructus, e.g. visnadin, sarnidin.
4. Dimeric coumarins
Drugs:
Daphne mezerei cortex, e.g. daphnoretin.
5. Ammi visnagae fructus occupies a special position, because it contains both benzo-a.-
pyrones and the isomerie benzo-y-pyrones or furanochromones.
145
I. Preparation of Drug Extracts and Pharmaceuticals for TLC
1. Drugs
Powdered drug (1 g) is extracted by shaking with 10 ml methanol for 30 min on
the water bath. The clear filtrate is evaporated to about 1 ml, and 20 J.lI are applied
to TLC.
DAB 8: Ammeos visnagae fructus: The drug (0.5 g) is extracted by shaking with
10 ml of 60% ethanol for 30 min on a water bath. The filtrate is evaporated to
about 5 ml, and 20 J.lI are applied to TLC.
2. Pharmaceutical preparations
Carduben35®: 1 dragee contains 35 mg visnadin.
One dragee is powdered and extracted by shaking with 5 ml methanol for 5 min
on the water bath. Five microlitres of the clear filtrate are applied to the chromato-
gram.
Application of 20 J.lg ensures the chromatographie detection of all coumarins.

All coumarin standards are prepared as 1 % methanolic solutions.
2. Adsorbent
3. SampIe size
The following quantities are applied to TLC: drug extracts (20 J.lI), standard solutions
(5 or 10 J.ll), extracts of pharmaceuticals (5 J.ll).
C-1 toluene-ether (1: 1, saturated with 10% acetic acid)
Toluene (50 ml) and ether (50 ml) are shaken 5 min with 50 ml of 10% acetic acid
in a separating funnel. The lower phase is discarded, and the toluene-ether mixture
is used for TLC.
C-l is a universally applicable TLC solvent for coumarin aglycones; it must
be freshly prepared.
C-2 ethyl acetate
Ammeos visnagae fructus extracts (DAB 8).
C-2 separates polar coumarins in a relatively higher Rf range in comparison with
C-1.
Remarks: Polar coumarins from Rutae herba extracts can also be separated in the solvent
system, ethyl acetate-forrnic acid-glacial acetic acid-water (100: 11: 11: 27) (see Flavonoids,
p. 164).
III. Detection
UV-254 nm
All coumarins show a distinct fluorescence quenching.
UV-365 nm
All simple coumarins show intense blue or blue-green fluorescence. Furanocouma-
rins sometimes show yellow, brown or blue fluorescence (detection limit ca. 1 J.lg).
146
Non-substituted coumarin fluoresces yellow-green in UV-365 nm only after treat-
ment with KOH-reagent.
Chromones show less intense fluorescence (e.g. visnagin, pale blue; khellin, weak
yellow-brown).
2. Spray reagents
a) Potassium hydroxide (KOR No. 21, p. 302)
Blue fluorescent zones are intensified by spraying with 5% ethanolic KOR. Ammo-
nia vapour has the same effect.
b) Natural products-polyethyleneglycol-reagent (NPjPEG No. 28, p. 303)
This reagent intensifies and stabilizes the existing fluorescence of the native couma-
nns.
c) Antimony III chloride (SbCI 3 No. 3, p. 299)
Visnagin gives alernon yellow fluorescence in UV-365 nm after treatment with this
reagent.
IV. List of Coumarin Drugs


F amily/Pharmacopoeia
A. Drugs from the family Apiaceae

2--4 Angelicae Radix Coumarins: 0.001 %--0.008%, consisting of
Angelica root angelicin (2' ,3': 7,8-furanocoumarin); bergap-
Angelica archangelica L. ten (5-methoxy-(2',3':7,6-FC)); imperatorin
(8-oxy-y-y-dimethyl-allyl-2' ,3': 7,6-FC); os-
ÖAB, 2. AB-DDR (essential oils) thenol (7 -oxy-8-(8,8-dimethylallyl)-coumarin)
and its methyl ester, osthol; oxypeucedanin
hydrate (hydrate of 5-oxy (3,3' -dimethyl-2,3-
epoxy-propano-2' ,3': 7,6-FC); xanthotoxin
(8-methoxy-2' ,3': 7,6-FC); xanthotoxol
(8-oxy-2' ,3': 7,6-FC); umbelliferone (7-hy-
droxycoumarin); umbelliprenin (farnesyl
ether of umbelliferone).
Angelicae silvestris radix This adulterant of Angelica archang. contains
Wild angelica only umbelliferone, isoimperatorin (5-oxy-(y-
Angelica sylvestris y-dimethyl-allyl-2',3': 7,6-FC)), oxypeuce-
danin and its hydrate.
Imperatoriae Radix Coumarins: oxypeucedanin, oxypeucedanin
Master-wort hydrate, imperatorin, isoimperatorin, osthol
Peucedanum ostruthium L. (cf. Angelica root); also ostruthin (6-(3-
methyl-6-dimethyl-2,5-hexene)-7 -oxycou-
marin), and ostruthol (angelic acid ester of
oxypeucedanin hydrate).
Levistici Radix Coumarins: The total coumarin content is low
Lovage compared with that of Angelicae and Impera-
Levisticum officinale KOCH toriae radix; bergapten, umbelliferone.
ÖAB, 2. AB-DDR (essential oils) Phthalide: 3-butylidenephtalide (ligusticum
lacton).
147
F amily jPharmacopoeia
Pimpinellae Radix Coumarins (in both species):

Burnet root bergapten, umbelliferone, umbelliprenin, sco-
Pimpinella saxifraga L. poletin (6-methoxy-7-oxy-coumarin); sphon-
Pimpinella major (L.) HUDS. din (6-methoxy-(2',3': 7,8-FC)); isobergapten
(5-methoxy-(2' ,3': 7,8-FC); pimpinellin (5,6-
Helv. VI dimethoxy-(2' ,3': 7,8-FC)); isopimpinellin
(5,8-dimethoxy-(2' ,3': 7,6-FC)).
Heraclei Radix The drug is an acceptable substitute for Pim-
Hogweed root pinellae radix. The coumarin content is ab out
Herac1eum sphondylium L. 20 times higher than that ofPimpinellae radix.
Pastinacae Radix An adulterant of Pimpinellae radix with a low

Wild parsnip root coumarin content. Bergapten, imperatorin
Pastinaca sativa L. and osthol are present.
9, 10 Ammi majoris Fructus Coumarins: bergapten, imperatorin, xantho-

Ammi fruit toxin (see Angelicae radix), ammajiin, a glu-
Ammi majus L. coside of marmesin.
Ammi visnagae Fructus Coumarins: visnagan group, with samidin, di-

Ammeos visnagae fructus hydrosamidin and visnadin.
Ammi visnaga fruits Furanochromones: (2-4%; DAB 8 specifies
Ammi visnaga (L.) LAMARCK not less than 1%): khellin (5,8-dimethoxy-2-
DAB8 methylfuranochromone) is the major constit-
uent (0.3-1%), accompanied by visnagin,
khellinol, khellol, khellol glucoside, ammiol
and visammiol.
7,8 Asa foetida A gum resin containing 25-65% asaresin. This

Asafetida in turn contains about 60% ferulic acid esters,
Ferula assa-foetida L. asaresitannol, and about 1.3% free ferulic
acid, which is converted to umbelliferone on
dry distillation. Umbelliferone derivatives are
also presen t.
B. Drugs from other plant ramilies

5,6 Asperulae Herba Unsubstituted coumarin.
Woodruff
Galium odoratum (L.) ScoP.
Rubiaceae
Meliloti Herba Unsubstituted coumarin, melilotoside.
Tall melilot Remarks: Bacterial action on the damp drug
Melilotus officinalis LAM. em. THUILL. produces dicoumarol (3,3' -methylene-bis-4-
Fabaceae hydroxy-coumarin).
Herniariae Herba Coumarins: herniarin, umbelliferone.
Rupture-wort Flavonoids; rutin, narcissin (see section on
Herniaria glabra L. Flavonoids, Fig. 6, p. 176).
Herniaria hirsuta L. Saponins: Herniaria saponins land II.
Caryophyllaceae
ÖAB
148
Rutae Herba Coumarins: scopoletin, umbe1liferone, ber-

Rue gapten, isoimperatorin, psoralene, xantho-
Ruta graveolens L. toxin, rutacultin, rutamarin, daphnoretin,
Rutaceae daphnoretin methyl ether.
Flavonoids: rutin (see Flavonoids, Fig. 16,
p.186).
Abrotani Herba Coumarins: umbeIliferone, scopoletin.
Southernwood
Artemisia abrotanum L.
Asteraceae
7, 8 Fraxini Cortex Coumarins: fraxidin (0.6%), isofraxidin

Ash bark (0.12%), fraxetin, fraxin (fraxetin glucoside),
Fraxinus excelsior L. fraxinol (6- hydroxy- 5, 7-dimethoxycoumarin;
Fraxinus ornus L. ca. 0.45%).
Oleaceae
Mezerei Cortex Coumarins: daphnetin, daphnin (7,8-dihy-

Mezereon bark droxy-coumarin-7 -O-glucoside), umbellife-
Daphne mezereum L. rone, scopoletin (traces).
Thyme1aeaceae
4 Scopoliae Radix Coumarins: scopoletin, scopoline (scopoletin

Scopolia rhizome glucoside).
Scopolia carniolica L. Alkaloids: hyoscyamine/atropine, scopol-
Solanaceae amine (see also Fig. 26, p. 90).
149
v. Formulae of Constituents of Coumarin Drugs
SIMPLE COUMARINS
R, R2 Rs R.
H H OH H Umbellifefone
(7-Hydroxycoumarin)
H H OCHs H Herniarin
(7-Methoxycoumarin)
H OH OH H Aesculetin
(6,7-Hydroxycoumarin)
H H OH OH Daphnoretin
(7,8-Hydroxycoumarin)
H OCHs OH H Scopoletin
(6-Methoxy-7-hydroxycoumarin)
H OCHs OH OCHs Isofraxidin
(7-Hydroxy-6,8-methoxycoumarin)
OCHs OH OCHs H Fraxinol
(6-Hyd roxy-5, 7-methoxycou marin)
C-PRENYLATED COUMARINS
R, R2 R3 R4
CH 3 CH 3
H H X H Umbelliprenin \ I
H X OH H Ostruthin X= C(=CH-CH 2 -CH 2 -Ch=CH-CH 2 0-
CHi
Rutacultin
Rutamarin
DIMERIC COUMARIN ~
H3CO~O 0 0 Daphnoretin
HoMo~o
150
FURANOCOUMARINS R1 R2 Furanocoumarin
H H Psoralen
7,6-Furanocoumarins OCH 3 H Bergapten
oQo
H OCH 3 Xanthotoxin
H OH Xanthotoxol
OCH 3 OCH 3 Isopimpineliin
/CH 3
H -OCH 2 -CH=C Imperatorin
10 7 0 0 "'CH 3
/CH3
R2 -OCH 2 -CH=C H Isoimperatorin
~CH3
/0 /CH3
-OCH 2 -CH-C H Oxypeucedanin
'CH 3
___ CH 3
-OCH 2 -CH-CH H Oxypeucedanin hydrate
I I-CH 3
OH OH
R290
7,8-Furanocoumarins
R1 R2 Furanocoumarin
H H Angelicin
OCH 3 H Isobergapten
o 8 0 0 H
OCH 3
OCH3
OCH3
Sphondin
Pimpineliin
PYRANOCOUMARINS (VISNAGAN GROUP)
R = -CO-CH=C-CH3 Samidin
tH 3
o R = -CO-CH 2 -CH-CH 3
I
Dihydrosamidin
CH 3
R = -CO-CH-CH 2 -CH 3 Visnadin
I
CH 3
FURANOCHROMONES
R, 0 R1 R2 R3 Chromone
~
OCH3 OCH3 CH 3 Khellin
OH OCH3 CH3 Kheliinol
o 0 R3 OCH 3
OCH 3
H
H
CH 3
CH2 0H
Visnagin
Kheliol
R2 OCH 3 OCH 3 CH 20H Ammiol
151
Coumarins - Chromatographie Standards
Tests Tl = daphnoretin T5 = herniarin T 9 = caffeic acid *
T2 = scopoletin T6 = xanthotoxin T10 = isopimpinellin
T3 = isofraxidin T7 = imperatorin T11 = isobergapten
T4 = umbelliferone T8 = ferulic acid * T12 = oxypeucedanin
Solvent C-l: toluene-ether (1: 1, saturated with 10% acetic acid)
system
Detection Without chemical treatment UV-365 nm Fig.l
Chromato- UV-365 nm:

gram light blue: daphnoretin, scopoletin, isofraxidin, umbelliferone.
1 violet: herniarin.
light green: xanthotoxin, isobergapten, oxypeucedanin.
yellow-green: isopimpinellin.
* Remarks: Coumarin drugs often contain plant acids, e.g. ferulic acid and caffeic acid, which
also give blue fluorescence. In methanolic extracts, caffeic acid (T9) always produces two
zones, corresponding to the free acid and its faster migrating methyl ester.
Pimpinellae Radix Heraclei Radix

Tracks 1 = Pimpinellae radix (P . saxifraga)
2=Pimpinellae radix (P. major)
3 = Heraclei radix
Tests TGl = scopoletin (Rf ca. 0.3), umbelliferone (Rf ca. 0.45), imperatorin (Rf ca. 0.6)
TG2 = umbelliferone (Rf ca. 0.45), xanthotoxin (Rf ca. 0.55)
system
Detection Without chemical treatment UV-365 nm Fig.2
Chromato-
gram For description of drugs see p. 148. Formulae p. 150-151
2 1,2 Pimpinellae radix: a strong blue fluorescent zone at the start in UV-365 nm, and
5 weaker blue fluorescent zones in the Rf range 0.1-0.55.
Scopoletin (TG1) and sphondin (direcdy below the xanthotoxin test) can be identi-
fied.
Remarks: Other coumarins e.g. isobergapten or isopimpinellin as described in the literature,
can be detected only in enriched extracts of the drug.
3 Heraclei radix: at least 10 blue or green-blue fluorescent zones in the Rf range
0.1-0.8.
Between the start and Rf 0.5, the chromatographie features are very similar
to those obtained with Pimpinella extracts; sphondin (main zone Rf ca. 0.5) shows
a distinct light blue fluorescence; a green-blue fluorescent zone of isopimpinellin
(Rf ca. 0.55), and a blue fluorescent zone of isobergapten (Rf ca. 0.75) are also
detectable and between these two zones lies pimpinellin, overlapped by bergapten.
The violet fluorescent zone at Rf ca. 0.8 is due to umbelliprenin.
152
Fig.l
Fig.2
153
Angelicae, Levistici, Imperatoriae, Scopoliae Radix
Tracks 1 = Angelicae radix 4 = Mei athamantici radix
2 = Levistici radix 5 = Scopoliae radix
3 = Imperatoriae radix
Tests Tl = xanthotoxin
T2 = umbelliferone (Rf ca. 0.4), imperatorin (Rf ca. 0.6)
T3 = scopoletin
Solvent C-l: toluene-ether (1: 1, saturated with 10% acetic acid) Fig. 3, 4
system
KOR reagent (No. 21, p. 302) UV-365 nm Fig.4
For description of drugs see p. 147-149. Formulae p. 150-151
Chromato- Angelicae (1) and Imperatoriae (3) radix contain many structurally similar coumarins,
gram which partly overlap on the chromatogram picture. Levistici radix (2) shows a lower
3,4 coumarin content.
Coumarins 1 2 3 Rf-range (approx.)
U mbelliprenin x 0.8
Bergapten x x x 0.6
Ostruthin x
Xanthotoxin x
Imperatorin
Angelicin
x
x
x I
0.5
Umbelliferone x x x 0.45
Scopoletin x x x 0.25
Oxypeucedanin hydrate
Plant acids
x
x x :} 0.2-0.1
1 Angelicae radix: at least 15 light blue, dark blue or yellow-green fluorescent zones
between the start and Rf 0.85. The prominent zones are in the Rf range 0.5-0.75;
these are the partly overlapping zones of angelicin, imperatorin, xanthotoxin, hergap-
ten and osthenol (see Table).
KOH treatment intensifies the fluorescence, especially in the Rfrange 0.7-0.75.
2 Levistici radix: 5-7 mostly very weak light or dark blue fluorescent zones in the
Rf range 0.25-0.9. Bergapten (Rf ca. 0.6, very elose to the imperatorin test) and
umhelliferone (cf. T2) can be identified. The main zone at Rf ca. 0.9 is due to
3-butylidenephtalide (ligusticum lactone).
3 Imperatoriae radix: the chromatogram is similar to that of Angelicae radix, but
differs by the presence of four especially strong, light blue fluorescent zones in
the Rf range 0.3-0.6. The strong zone above the umbelliferone test (cf. T2) is due
to ostruthin. Imperatorin (cf. T2) gives a green-blue fluorescence after KOR treatment
(Fig.4).
4 Mei athamantici radix: the single strong blue fluorescent zone is due to ligustilide.
Mei atham. radix can be used as a test standard for Levistici radix.
5 Scopoliae radix: the chromatogram is characterized by the presence of scopoletin
(T3, Rf ca. 0.25) and scopoline (scopoletin glucoside), which remains near the start
(see also Alkaloids, p. 90, Fig. 26).
154
Fig.3
Tl 2 3 T2 2 4
FRONT
Rf
START
Fig.4
Tl 2 3 T2 5 T3
155
Herniariae, Meliloti, Asperulae, Abrotani, Rutae Herba
Tracks 1 = Herniariae herba
2 = Meliloti herba
3 = Asperulae herba
4 = Abrotani herba
5 = Rutae herba
Tests Tl = herniarin
T2 = coumarin
T3 = scopoletin (Rf ca. 0.25), umbelliferone (Rf ca. 0.4)
Solvent
system C-1: toluene-ether (1: 1, saturated with 10% acetic acid)
KOH reagent (No. 21, p. 302) UV-365 nm Fig.6
Chromato- In drugs 1-4, the red fluorescent zones are due to various chlorophyll derivatives.
gram
1 Herniariae herba is characterized in UV-365 nm by the intense violet fluorescent
5, 6
zone of herniarin (cf T1), and two weak blue-violet fluorescent zones at Rf 0.35-0.4.
Umbelliferone migrates at Rf ca. 0.4.
The fluorescence of herniarin and umbelliferone is intensified by KOH treatment
(Fig. 6) (see also Flavonoids, p. 176).
2 Meliloti herba and
3 Asperulae herba
A large number of red fluorescent zones are seen in UV-365 nm. Otherwise there
are only 4 weak blue or violet fluorescent zones in the Rf range 0.25--0.5. Coumarin
(fluorescence quenching in UV-254 nm!) is clearly detectable only after KOH treat-
ment, when it forms an intense green-yellow main zone at Rf ca. 0.65 (Fig. 6, cf.
T2). Both drugs also show weak zones of scopoletin and umbelliferone (cf. T3).
Meliloti herba can be differentiated from Asperulae herba by the presence of
an additional blue fluorescent zone at Rf ca. 0.5.
After KO H treatment, only the chromatogram of Asperulae herba shows a strong
blue fluorescence at the start.
4 Abrotani herba is characterized by strong blue fluorescent zones, which correspond
to scopoletin and umbelliferone in the test mixture T3. Directly below scopoletin
is another, equally strong, blue fluorescent zone.
5 Rutae herba shows at least 12 blue fluorescent zones between the start and the
solvent front. The furanocoumarins, xanthotoxin, psoralene, bergapten und iso-
imperatorin (cf. Apiaceae) migrate in the upper half of the chromatogram (Rf
0.5-0.8).
Scopoletin and umbelliferone (Rf 0.25-0.4, cf. T4), rutaretin, daphnoretin and
daphnoretin methyl ether can be identified in the lower half of the chromatogram.
A TLC separation of Ruta-Flavonoids is described in the section on Flavonoids,
p. 186, Fig. 16.
156
FRONT
Rf
Fig.5
Tl 2 3 T2 4 T3 5
FRONT
Rf
START
Fig.6
Tl 2 3 T2 4 T3 5
157
Mezerei, Fraxini Cortex Asa foetida
Tracks 1 = Mezerei cortex
2 = Fraxini cortex
3 = Asa foetida
Tests Tl = scopoletin
T2 = umbelliferone
system
For description of drugs see p. 148-149. Formulae p. 150-151
Chromato- 1 Mezerei cortex is characterized in UV-365 nm by about 5 blue fluorescent zones

gram in the Rf range 0.2-0.75.
7, 8 NP/PEG reagent intensifies the fluorescence, which becomes light blue; in addi-
tion, yellow-green fluorescent zones appear at Rf 0.3 and above the start. Coumarin
glucosides, e.g. daphnetin glucoside, remain at the start. After NP/PEG treatment,
the zones at RF 0.35-0.45 are intensified, which is typical of ferulic and caffeic
acid.
2 Fraxini cortex shows about 6 blue fluorescent zones in the lower and intermediate
Rf range. The four c10sely neighbouring zones at Rf 0.25-0.4 are due to fraxetin,
fraxidin, isofraxidin and fraxinol, all of which are coumarins with very similar pat-
terns of substitution. Coumarin glycosides (e.g. fraxin) remain at or ne ar the start.
Intensification of fluorescence in the lower Rf range, observed after treatment
with NP/PEG reagent, is typical of this drug
3 Asa foetida. In UV-365 nm, the chromatogram shows a characteristic series of at
least 10 blue or violet fluorescent zones between the start and Rf 0.8. The main
zone corresponds chromatographically with the umbelliferone test (T2), and it con-
sists of umhelliferone plus ferulic acid. The other fluorescent zones are due to umbelli-
ferone derivatives.
158
Fig.7
Fig.8
159
Ammi (Ammeos) Fructus
Tracks 1 = Ammi fructus (A. major)
2 = Ammi fructus (A. visnaga)
Tests Tl =visnadin (extracted and concentrated from Carduben®)
Rf ca. 0.6 (solvent C-l), Rf ca. 0.9 (solvent C-2)
T2=khellin
T3 = visnagin
Solvent C-l: toluene-ether (1: 1, saturated with 10% acetic acid) Fig. 9A, B
system C-2: ethyl acetate Fig. lOA, B
Detection Without chemical treatment UV-365 nm Fig.9A
KOR reagent (No. 21, p. 302) UV-365 nm Fig. 9B; lOA
SbCl 3 reagent (No. 3, p. 299) UV-365 nm Fig. lOB
Chromato- 1 Ammi majoris fructus. In UV-365 nm the chromatogram is characterized by aseries

gram of at least 12 intense, light blue fluoreszent zones between the start and Rf ca.
Solvent C-I 0.7.
9 A, B The fluorescence is intensified by KO H treatment (Fig. 9 B).
The furanocoumarins, bergapten, xanthotoxin, isopimpinellin and imperatorin
(cf. tests, Fig. 1, p. 152) migrate above RfO.5, overlapping to some extent. According
to the literature, the zones below Rf 0.5 are due to marmesin, marmesinin and
ammaJllll.
2 Ammi (Ammeos) visnagae fructus. In UV-365 nm the chromatogram shows only
a few weak violet and pale blue fluorescent zones at and near the start, and at
Rf 0.4-0.6 with visnadin as the main one. The zones also show a distinct fluorescence
quenching in UV-254 nm.
KOH treatment intensifies the fluorescence, especially in the Rf range 0.4-0.6.
Visnagin and khellin (cf. T3 and T2) show prominent fluorescence quenching
in UV-254 nm, and light blue (visnagin) and weak green-blue (khellin) fluorescence
in UV-365 nm, which is hardly intensified by treatment with KOR reagent.
In solvent C-2, ethyl acetate, all coumarins and chromones have rather high Rf
values.
10A 1 Ammi majoris fructus. There are many overlapping coumarin zones, especially in
the Rf range 0.7-0.9.
2 Ammi visnagae fructus shows a better separation of khellin and visnagin in the
solvent system C-2.
10 B After treatment with SbCI3 , visnagin gives a typical yellow-green fluorescence. Khel-
lin, however, appears black-brown and is no longer c1early defined against the back-
ground. Khellol glucoside, khellol and khellinol mi grate at Rf 0.1-0.25 as blue
fluorescent zones. Visnadin (Tl) forms a blue-violet fluorescent zone be10w the zones
of samidin and dihydrosamidin at the solvent front.
160
FRONT
Rf
START
Fig.9
Tl 2 Tl 2 T2 T3
Fig.10
T 2 T2 T3 Tl 2
161
Flavonoid Drugs
The main eonstituents of flavonoid drugs are 2-phenyl-y-benzopyrones (2-phenyl-

ehromones) or strueturally related, mostly phenolie, eompounds.
The various types of flavonoid strueture differ in the degree of oxidation of
ring C, and in the pattern of substitution in the A and/or B rings (see formulae,
p. 170).
Most of these eompounds are present in the drugs as mono-or diglyeosides.

Powdered drug (1 g) is extraeted with 10 ml methanol for 5 min on a water bath
at about 60° C. The clear filtrate is used for ehromatography.
This rapid method also extraets both lipophilie and hydrophilie flavonoids.
Exeeptions:
Cardui mariae fructus (Silybi fruetus): powdered drug (1 g) is first defatted by heating
under reflux for 30 min with 50 ml light petroleum. The petroleum extraet is dis-
earded and the drug residue is heated under reflux for 15 min with 10 ml methanol.
The filtrate is eoneentrated to 5 ml, and 30 111 are used for ehromatography.
Orthosiphonisfolium, DAB 8: powdered drug (1 g) is extraeted by shaking for 15 min
with 10 ml diehloromethane. The clear filtrate (30 111) is used for ehromatography.
Farfarae folium, Petasites folium (test for petasins): powdered drug (2 g) is extraeted
by heating under reflux for about 20 min with about 40 ml light petroleum on
a water bath. The clear filtrate is eoneentrated to about 1 ml, and 30 111 are used
for ehromatography.

Standard eompounds are prepared as 0.05% solutions in methanol, and about 10 f..ll
are used for ehromatography. The average deteetion limit for flavonoids is 5~10 f..lg.
Unless otherwise stated, a mixture of standards (Tl) of rutin, ehlorogenie aeid and
hyperoside is used.
2. Adsorbent
Siliea gel 60F 254 pre-eoated TLC plates (Merek, Darmstadt).
Provided the flavonoid eontent ofthe drug is between 0.5 and 1.5%, 25~30 f..ll extraet
are suffieient. For pharmaeeutical preparations, the quantity of extract used for
TLC must be adjusted in proportion to the flavonoid eontent.
163
F -1 Ethyl acetate-formic acid-glacial acetic acid-water (100: 11: 11: 27)
The ethyl acetate, formic acid and glacial acetic acid are mixed first, and the
water is added gradually with vigorous shaking. If the ethyl acetate is technical
grade, the composition 100: 11 : 11 : 26 should be used; separations with this
mixture show very slightly different Rf values, but no change in the order
of separation. F -1 is suitable as a screening system for the TLC investigation
of flavonoid glycosides.
Solvent systems specified in the official methods are variations of similar
system of ethyl acetate-glacial acetic acid-water:
Arnicae flos, DAB 8 (66: 15:20)
Tiliae flos, DAB 8 (67: 13: 20)
Aurantii pericarpium, DAB 8 (67: 7: 26)
Crataegi folium cum flore, DAB 8 (67: 7: 26)
F-2 Ethyl acetate-formic acid-glacial acetic acid-ethylmethyl ketone-water
(50:7:3:30:10)
Certain separation problems have been overcome by addition of ethylmethyl
ketone to F-1, e.g. for the constituents of Crataegi folium or flos (see p. 179,
Fig.8).
F-3 Chloroform-acetone-formic acid (75: 16.5: 8.5)
For the separation of the flavanolignans of Cardui mariae fructus.
F-4 Chloroform-ethyl acetate (60:40)
For the separation of the flavonoid aglycones of Orthosiphonis folium.
Remarks: Flavonoid aglycones can also be separated in benzene-pyridine-formic
acid (72: 18: 10) or in toluene-ethyl formate-formic acid (50: 40: 10).
F-5 n-Butanol-glacial acetic acid-water (40: 10: 50) (upper phase).
For the separation of flavonoid glycosides on cellulose plates according to
DAB8.
F-6 Analytical grade chloroform (with chamber saturation)
For the detection of petasins. Petasites folium is an adulterant of Farfarae
folium.
IH. Detection
Residual solvent (acids) must be thoroughly removed from the silica gel layer with
the aid of a hot air blower.

UV-254 nm: All flavonoids cause fluorescence quenching, which is seen as dark
blue zones on the yellow background of the TLC plate.
UV-365 nm: Depending on the structural type, flavonoids fluoresce yellow, blue
or green.
Intensification and greater differentiation of fluorescence in UV-365 nm can be
achieved by the use of various spray reagents.
Flavonoid extracts often contain other materials, such as plant acids and couma-
rins, which form blue fluorescent zones (e.g. Rutae herba).
2. Spray reagents
a. Natural products reagent (NP/PEG No. 28, p. 303)
Typical intense fluorescent colours in UV-365 nm are produced immediatelyon
spraying, or after about 15 min. Addition of PEG lowers the detection limit from
10 Ilg to 0.5 Ilg·
164
Fluorescence behaviour is structure-dependent:
Flavonols:
Glycosides of quercetin and myricetin -> orange
Glycosides of kaempferol and isorhamnetin -> yellow-green
Flavones:
Glycosides of luteolin -> orange
Glycosides of apigenin -> yellow-green
b) Fast blue saft B (FBS No. 12, p. 301)
Blue or blue-violet (vis.) azo-dyes are formed in daylight. To some extent, these
can be intensified by further spraying with 0.1 M sodium hydroxide or 10% potassi-
um hydroxide.
IV. List of Flavonoid Drugs

Grouping of drug chromatograms according to plant parts:

Flos: Figs. 3-10
Folium! Herba: Figs. 11-16, 22
Gemma! Pericarpium: Figs. 15, 17
Drugs containing predominantly flavonoid aglycones: Figs. 18-20
Fig. . Drug/Plant source Main constituents

F amily /Pharmacopoeia Adulterants
5 Arnicae Flos Quercetin-3-0-glucoside,

Arnica flowers Q-3-0-glucogalacturonide,
Arnica montana L. luteolin-7-0-glucoside,
Asteraceae kaempferol-3-0-glucoside.
DAB 8, Helv. VI, ÖAB Adulterants : Calendulae flos, Heterothecae
inuloidis flos, Farfarae flos, Taraxaci flos.
Arnica chamissonis LESS.
2. AB-DDR
10 Acaciae Flos Kaempferol-3-0-rhamnosylgalactosyl-7-

Acacia flowers rhamnoside (robinin), acacetin-7-0-rutino-
Robinia pseudoacacia L. side, acaciin.
Fabaceae Adulterant: Pruni spinosae flos
11 Anthemidis Flos Apigenin-7-0-glucoside, luteolin-7-0-gluco-

12 Chamomile flowers side.
13 Chamaemelum nobile (L.) Essential oil (see Essential Oil Drugs, Fig. 11,
ALLIONI (syn. Anthemis p.32)
nobilis L.)
Asteraceae
Ph. Eur. III, ÖAB
6 CactiFlos Isorhamnetin glycosides:

Night-blooming Cereus 1-3-0-galactoside (cacticin),
Cereus grandiflorus MILL. 1-3-0-galactosyl-rutinoside,
Cactaceae I-3-0-rutinoside (nareissin).
Rutin
165
Family /Pharmacopoeia Adulterants
5, 6 Calendulae Flos Isorhamnetin glycosides:

Marigold flowers I-3-0-glucoside,
Calendula officinalis L. I-3-0-rutinoside (narcissin),
Asteraceae I -3-0-rutinorhamnoside.
2. AB-DDR Quercetin-3-0-glucoside and Q-3-0-gluco-
rhamnoside,
Adulterant: Arnicae flos, Anthemis tinctoria L.,
Inula spp.
7,8 Crataegi Flos Glycosides of quercetin and apigenin

Hawthorn flowers Hyperoside, rutin, quercetin rhamnogalacto-
Crataegi Folium c. Flore side, vitexin, vitexin-2" -O-rhamnoside and
Hawthorn leaves with flowers other flavone-C-glycosides in varying concen-
DAB8 trations in all parts of the drug.
Crataegi Folium Adulterant: Acaciae flos
Hawthorn leaves
Helv. VI
Crataegi Fructus
Hawthorn fruits
Crataegus monogyna JAQUIN
emend. LIND MANN
Crataegus pentagyna, C. nigra,
C. azarolus L.
Rosaceae
5 Farfarae Flos Quercetin glycosides: rutin, hyperoside and

Coltsfoot flowers isoquercetin in varying concentrations in both
drug parts.
21,22 Farfarae Folium Adulterants: Petasites folium (Petasites hybri-
Coltsfoot leaves dus, P. albus, P. paradoxus): petasin, isopeta-
Tussilago farfara L. sin in varying concentrations.
Asteraceae
DAB 8 (leaves), Helv. VI (flowers)
13 Matricariae Flos 0.5--3% total flavonoids: quercimeritrin, api-

(Chamomillae Flos) genin-7-glucoside, luteolin-7-0-glucoside, pa-
German chamomlIe flowers tuletin-7-0-glucoside and more than 7 agly-
cones.
Chamomilla recutita (L.) St. RAu-
SCHERT Adulterant: Anthemidis flos.
(syn. Matricaria chamomilla L.)
Essential oil (see Essential Oi! Drugs, Figs. 11
Asteraceae & 12, p. 32).
Ph. Eur. III, 2. AB-DDR,
Helv. VI, ÖAB
6 Primulae FIos Glycosides of quercetin or gossypetin, kaemp-

Official primrose flowers ferol-dirhamnoside (Primula-flavonoside) and
K -3-0-gentiotrioside.
Primula veris L.
Primula elatior (L.) HILL. Adulterant: Verbasci flos.
Primulaceae
Remarks: for Primulae radix, see under Sa-
ponin drugs, Fig. 3, p. 236.
166
F amily/Pharrnacopoeia Adulterants
9 Pruni spinosae Flos Quercetin glycosides: rutin, avicularin (Q-3-

Blackthorn flowers O-arabinoside).
Prunus spinosa L. Kaempferol glycosides: K-3,7-0-dirhamno-
Rosaceae side, K-3-0-rhamnoside, K-3-arabinoside.
Adulterant: Robiniae flos.
7 Sambuci Flos Quercetin glycosides 1.5-3%: hyperoside,

Eider flowers isoquercitrin, rutin.
Sambucus nigra L.
Caprifoliaceae
2. AB-DDR, Helv. VI, ÖAB
9 Spiraeae Flos Spiraeoside (quercetin-4' -O-glucoside), hyper-

Meadow-sweet flowers oside, avicularin (quercetin-3-0-arabinoside).
Filipendula ulmaria L. Adulterant: Sambuci flos.
Rosaceae
7 Stoechados Flos N aringenin-5-monoglucoside (salipurposide),

(syn. Helichrysi flos) kaempferol-3-0-glucoside, K-3-0-digluco-
Cat's-foot flowers side, apigenin-7 -glucoside, luteolin-7 -O-glu-
coside.
Helichrysum arenarium (L.) DC
Asteraceae
Helv. VI
10 Tiliae Flos Quercetin glycosides: Q-3-0-g1ucoside, Q-3-

Lime flowers O-rhamnoside, Q-3-0-glucosyl-7-0-rhamno-
side.
Tilia cordata MILL.
Tilia platyphylla ScoP. Kaempferol glycosides: K-3-0-glucoside, K-3-
Tiliaceae O-rhamnoside, K -3-0-glucosyl-7 -O-rhamno-
side, K-3,7-0-dirhamnoside, K-p-coumaroyl-
DAB 8, Helv. VI, ÖAB,
glucoside (tiliroside).
2. AB-DDR
Myricetin glycosides: M-3-0-glucoside, M-3-
O-rhamnoside.
Adulterant: T. argentea.
7 Verbasci Flos 2-4% total flavonoids. The main flavonoids

Mullein flowers are rutin, hesperidin and other flavonol glyco-
sides.
Verbascum phlomoides L.
V. thapsiforrne SCHRADER Adulterants: Primulae flos, and other species
Scrophulariaceae ofVerbascum and Genista
Helv. VI, ÖAB
11 Betulae Folium About 1.5% Quercetin glycosides:

Birch leaves Q-3-0-arabinoside, Q-3-0-rhamnoside (quer-
citrin), Q-3-0-galactoside (hyperoside), Q-3-
Betula pendula ROTH.
O-rutinoside (rutin).
B. pubescens ERHART
Betulaceae Myricetin-3-digalactoside
Kaempferol-3-0-glucoside,
K-3-0-rhamnoside, Hesperidin.
2. AB-DDR
167
F amily/Pharrnacopoeia Adulterants
12 Juglandis Folium Hyperoside (ca. 0.2%) and other flavonol gly-

Walnut leaves cosides.
J uglans regia L.
Juglandaceae
15 Anserinae Herba Quercetin-3-0-glucoside, Q-3-0-rhamnoside.

Silverweed
Myricetin and myricetin rhamnoside.
Potentilla anserina L.
Rosaceae
16 Equiseti Herba Flavonoids: Luteolin-5-0-glucoside (galuteo-

Common horsetail lin), isoquercitrin, kaempferol-7-0-digluco-
side (equisetrin).
Equisetum arvense L.
Equisetaceae Saponins: Equisetonin (HI 660).
2. AB-DDR
6 Herniariae Herba Flavonoids: Rutin, narcissin.

See drug list on p. 148.
Coumarins: see Figs. 5 & 6, p. 156.
16 Leonuri Herba Rutin

Motherwort
Adulterant: Leonurus glaucescens.
Leonurus cardiaca L.
Lamiaceae
16 Rutae Herba Rutin

See drug list on p. 149. Coumarins: see Figs. 5 & 6, p. 156.
15 Sarothamni scop. Herba Scoparin (3' -O-methyl-orientin), vitexin.

Broom
Adulterant: Spartium junceum.
Sarothamnus scoparius (L.) WIM-
Alkaloids (see p. 86, sparteine).
MER
Fabaceae
DAC
16 Veronicae Herba Luteolin, L-7-0-glucoside, rutin.

Common speedweIl
Adulterant: Stachys alpina.
Veronica officinalis L.
Scrophulariaceae
14 Vigaureae Herba Quercetin glycosides:

(Solidaginis virgaureae herba) Q-3-0-rutinoside (rutin),
Golden-rod Q-3-0-rhamnoside (quercitrin),
Q-3-0-glucoside (isoquercitrin).
Solidago virgaurea L.
Asteraceae Kaempferol-3-0-glucoside (astragalin).
Adulterants: S. canadensis L., S. gigantea L.
15 Violae tricoloris Herba Quercetin glycosides: high content of rutin

Wildpansy (" Viola-quercitrin ").
Viola tricolor L. Adulterant: V. tricolor var. vulgo
Violaceae, ÖAB
168
F amily/Pharmacopoeia Adulterants
15 Sophorae Gemma Rutin (ca. 20%) and other flavonol glycosides.

Sophora buds
Sophora japonica L.
Fabaceae
17 Aurantii Pericarpium Eriocitrin, rutin, naringenin, naringin, hesperi-

Seville orange peel din, neohesperidin, sinensetin.
Citrus aurantium L. ssp. aurantium Adulterant: Aurantii albedo.
Rutaceae
Bitter principles: see Fig. p. 132.
Essential oiIs: see Fig. p. 44.
Helv. VI
Citri pericarpium Eriocitrin (eriodictyol-7 -O-rutinoside), rutin,
Lemonpeel naringenin -7-0- hesperoside, neohesperidin,
hesperidin, apigenin-C-glucoside; aureusidin,
Citrus media L.
Au-6-glucoside, Au-6-rhamnoglucoside; iso-
Rutaceae rhamentin-3-arabinoglucoside; limocitrol, L-
3-glucoside, L-3-arabinoglucoside; limocitrin,
L-3-g1ucoside, L-3-arabinoglucoside; luteo-
lin-7-rutinoside.
Bitter principles and essential oils: see p. 132
and p. 44.
Drugs containing predominantly flavonoid aglycones:

18 Eriodictyonis Herba Homoeriodictyol ( = eriodictyone), eriodictyol,
Yerba Santa (herba) chrysoeriodictyol, xanthoeriodictyol.
Eriodictyon glutinosum BENTH. Adulterant: Eriodictyon crassifolium Benth.
Hydrophyllaceae
Orthosiphonis Folium About 0.2% flavonoids:
Orthosiphon Ieaves sinensetin (3',4',5,6,7 -pentamethoxyflavone),
scutellarein tetramethyl ether and eupatorin
Orthosiphon spicatus (THUNB.)
(3' ,5-dihydroxy-4' ,6, 7-trimethoxyflavone).
Bak.
Lamiaceae
DAB 8, Helv. VI
19, 20 Cardni mariae Fructus Flavanolignans:

Milk-thistle fruits silybin, silychristin and silydianin.
Silybum marianum GAERTNER Adulterant: other Silybum spp.
Asteraceae
DAB8
169
v. Formulae of Constituents of Flavonoid Drugs
FLAVONES R R Aglycones Glycosides
OH H Apigenin Apigenin 8-C-glucoside

(=Vitexin)
Vitexi n-2" -O-rhamnoside
HO OH Luteolin Luteolin-8-C-glucoside
(= Orientin)
OH 0
FLAVONOLS R, R2 Aglycones Glycosides
OH OH H Quercetin Q-3-0-galactoside (Hyperoside)

Q-3-0-glucoside (Isoquercitrin)
HO Q-3-0-rhamnoside (Quercitrin)
R2 H H
Q-3-0-rutinoside (Rutin)
Kaempferol K-3-0-glucoside (Astragalin)
OH OH Myricetin M-3-0-digalactoside
OCH a H Iso- 1-3-0-rutinoside (Narcissin)
rhamnetin
OH 0
FLAVANON(OL)S R, R2 Ra Aglycones Glycosides

R3 H H OH Naringe- Nari ngeni n-7 -O-neohespe-
nin ridoside (Naringin)
HO H OH OH Eriodic- Eriodictyol-7 -O-ruti no-
tyol side (Eriocitrin)
H OCH a OH Homoerio-
dictyol
H OH OCH a Hespere- Hespereti n-7 -O-neohes-
OH 0 tin peridoside (Neohesperidin)
Hespereti n-7 -O-ruti noside
(Hesperidin)
OH OH OH Taxifolin
HO rAr0 .CH 20H
OH
HOuO'(~OCH'
~OH OH
HO 0
Amentoflavone Silybin
170
~OH
H°ItY°i···OOH
~OHOH 0 H
Sinensetin Taxifolin Coniferyl alcohol
Caffeic acid Quinic acid PHENOL CARBOXYLIC ACIDS

. -______~A~_ _ _ _ _, , - -_ _--'A\.-_ _----, Chlorogenic acid 3-Caffeoylquinic acid
Neochlorogenic acid 5-Caffeoylquinic acid
4-Caffeoylquinic acid
HO~ R: ;-l!}0H
1-Caffeoylquinic acid
H0-O--CH=CH-C+-O~ Isochlorogenic
acid
j1,3-DicaffeOYlqUinic
3,5-Dicaffeoylquinic
3,4-Dicaffeoylquinic
aicd
acid
acid
HO OH 4,5-Dicaffeoylquinic acid
Chlorogenic acid Cynarin (native) 1,3-Dicaffeoylqu inic acid
Cynarin (isoL) 1,5-Dicaffeoylquinic acid
m
PETASINS
~""OR
'O~ OR
CH3
I
o11 o11
CH3
I
Petasin (I)R=C-C=CH (IV) R= C-C=CH Isopetasin
I I
CH3 CH3
o11
(V) R = H (Isopetasol)
S-Petasin (11) R =C-CH=CHSCH3
( !!!) R =H (Petasol)
171
Reference Compounds Flavones, Flavonols, Flavanones, Phenol carboxylic acids
Test series A:
Tracks 1 = quercetin-3-0-gentiobioside
2 = kaempferol-3-0-gentiobioside
Fig. 1 3 = quercetin-3-0-rutinoside (rutin)
4 = vitexin-2" -O-rhamnoside
5 = naringin and neo hesperidin
6 = chlorogenic acid (Rf ca. OA5)
7=orientin
8=vitexin
9= isorhamnetin-3-0-glucoside (with isoquercitrin)
10= chlorogenic acid, isochlorogenic acid (Rf ca. 0.8), caffeic acid
(Rf ca. 0.9)
11 = isorhamnetin-3-0-galactoside
12 = quercetin-3-0-rhamnoside (with traces of astragalin)
13 = kaempferol-7-0-rhamnoside
14 = caffeic acid and ferulic acid (Rf 0.9-0.95)
15 = rutin (Rf ca. OA), chlorogenic acid (Rf ca. OA5), hyperoside (Rf ca. 0.6)
(standard mixture Tl; these are the three main compounds
for standardization of flavonoid chromatograms)
Test series B:
Tracks 1 = quercetin-3-0-gentiobioside
2 = quercetin-3-0-sophoroside
Fig.2 3 = quercetin-3-0-galactosyl-7-0-rhamnoside
4 = kaempferol-3-0-gentiobioside
5 = quercetin-3-0-rutinoside (rutin)
6 = kaempferol-3-0-rhamnoglucoside
7 = quercetin-3-0-glucuronide
8 = quercetin-3-0-galactoside (hyperoside)
9 = quercetin-3-0-glucoside (isoquercitrin)
10= kaempferol-3, 7-0-dirhamnoside
11 = quercetin-3-0-rhamnoside (quercitrin)
12 = kaempferol-3-0-arabinoside
13 = quercetin
14 = kaempferol
15=mixture of 1-14
Solvent F-l: ethyl acetate-formic acid-glacial acetic acid-water
system (100:11:11:27)
(NPjPEG No. 28, p. 303) UV-365 nm Fig. 1,2
Fig.l shows glycosides of flavones, flavonols and flavanones, which fluoresce orange,
yellow-green and dark green in UV-365 nm after treatment with NP/PEG reagent.
Phenol carhoxylic acids, which frequently occur in flavonoid drugs, appear as
intense, light blue zones.
Fig.2 shows flavonol glycosides of the aglycones, quercetin and kaempferol.
Orange or yellow-green fluorescence in UV-365 nm, following NP/PEG treat-
ment, is re1ated to the specific substitution pattern in ring B:
Two adjacent hydroxyl groups in ring B (e.g. quercetin) give rise to orange fluo-
rescence, whereas a single free hydroxyl group (e.g. kaempferol) results in yellow-
green fluorescence.
172
-FRONT
Rf
START
Fig.l
2 3 4 5 6 7 8 9 10 11 1213 14 15 TestreiheA
Test series A
- .. •
• •
•
e •
•
.t . ,
•• • • •I
..
Fig.2
1 2 3 4 5 6 7 8 9 1011 12 13 14 15 TestreiheB
Test series B
173
Flower Drugs I TLC Synopsis
Tracks 1 = Arnicae flos
2 = Stoechados flos
3 = Sam buci flos
4 = Pruni spinosae flos
5 = Tiliae flos
6 = Verbasci flos
7 = Calendulae flos
8 = Cacti flos
9= Primulae flos
Solvent F-1: ethyl acetate-formic acid-glacial acetic acid-water
system (100:11:11:27)
Detection Natural products reagent (NP No. 28, p. 303) UV -365 nm Fig. 3
For description of drugs see pp. 165-167. Formulae p. 170
Chromato- Each drug shows a characteristic order and number of yellow, green or blue fluores-
gram cent zones.
3,4 In drugs 1-6, flavonoids are absent in the lower Rf-range of the chromatogram.
The main flavonoids and acids are found between Rf 0.35 and the solvent front.
Drugs 7-9 show flavonoid glycosides especially at Rf 0.1-0.4. Drugs 7 and 8,
but not 9, also show a few weaker zones in the higher Rf range.
The numerous blue fluorescent zones of phenol carhoxylic acids are characteristic
of drugs 1-4; these are almost absent from drugs 5, 8 and 9, and only weakly
represented in drugs 6 and 7.
Figs. 3 and 4 show pronounced differences in the fluorescence colours of the
flavonoids. These gradations of colour amongst yellow, green and orange become
more distinct after treatment with NP/PEG reagent.
Characterization of these individual drugs 1-9, with identification of the main
flavonoids, is described on pp. 176 and 180, Figs. 5-10.
174
-FRONT
Rf
0 .5
START
Fig.3
2 3 4 5 6 7 8 9
-: -, -- -
-FRONT
Rf
- . · .. -tr
---
-0.5
-_.. ...-
: _ 11 1
• -
-
-
START
Fig.4
2 3 4 5 6 7 8 9
175
Flower Drugs 11
Tracks 1 = Arnicae flos
2 = F arfarae flos
3 = Calendulae flos (patterns land 11)
4 = Herniariae herba
5 = Cacti flos
6 = Primulae flos
Tests Tl = rutin (Rf ca. 0.3), chlorogenic acid (Rf ca. 0.4), hyperoside (Rf ca. 0.55)
Solvent F-l: ethyl acetate-formic acid-glacial acetic acid-water (100: 11: 11 :27)
system
(NPjPEG No. 28, p. 303) UV -365 nm Fig. 5, 6
For description of drugs see p. 165-168. Formulae p. 170
Chromato- 1 Arnieae jlos

gram In UV -265 nm, three orange fluorescent zones are seen at Rf 0.45-0.6: isoquercitrin
5 directly above hyperoside test, luteolin-7-0-glueoside at the same Rf as for the stan-
dard hyperoside test, and a third flavonol glycoside at Rf 0.45 (cf. Tl). Also present
are the weak, green fluorescent zone of a kaempferol monoglycoside (Rf ca. 0.7)
and two intense, blue fluorescent zones due to ehlorogenie acid (Rf ca. 0.4, cf.
Tl) and eajJeie acid (Rf ca. 0.9).
2 Farfarae jlos
This drug is characterized by three orange fluorescent zones. The main zone is
due to rutin (Rf ca. 0.3, cf. Tl); the other two, which migrate at Rf 0.6-0.7 (above
the hyperoside test), are due to flavonol monoglycosides. The blue fluorescent zones
are due to ehlorogenie acid (Rf ca. 0.4, cf. Tl), isoehlorogenie aeid (Rf ca. 0.75)
and cajJeie acid (Rf ca. 0.9).
3 Calendulae jlos
The chromatogram shows orange and yellow-green fluorescent zones in UV-365 nm,
which are most intense between Rf 0.15 and 0.4. Rutin (cf. Tl) lies between two
yellow-green fluorescent zones, which are due to narcissin (isorhamnetin-3-0-rutino-
side) (above rutin) and isorhamnetin rutinorhamnoside (below rutin). In addition,
there is a weak, green fluorescent zone of isorhamnetin-3-0-glueoside (Rf ca. 0.7)
and a weak, blue fluorescent zone in the same Rf region as eh/orogenie acid (cf.
Tl).
Differentiation
The main zone of rutin and nareissin in Calendulae jlos, and the zones of rutin
and isoehlorogenie acid in Farfarae jlos are easily recognized, even in mixtures with
Arnicae jlos.
6 4, 5, 6 Herniariae herba, Caeti jlos, Primulae jlos
Extracts of Herniariae herba and Caeti jlos, like those of Calendulae jlos (3), show
the typical zone of rutin and narcissin at Rf 0.3-0.35.
The orange fluorescent zones from Primulae jlos are due to glycosides of querce-
tin and gossypetin; the green fluorescent zones are due to kaempferol diglycosides.
In the lower Rf region a high concentration of flavonol triglycosides is evident.
176
FRONT
Rf
Fig.5
2 3
FRONT
Rf
Fig.6
4 5 6
177
Flower Drugs 111 Crataegi Folium, Fructus, Flos
Tracks 1 = Stoechados flos
2 = Sambuci flos
3 = Crataegi flos
4 = Verbasci flos
5 = Crataegi folium
6 = Crataegi fructus
Tests Tl = rutin (Rf ca. 0.35), chlorogenic acid (Rf ca. 0.45), hyperoside (Rf ca. 0.55)
T2 = vitexin-2" -O-rhamnoside (Rf ca. 0.35), vitexin (Rf ca. 0.7)
Solvent F -1: ethyl acetate-formic acid-glacial acetic acid-water
system (100 : 11 : 11 : 27) Fig. 7
F-2: ethyl acetate-formic acid-glacial acetic acid-
ethylmethyl ketone-water (50:7:3:30:10) Fig.8
Detection Natural products-polyethyleneglycol
reagent (NP/PEG No. 28, p. 303) UV- 365 nm Fig. 7, 8
Chromato- 1 Stoeehados flos

gram The flavanone glycoside, ( - ) or ( + ) naringenin-5-0-glueoside (salipurposide), which
7 is characteristic of this drug extract, appears as a black-brown zone at Rf ca. 0.8
(cf. Fig. 3, p. 175). Apigenin-7-0-glueoside (green-yellow fluorescence) and luteolin-7-
O-glueoside (orange fluorescence) migrate above the hyperoside test (cf. Tl) at Rf
0.6-0.7. The green-yellow fluorescent zone of kaempferol-3-0-glueoside lies directly
below the intense, blue fluorescent zone of eaffeie acid (Rf ca. 0.9). Other blue
fluorescent zones are due to ehlorogenie aeid (cf. Tl) or isoehlorogenie aeid (Rf
ca. 0.7).
2 Samhuci flos
The chromatogram shows two orange fluorescent zones of about equal intensity,
due to rutin (Rf ca. 0.35) and isoquercitrin (Rf ca. 0.6). Each is accompanied by
a weak, yellow-green fluorescent zone directly above it. Chlorogenie acid (cf. T1)
and eaffeie acid are present, as in drugs 1 and 3.
3 Crataegi flos
The chromatogram characteristically shows two strong, orange fluorescent zones
in the region of the hyperoside test due to hyperoside and a flavonol monoglycoside,
two strong, blue fluorescent zones in the region of the chlorogenic acid test (cf.
T1) (due to ehlorogenie aeid and neoehlorogenie aeid), a weak orange zone of rutin
(cf. Tl) and a blue fluorescent zone of eaffeie acid (Rf ca. 0.9). Rutin and vitexin-2"-
O-rhamnoside can be separated in solvent system F-2 (see Fig. 8).
4 J7erhaseiflos
There are three orange fluorescent flavone glycosides in the Rf region of the rutin
test, and above and below the hyperoside test. Phenol carboxylic acids are absent,
and an intensive yellow zone (flavonoid aglycones) is seen at the solvent front.
The yellow-green zone at Rf ca. 0.6 is due to hesperidin.
8 5,6 Crataegifolium (eumflore), Crataegifruetus
Using solvent system F-2 (a modified system, based on the addition of ethyl methyl
ketone), vitexin and vitexin-2"-O-rhamnoside (cf. T2) are separated from hyperoside
and rutin, respectively. Crataegi folium and flos contain weil detectable quantities
of these two compounds, whereas little or none is present in Crataegi fruetus, which
has a relatively low total flavonoid content. Chlorogenie acid and other phenol
carboxylic acids are present in all parts of the drug.
178
FRONT
Rf
Fig.7
2 3 4
FRONT
Rf
TART
Fig.8
5 6 3
179
Flower Drugs IV, with Their Most Common Adulterants
Tracks 1 = Pruni spinosae flos 4 = Robiniae (Acaciae) flos
2 = Sambuci flos 5 = Acaciae verticil. flos
3 = Spiraeae flos 6-10= Tiliae flos (patterns from official commercial
drugs I-V)
Tests T1 = rutin (Rf ca. 004), chlorogenic acid (Rf ca. 0.5), hyperoside
(Rf ca. 0.6)
system (100: 11 : 11: 27)
(NP/PEG No. 28, p. 303) UV-365 nm Fig. 9, 10

Chromato-
gram 1, 4 Pruni spinosae flos, A eaciae flos
1 Pruni flos' chromatogram is similar to that of Tiliae flos (Fig. 10) m showing
9 at least 8 strong, orange fluorescent zones in the Rf range 0.4-0.9:
rutin Rf ca. 0.35
kaempferol diglycoside ca. 0.4
isoquercitrin (Q-3-0-glucoside) ca. 0.55
kaempferol-3,7-dirhamnoside ca. 0.6
avicularin (Q-3-0-arabinoside) ca. 0.85
kaempferol-3-0-arabinoside ca. 0.9
In addition, there are two powerful, blue fluorescent zones in the same Rf range as the chIoro-
genie acid test.
4 Rohiniae (Aeaciae)flos
Predominantly green-yellow fluorescent zones in the lower third of the chromato-
gram. The main compound is rohinin (kaempferol-3-0-rhamnosyl-galactosyl-7-
rhamnoside) at Rf ca. 0.2. Aeaeetin-7-0-rutinoside migrates directly above rutin.
5 Extracts of flowers of Acacia verticil. show additional orange zones at Rf 0.6-0.75.
2, 3 Samhuci flos, Spiraeae flos
2 Samhuci flos is characterized by the presence of rutin, ehlorogenie acid (cf. T1) and
isoquercitrin (Rf ca. 0.65) (cf. Fig. 7, p. 178). .
3 Spiraeae flos is characterized chiefly by two main blue fluorescent zones in the
Rf region of the hyperoside test. The upper zone overlaps the green fluorescent
zone of spiraeoside (quercetin-4' -O-glucoside).
10 6-10 Tiliae flos
The flavonoid pattern of Tiliae flos extracts consists of at least 8 glycosides of
quercetin, myrieetin and kaempferol:
Rf ca. 0.9 tiliroside

ca. 0.8 Q-3-0-rhamnoside M-3-0-rhamnoside K-3-0-rhamnoside
{ Q-3-0-glucoside M-3-0-glucoside K-3-0-glucoside
ca. 0.7
Q-3,7-dirhamnoside K-3,7-dirhamnoside
ca. 0.4 rutin
Tiliae flos eommercial drugs show qualitative differences in flavonoid composition.

The flavone zone in the Rf range of the chlorogenie acid test is occasionally absent.
The adulterant, Tilia argentea, is recognizable by the presence of an additional
zone below the standard rutin test, and by the absence of rutin itself.
180
FRONT
Rf
Fig.9
2 3 4 5
FRONT
Rf
Fig.1O
7 8 9 10
181
Betulae, J uglandis Folium Anthemidis Flos
Tracks 1 = Betulae folium
2 = Juglandis folium
3 = Anthemidis flos
Tests Tl = rutin (Rf ca. OA), chlorogenie acid (Rf ca. 0.5), hyperoside (Rf ca. 0.6),
quercitrin (Rf ca. 0.8), kaempferol arabinoside (Rf ca. 0.9)
T2 = luteolin-7-0-glucoside
T3 = apigenin-7-0-glucoside
T4 = isochlorogenie acid (Rf ca. 0.75), chlorogenie acid,
caffeic acid (Rf ca. 0.85)
T5 = rutin (Rf ca. OA), chlorogenie acid (Rf ca. 0.5), hyperoside (Rf ca. 0.6)
T6 = rutin, hyperoside
T7 = rutin, hyperoside, caffeic acid
Adsorbent Silica gel 60F 254 pre-coated plates (Merck, Darmstadt) Fig. HA, B
Cellulose F 254 pre-coated plates (Merck, Darmstadt) Fig. 12A, B, C, D
Solvent F -1: ethyl acetate-formic acid-glacial acetic
system acid-water(100:11:11:27) Fig. HA, B
F -7: n-butanol-glacial acetic acid-water
(40: 10: 50), upper phase Fig. 12A, B, C, D
Detection Natural products-polyethyleneglycol UV-365 nm Fig. HA, B; 12B, D
reagent (NP/PEG No. 28, p. 303) VlS. Fig. 12A, C
Chromato- Betulae folium, Juglandis folium

gram Both drug extracts show a very similar pattern of flavonoids in the intermediate
11 A and upper Rf range.
1 Betulae folium shows hyperoside, quercitrin (cf. Tl), quereetin-3-0-arabinoside (Rf
ca. 0.9), traces of rutin, and a blue fluorescent zone of chlorogenie acid (cf. Tl).
There are also two further orange zones, one above and one below quercitrin.
11 B 2 Juglandis folium shows, in addition, a yellow-green zone at Rf 0.95, and an orange
zone above hyperoside. Rutin and chi orogenie acid are absent, but the neochloro-
genie acid is present.
12A, B The TLC separation of Betulae folium extract on cellulose, as specified by DAB 8,
shows main orange (vis.) zones in the Rf range 0.4-0.85.
3 Anthemidis flos
The chromatogram is characterized by the intense, light blue fluorescent zones of
"isochlorogenic acid" and chlorogenie acid (cf. T4 and T5), the intense, yellow-green
fluorescent zone of apigenin-7-0-glueoside (Rf ca. 0.7, cf. T3), the orange zone of
luteolin-7-0-glueoside (cf. T2) and a strong, yellow fluorescent zone of flavonoid
aglyeones at the solvent front (cf. also Fig. 13, p. 184). Apiin (apigenin apiosyl gluco-
side), a constituent reported in the literature, may be present in traces (Rf ca. OA5).
12C, D The TLC separation on cellulose, as specified by DAB 8, shows two orange (vis.)
zones and one yellowish (vis.) zone in the upper Rf range. In UV-365 nm, these
appear as almost white or dark blue fluorescent zones, respectively.
182
-• ..-- • • •11 ....
A e
~ -FRONT
Rf
• - -
~
.....
..., • I
-
• :
..•
.....
• 5
t'
TARl
Fig.11
Tl 2 T2 3 T3 T4 T5
A B c D
-FRONT
RI
-0.5
-START
Fig.12
T6 T6 T7 3 T7 3
183
Matricariae Flos "Herba Drugs" TLC Synopsis
Tracks 1-3 = Matricariae flos (commercial pattern) Fig.13
4 = Anthemidis flos
5 = Anserinae herba Fig.14
6 = Leonuri herba
7 = Virgaureae herba
8 = Sarothamni scopariae herba
9= Veronicae herba
10= Violae tricoloris herba
Test T1 = rutin (Rf ca. 0.4), chlorogenie acid (Rf ca. 0.45),
hyperoside (Rf ca. 0.55), caffeic/ferulic acid (Rf 0.9-0.95)
T2 = rutin (Rf ca. 0.4), chi orogenie acid (Rf ca. 0.5), hyperoside (Rf ca. 0.6),
"isochlorogenie acid" (Rf 0.75-0.85), caffeic acid (Rf ca. 0.9)
system (100: 11: 11:27)
(NP/PEG No. 28, p. 303) UV-365 nm Fig. 13, 14

Chromato-
gram 1-3 Matricariae (Chamomillae)flos
13 Quercimeritrin, luteolin- and patuletin-7-0-glucoside (constituents reported in the Iit-
erature) migrate at Rf ca. 0.6 in the same region as for the hyperoside test. A
weak, green fluorescent zone at Rf ca. 0.7 shows the same Rf as apigenin-7-0-
glucoside (cf. Anthemidis flos, Fig. 11, p. 183). A narrow, orange-yellow zone of
flavonoid agIycones is found at the solvent front.
Commercial drugs show variations in the concentrations of constituents in the
Rf-range 0.4 and 0.6.
The blue fluorescent zones are due to phenol carboxylic acids (chlorogenie, neo-
chlorogenie, isochlorogenie, caffeic and ferulic acid respectively).
4 Anthemidis flos can be distinguished from Matricariae flos, because of the high amount of
flavonoid aglycones (solvent front) and apigenin-7-0-glucoside (Rf ca. 0.7), and the presence
of luteolin-7-0-glucoside (below apigenin-7-0-glucoside). Both extracts have a similar pattern
of "acids", but Anthemidis flos also shows two very distinct blue fluorescent zones, one
directly above chlorogenie acid and the other at Rf ca. 0.8 (cf. Essential Oils, Figs. 11 and
12, p. 32).
14 5-10 TLC synopsis 0/" Herba dl'ugs" (see p. 186, Figs. 15 and 16)
Extracts of Anserinae herba (5) and Virgaureae herba (7) show similar patterns of
flavonoids, consisting of orange fluorescent quercetin monoglycosides (Rf 0.6-0.8)
and diglycosides (Rf 0.35-0.45).
Violae tricol. herba (10) is characterized by a high content of flavonoid di-, and
triglycosides at Rf 0.1-0.5, and by the absence of flavonoids in the upper Rf range.
Leonuri herba (6) and Veronicae herba (9) show similar patterns of "acids" at Rf
0.1-0.9.
Sal'othamni (Spartii) herba (8) is characterized by four yellow-green fluorescent zones
at Rf 0.6-0.85.
184
FRONT
Rf
START
Fig.13
Tl 2 3 4
FRONT
Rf
START
Fig. 14
T2 5 6 7 8 9 10
185
Herba Drugs
Tracks 1 = Anserinae herba 5 = Leonuri herba
2 = Violae tricoloris herba 6 = Veronicae herba
3 = Sarothamni herba 7= Rutae herba
4 = Sophorae gemma 8 = Equiseti herba
Tests Ti = rutin, chlorogenie acid, hyperoside
T2 = rutin (Rf ca. 0.35), chI orogenie acid (Rf ca. 0.45), hyperoside (Rf ca. 0.6),
isochlorogenie acid (Rf ca. 0.8), caffeic acid (Rf ca. 0.95)
system (100: 11 : 11: 27)
(NPjPEG No. 28, p. 303) UV-365 nm Fig. 15, 16
Chromato- 1 Anserinae herba

gram Extracts show 8 strong fluorescent orange zones, due to glycosides of quercetin
15 and myricetin, in the Rf range 0.3-0.9. Quercetin-3-0-glucoside (Rf ca. 0.6) and
myricetin- and quercetin-3-0-rhamnoside (Rf 0.65--0.7) mi grate above the hyperoside
test (T1), while the corresponding diglycosides migrate in the same region as the
rutin test (Ti).
2, 4 Violae herba, Sophorae gemma
Both drugs are characterized by a high content of rutin (cf. Ti), and by aseries
of predominantly orange fluorescent flavonol di-, and triglycosides in the Rf range
0.1-0.4.
3 Sarothamni scopariae (Spartii) herba
Chromatograms characteristically show a main yellow-green fluorescent zone of
scoparin (Rf ca. 0.7) and a green-yellow fluorescent zone of vitexin (Rf range of
the hyperoside test, cf. T1), together with three other weak, green fluorescent zones
(Rf 0.7-0.9) and 4-5 weak, blue fluorescent zones (Rf 0.2-0.4).
16 5,6 Leonuri herba, Veronicae herba
The chromatograms of these drug extracts show similar patterns of predominantly
blue fluorescent zones. Rutin is only detectable as a weak zone (cf. T2), but the
orange fluorescent zone of aglycones at the solvent front is more pronounced.
The blue fluorescent zones are due partly to phenol carboxylic acids (cf. T2, chloro-
genie and isochlorogenic acid).
7 Rutae herba
The drug is characterized by the orange main zone of rutin, and the blue-violet
fluorescent zones of coumarins (see also Coumarin drugs, Figs. 5 and 6, p. 156).
8 Equiseti herba
The orange fluorescent, main zone is due to isoquercitrin (Rf ca. 0.7). Galuteolin
(luteolin-5-0-glucoside), ferulic acid and caffeic acid appear as blue fluorescent zones
in the upper Rf range. About six more very weak blue or blue-green fluorescent
zones are present in the lower and intermediate Rf range.
Remarks: Red fluorescent zones at the solvent front are due to the chlorophyll fraction.
186
FRONT
RI
TART
Fig.15
2 3 4
FRONT
RI
Fig.16
T2 5 6 7 8
187
Citri, Aurantii Pericarpium
Eriodictyonis Herba, Orthosiphonis Folium
Tracks 1 = Citri pericarpium 3 = Eriodictyonis herba
2 = Aurantii pericarpium 4, 5 = Orthosiphonis folium
Tests Tl = rutin T3 = eriodictyol
T2 = homoeriodictyol T 4 = sinensetin
system (100: 11: 11 :27) Fig.17A, B
F-3: chloroform-acetone-formic acid
(75: 16.5: 8.5) Fig.18A
F-4: chloroform-ethyl acetate (60:40)
with and without chamber saturation Fig. 18B, C
Detection Natural products-polyethyleneglycol reagent UV-365 nm Fig. 17 A; 18A
(NP/PEG No. 28, p. 303) VlS. Fig.17B
Without chemical treatment UV-365 nm Fig. 18B, C
Chromato-
gram 1,2 Citri pericarpium, Aurantii pericarpium
17A, B After treatment with NPjPEG reagent, observation in UV-365 nm reveals the red-
orange fluorescent zone of eriocitrin (Rf ca. 0.4) and the yellow zone of rutin (cf.
Tl).
The broad zone of naringin, neohesperidin and hesperidin (dark green fluorescence
in UV-365 nm; ochre in vis.), which migrates directly above the eriocitrin zone,
can be used to distinguish between these two drugs; Citri pericarp. contains only
traces of neo hesperidin and hesperidin. The higher proportion of flavanones in
Aurantii pericarp. is indicated by two powerful, ochre (vis.) zones directly above
the violet zone of eriocitrin.
Aurantii pericarp. also shows additional, blue fluorescent zones in the Rf range
0.5-0.9; these are due to -methyl anthranilate, flavonoids (e.g. sinensetin) and couma-
rins.
Remarks: The eriocitrin zone becomes intensely red after 15 min exposure to UV-365 nm.
18A 3 Eriodictyonis herba (Yerba Santa)
After NP/PEG treatment and observation in UV-365 nm, the chromatogram (run
in solvent system F-3) characteristically shows yellow-green (homoeriodictyol (cf.
T2) and chrysoeriodictyol) and yellow-orange (xanthoeriodictyol) fluorescent zones
in the Rf range 0.55-0.75, and the zone of eriodictyol (cf. T3) at Rf ca. 0.4.
18 B 4 Orthosiphonis folium
U sing solvent system F -4, observation of the chromatogram in UV -365 nm without
chemical treatment shows 3-4 light blue fluorescent zones of flavone aglycones:
sinensetin (cf. T4) is the main zone directly below scutellarein methyl ether. Eupatorin
and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone are found at Rf 0.3-0.4.
18 C 5 Without chamber solvent saturation, the characteristic flavones of Orthosiph. folium
migrate at lower Rf values (Rf 0.1-0.4).
The chlorophyll fraction of the fresh drug (5) fluoresces red in UV-365 nm.
188
FRONT
Rf
-0 .5
-START
Fig.17
Tl 2 Tl 2
FRONT
RI
START
Fig.18
T2 3 13 T4 4 5
189
Cardui mariae (Silybi) Fructus
Tracks 1-4 = Cardui mariae fructus
Tests Tl = taxifolin (Rf ca. 0.4), silybin (Rf ca. 0.6)
T2 = silychristin
Solvent F-3: chloroform-acetone-formic acid (75: 16.5: 8.5)
system
(NPjPEG No. 28, p. 303) UV-365 nm Fig.19
Fast blue salt reagent (FBS No. 12, p. 301) VIS. Fig.20

Chromato-
gram 1-4 Cardui mariae fructus
19 After treatment with NP/PEG reagent, chromatograms are characterized in UV-
365 nm by the two intense, light yellow fluorescent zones of silybinjisosilybin (Rf
ca. 0.6, cf. Tl) and silychristin (Rf ca. 0.35, cf. T2), and by the yellow-orange
fluorescent zone of taxifolin (Rf ca. 0.4, cf. Tl). A third, pale yellow fluorescent
zone of silydianin, between taxifolin and silybin, is not always present in commercial
drugs. 1 The fluorescent zones above silybin are due to dehydro-derivatives of silybin
and isosilybin.
20 The same main zones become red-brown (vis.) after treatment with FBS/KOH re-
agent.
1 Remarks: There are chemical races of Silybum marianum, e.g. with high content of silydianin
and relatively low content of silybin and silychristin.
190
FRONT
RI
Fig.19
Tl T2 2 3 4
-FRONT
RI
-0.5
-START
Fig.20
2 3 4
191
Farfarae Folium Petasites Folium
Tracks 1 = Farfarae folium (light petroleum extract, p. 163)
2 = Petasites hybridi folium (light petroleum extract, p. 163)
3 = Petasites albi folium (light petroleum extract, p. 163)
4 = Petasites paradoxi folium (light petroleum extract, p. 163)
5 = Farfarae folium c. flore (MeOH extract, p. 163)
6 = Farfarae folium (MeOH extract, p. 163)
7 = Petasites folium (MeOH extract, p. 163)
Tests Tl =isopetasin (Rfca. 0.47)
T2=petasin
T3 = rutin (Rf ca. 0.4), chlorogenic acid (Rf ca. 0.5), hyperoside (Rf ca. 0.6)
T4 = chlorogenic acid, isochlorogenic acid (Rf ca. 0.8), caffeic acid (Rf ca. 0.9)
Solvent F-6: chloroform p.A. Fig. 21, 22A
system F-l: ethyl acetate-formic acid-glacial acetic acid-water Fig.22B
(100: 11: 11 :27)
Detection Anisaldehyde-acetic acid reagent (AA No. 1, p. 299) UV-365 nm Fig.21A
vis. Fig. 21 B
Sulphuric acid (H 2 S0 4 conc. No. 34C, p. 303) UV-365 nm Fig.22A
(NP/PEG No. 28, p. 303) UV-365 nm Fig.22B
Chromato- Farfarae folium, Petasites folium

gram After treatment with AA reagent or H 2 S0 4 and observation in UV-365 nm, petro-
21A, B leum extracts of the leaf drugs of Tussilago farfara (1) and Petasites hybridus,
22A P. albus and P. paradoxus (2, 3, 4) show similar chromatographic patterns, except
in the Rf range 0.35-0.45.
Petasin and isopetasin (cf. T2 and Tl) cause a distinct quenching of fluorescence
in UV-254 nm before chemical treatment. After treatment, they show green fluores-
cence in UV-365 nm, and a weak grey in the vis. Petasins are absent from Farfarae
folium, but present in Petasites in species-dependent concentrations:
2 Petasites hybridus: medium concentration of petasin and isopetasin.
3 Petasites albus: practically no petasin; albopetasin * is present.
4 Petasites paradoxus: rather high content of petasin and isopetasin.
* Albopetasin forms a reddish fluorescent zone (Rf 0.75-0.8) in UV-365 nm after treatment
with SbCl 3 reagent (No. 3, p. 299).
22B To a limited extent, Farfarae folium and Petasites spp. can also be distinguished
by the differentJlavone and acid pattern of their methanol extracts.
5,6,7 Flavonoids: Rutin is clearly demonstrable in Farfarae folium only when flower heads
are present in the drug (5). Petasites drugs show yellow to yellow-orange zones
(e.g. isoquercitrin) in or above the Rf-region of standard hyperoside; these may
also be present in lower concentration in Farfarae folium extracts. These zones
become more distinct when larger quantities (ca. 40 J.lI) of extract are applied to
the TLC; under these conditions, rutin also becomes detectable in both Farfarae
folium and Petasites folium (cf. Fig. 5, p. 176).
Phenol carboxylic acids: Farfarae folium and Petasites hybr. folium show practically
identical patterns of "acids" (chlorogenic, isochlorogenic, caffeic, ete.). Extracts
of Petasites paradoxus show an additional zone (Rf ca. 0.8) directly above that
of chlorogenic acid; this zone may be absent from other Petasites spp.
192
-FRONT
Rf
-0.5
START
Fig.21
Tl T2 2 3 4 T2 3 4
Fig.22
T2 2 3 4 T3 5 6 7 T4
193
Cardiac Glycoside Drugs
These drugs contain steroid glycosides which specifically affect the dynamics and
rhythm of the insufficient heart musc1e.
The steroids are structurally derived from the tetracyclic 10,13-dimethylcyclopen-
tanoperhydrophenanthrene ring system. They posses a y-lactone ring (cardenolides)
or a J-lactone ring (bufadienolides) attached in the ß-position at C-17. The sugar
residues are typically derived from deoxy-, and/or C-3-0-methylated sugars, and
they are linked glycosidically via the C-3-0H groups of the steroid aglycones.
I. Preparation of Drug-extracts for TLC

Powdered drug (1 g) is extracted by heating for 15 min under reflux with 20 ml
of 50% ethanol, with the addition of 10 ml of 10% lead(II) acetate soln. After
cooling and filtration, the c1ear soln. is treated with a small quantity of acetic acid,
then extracted by shaking with three 15 ml quantities of dichloromethane; shaking
must be gentle to avoid emulsion formation.
The combined lower phases are filtered over anhydrous sodium sulphate and
finally evaporated to dryness. The residue is dissolved in 1 ml dichloromethane/
ethanol (1: 1), and this soln. is used for chromatography.
All cardiac glycoside drugs can be extracted by this method. Since Strophanthi
semen has a high cardenolide content, a simplified extraction procedure can be
applied to this drug.
Strophanthi semen
Finely ground seeds (2 g) are defatted by heating for 1 h under reflux with light
petroleum. The defatted and dried seed powder (1 g) is extracted for 5 min with
10 ml ethanol at ca. 60° C. The filtered soln. is used directly for chromatography.

a) Pure reference substances
Convallatoxin: 30 mg are dissolved, in 10 ml of 80% ethanol on a water bath.
Gitoxin: 10 mg are shaken with 0.1 ml pyridine, then brought completely into solu-
tion by adding 2 ml methanol at 60° C.
Digoxin, lanatoside A, B, C, oleandrin, g- and k-strophanthin: separate solutions
of each compound are made by dissolving 5 mg in 2 ml methanol at 60° C.
b) Standard compounds from proprietary pharmaceuticals
Digitalis glycosides
Ten tablets or dragees (see Table I) are finely powdered in a mortar, than extracted
by heafing in a flask at 60° C for 5 min with 5-15 ml (depending on the weight
of powder) dichloromethane/ethanol (1: 1). The c1ear filtrate is evaporated to ca.
2 ml, and 20 1-11 of tbis soln. is used for chromatography.
195
Table 1
Constituent Examples of appropriate mg per tablet

pharmaceuticals ® or dragee
(*) see remark p. 52)
digitoxin Digimerck 0.1

pen taacety 19i toxin Cordoval 0.4
Carnacid
acetyldigitoxin Acylanid 0.2
acetyldigoxin Novodigal 0.2
methyldigoxin Lanitop 0.1
lanatoside A, B, C Digilanid 0.25
digoxin Lanicor 0.25
lanatoside C Cedilanid 0.25
Allocor 0.2
Strophanthus glycosides
Six tablets of Strophoral®, or 10 tablets of Purostrophan®, or 12 tablets of Stro-
phanon ® are finely powdered and extracted with 10 ml methanol for 5 min on
the water bath. 20 fJ.l of each filtrate are used for chromatography.
Scilla glycosides
Twenty dragees of Talusin ®, Sandoscill ®, Scilloral ®, or Scillaren ® are finely pow-
dered and extracted with 10 ml methanol for 5 min at ca. 60° C. 20 fJ.l of each
c1ear filtrate are used for chromatography.
Uzara and Thevetia glycosides
Five dragees of Uzara ® (total glycosides) or Encordin ® (peruvoside) are fine1y pow-
dered and extracted with 10 ml methanol for 5 min at 60° C. 20 fJ.l of each c1ear
filtrate are used for chromatography.
2. Adsorbent
Si li ca gel 60F 254 pre-coated plates (Merck, Darmstadt)
Depending on the total cardenolide or bufadienolide concentration, 30-50 fJ.l of
drug extracts are applied to the chromatogram. Solutions of test substances prepared
from the individual compounds: 5 fJ.1.
Reference solutions prepared from pharmaceuticals: 20 fJ.l
H-1 Ethyl acetate-methanol-water (100: 13.5: 10) ~ (81: 11 :8).
A generally applicable solvent system for cardiac glycosides.
H-2 Ethyl acetate-methanol-ethanol-water (81: 11: 4: 8). The addition of ethanol in-
creases the Rf values of strongly polar compounds, e.g. k-strophanthoside.
H-3 Methylethyl ketone-toluene-water-glacial acetic acid (40: 5: 3: 2.5: 1). Separa-
tions in this system are similar to those in H-1 and H-2. It is suitable for
the separation of Scilla glycosides.
H-4 Chloroform-methanol-water (65: 35: 10); lower phase. For chromatography of
Hellebori radix extracts.
III. Detection
Fluorescence quenching by cardenolides is only very weak in UV-265 nm, but more
distinct zones of fluorescence quenching are produced by bufadienolides.
Cardiac glycosides do not fluoresce in UV-365 nm.
196
2. Spray reagents
a) Specific detection oJ the y-Iactone ring (cardenolides)
Kedde reagent (No. 23, p. 302)
Immediatelyon spraying, cardenolides form a pink or blue-violet (vis.) colour. Bufa-
dienolides do not react.
The colour fades after a few minutes, but can be regained by repeated spraying
even after several days.
Remarks:
LEGAL reagent (alkali ne sodium nitroprusside soln.)
BALlET reagent (alkaline picric acid soln.)
RAYMOND reagent (alkali ne m-dinitrobenzene soln.)
They give red, red-orange or violet (vis.) colours with cardenolides.
b) General detection methods Jor cardenolides and buJadienolides
IX) Antimony (III) chloride reagent (SbCl 3 No. 3, p. 299)
The TLC plate is sprayed with at least 10 ml SbCl 3 reagent, heated at 100° C for
about 6 min, then observed immediately in UV-365 nm (see Table 11). Changes are
observed in the fluorescence response, if the sprayed plate is allowed to stand for
a longer time. In visible light, the zones appear mainly violet or brown.
Table 2
Cardiac glycoside Fluorescence in UV-365 nm

(after SbCI 3 and 100° C)
K- and g-strophanthidine derivatives

K-strophantoside, k-strophanthidin-ß } orange, pale brown or
Cymarin, helveticoside, erysimoside, yellow-green
G-strophanthin, convallatoxin
Digitoxin, acetyldigitoxin }
dark blue or dark brown
Purpurea glycoside A, lanatoside A
Gitoxin, digoxin }
light blue
Purpurea glycoside B, lanatoside BjC
Oleander glycosides light blue
Bufadienoüdes
Proscillaridin, scillaren A, glucoscillaren yellow-brown
Scilliroside, glucoscilliroside pale green
Hellebrin, helleborogenone yellow
ß) Chloramine-trichloroacetic acid reagent (CT AN o. 7, p. 300)

Blue, yellow or yellow-green fluorescent zones are observed in UV -365 nm, similar
to those obtained with SbCl 3 reagent. Only weak, unspecific colours are seen in
visible light.
y) Sulphuric acid reagent (conc. H 2 S0 4 No. 34, p. 303)
The TLC plate is sprayed with ca. 5 ml of reagent, then heated for 3-5 min at
100° C under observation.
Blue, brown, green and yellowish fluorescent zones are seen in UV-365 nm;
the same zones appear brown or blue in daylight.
Remarks: Liebermann-Burchard reagent (LB No. 16, p. 301) can also be used for detection
of cardiac glycosides.
197
IV. List of Cardiac Glycoside Drugs
F amily /Pharmacopoeia
5,6, 7, 8 Digitalis lanatae Folium Total cardenolide content ca. 1%, comprising
White foxglove leaves over 60 different compounds.
Digitalis lanata ERHARD Lanatosides A and C constitute ca. 50% of
Scrophulariaceae total cardenolides. Lanatosides B, D, E, di-
USP XX, DAB 8, ÖAB goxin and digitoxin are present only in lower
concentrations.
DAB 8: Digitalis lanata powder
standardized at ca. 0.5% digoxin
activity
Digitalis purpureae Folium Total cardenolide content 0.15-0.4%.
Red foxglove leaves (Ph. Eur. III specifies not less than 0.3% re-
Digitalis purpurea L. lated to digitoxin); ab out 30 glycosides are
Scrophulariaceae present.
Purpurea-glycosides A and B constitute ca.
Ph. Eur. III, Helv. VI, ÖAB 60% of the mixt ure ; digitoxin ca. 12 %; gi-
DAB 8: Digitalis purpurea powder toxin and gitaloxin 10% each.
standardized at ca. 1 % digitoxin The major cardenolides of both species are
activity derivatives of digitoxigenin, gitoxigenin and
digoxigenin.
9, 10 Nerii (oleandri) Folium Total cardenolide content 1-1.5%

Oleander leaves Odoroside A and H (derived from the agly-
Nerium oleander L. cone, digitoxigenin) ; oleandrin, oleandrin
Apocynaceae monoglucoside (glucosyl oleandrin), gentio-
DAC biosyl oleandrin and nerigoside (all derived
from the aglycone, oleandrigenin).
Oleasides A and E are glycosides of the olea-
genin series. Adynerin is derived from adyneri-
genin.
11,12 Adonidis Herba Total cardenolide content ca. 0.25%, compris-

15, 16 Adonis ing about 20 glycosides.
Adonis vernalis L. Adonitoxin (adonitoxigenin-3-0-rhamnoside)
Ranunculaceae (0.07%) is one of the main glycosides, accom-
DAB8 panied by k-strophanthidin glycosides (e.g.
cymarin, 0.02%)
DAB 8: Adonis powder standard-
ized at ca. 0.2% cymarin activity Flavonoid: adonivernith (a flavone C-glyco-
side)
Convallariae Herba Total cardenolide content 0.2-0.3%, compris-
Lily of the valley ing about 20 glycosides.
Convallaria majalis L. The major glycosides, convallatoxin, convallo-
Liliaceae side and glucoconvalloside are derived from
DAB8 k-strophanthidin (=convallatoxigenin). Con-
vallatoxol, convallatoxoloside and glucocon-
DAB 8: lily of the valley powder vallatoxoloside are derived from k-strophan-
standardized at ca. 0.2% convalla- thidol.
toxin activity
Convallatoxin is the main glycoside in drugs
of western and northern European origin and
represents 40-45% of the glycosides. In mid-
dIe European drugs, lokundjoside predomi-
nates.
198
F amily jPharmacopoeia
11,12 Strophanthi grati Semen Total cardenolide content 4-7%, with 90-95%
13,14 Strophanthus seeds g-strophanthin, together with small quantities
Strophanthus gratus (WALL et of sarmentosides A, D, E.
HOOK.) FRANCHET
Apocynaceae
Strophanthi kombe Semen 5--10% cardenolides, derived from k-stro-
Strophanthus seeds phanthidin.
Strophanthus kombe Oliver The glycoside mixture, called "k-strophan-
Apocynaceae tin", consists of 80% k-strophanthoside and
k-strophanthin-ß, and 10-15% erysimoside
and cymarin.
Minor glycosides are cymarol, helveticosol
and periplocymarin.
17 Xysmalobii Radix The main glycosides are the mono- and diglu-
U zarae radix cosides of the aglycones, xysmalogenin (5,6-
Uzara root dehydrodigitoxin) and uzarigenin. The diglu-
Xysmalobium undulatum cosides, uzarin and xysmalorin, are the main
R.BROWN compounds. Uzarigenin differs from digitoxin
Asc1epidiaceae in possessing a trans linkage of rings A and
B.
18 Hellebori Radix The bufadienolide content and glycoside pat-

Hellebore root tern depend on the origin of the drug.
Helleborus niger L. The main glycoside in H. viridis and H. odor-
Helleborus odorus W ALDST. et KIT. us is hellebrin (ca. 0.5%), which is a gluco-
Helleborus viridis L. and other rhamnoside of hellebrigenin.
Helleborus spp. Other Helleborus spp. are very often free of
Ranunculaceae hellebrin.
19,20 Scillae Bulbus White variety: 0.2-0.4% bufadienolides, com-

White squill (var. alba) prising about 15 glycosides derived from scil-
Red squill (var. rubra) larenin.
Urginea maritima L. Main glycosides are proscillaridin
var. alba BAKER (0.005--0.05%), scillaren A (ca. 0.06%) and
Liliaceae glucoscillarin (ca. 0.005%). Scilliglaucoside,
DAB 8, Helv. VI scillaphaeoside and scillacyanoside are minor
constituents.
DAB: squill powder standardized
at ca. 0.5% proscillaridin activity Red variety: 0.04-0.1 % bufadienolides.
Main glycosides are scilliroside and glucoscil-
liroside, which are derived from scillirosidin.
As in the white variety, proscillaridin and scil-
laren Aare present but in low concentration.
199
v. Formulae of Constituents of Cardiac Glycoside Drugs
HO HO H
H
Cardenolide: Digitoxigenin Bufadienolide: Bufalin
DIGITALIS GL YCOSIDES
Purpureaglycoside A Digitoxigenin -Dox-Dox-Dox-G Iucose

Lanatoside A Digitoxigenin -Dox-Dox-(Dox-Ac)-Glucose
Digitoxin Digitoxigenin -Dox-Dox-Dox
ex/ß-Acetyldigitoxin Digitoxigenin -Dox-Dox-(Dox-Ac)
Purpureaglycoside B Gitoxigenin -Dox-Dox-Dox-Glucose
Lanatoside B Gitoxigenin -Dox-Dox-(Dox-Ac)-Glucose
Gitoxin Gitoxigenin -Dox-Dox-Dox
ex/ß-Acetylgitoxin Gitoxigenin -Dox-Dox-(Dox-Ac)
Lantoside C Digoxigenin -Dox-Dox-(Dox-Ac )-G Iucose
ex/ß-Acetyldigoxi n Digoxigenin -Dox-Dox-(Dox-Ac)
Digoxin Digoxigenin -Dox-Dox-Dox
Dox = Digitoxose
Dox-Ac = Acetyldigitoxose
Purpureaglycoside A: R, = R2 = H Lanatoside A: R, = H; R2 = Acetyl

Purpureaglycoside B: R, = OH; R2 = H Lanatoside B: R, = OH; R2 = Acetyl
200
ILanatoside AI IPurpureaglycoside AI IPurpureaglycoside 8 I ILanatoside 81
t
I Acetyl-Digitoxin
t
1--1 Digitoxin I
t t
IGitoxinl- I Acetyl-Gitoxinl
~ t .-----'----.t ~
I Digitoxigenin I IGitoxigenin I
o
HO HO
I Lanatoside cl IGlucogitaloxinl
t
IAcetyl-Digoxin I IGitaloxi nl
t t
I Gitaloxigeninl
IDi90xigenini
o
HO HO
201
o o
OCOCH3
Oleandrosyl-O oI H
H
Thevetosyl
Oleandrin Peruvoside
o
o
p
K-Strophanthidin: R = -C~
H
HO Strophanthidol: R = -CH 20H
Rha-O OH Periplogenin: R = - CH 3
OH
g-Strophanthin
Rha-O R, R2 R3
Convalloside CHO H Rhamnosyl-Glucosyl
Convallatoxin CHO H Rhamnosyl
Adonitoxin Convallatoxol CH,oH H Rhamnosyl
Convallatoxoloside CH,oH H Rhamnosyl-Glucosyl
Lokundjoside CH 3 OH Rhamnosyl
202
o
Gluc- Gluc-Rha-O Gluc-O

1Gluc -1Gluc
I Glucoscillaren AI ---- Scillaren A-
- - Proscillaridin A ~ Scillarenin GI UCOSCI"11""d
Irosl e-1GIUC " I ----
- - - lsCI"li"Iroslde 1Gluc SClllirosidin
RO RO
Gluc-Rha-O
OH
Hellebrin Uzarigenin: R = H Xysmalogenin: R = H
Uzarin: R = Gluc - Gluc Xysmalorin: R = Gluc - Gluc
203
TLC Synopsis of Cardiac Glycosides 11
Tracks 1 = g-strophanthin 8=digoxin
2 =" k-strophanthin" 9= gitoxin
3 = convallatoxin 10=digitoxin
4=cymarin 11 =cymarol
5 = lanatoside A 12 = peruvoside
6 = lanatoside B 13 = oleandrin
7 = lanatoside C
Solvent H-1: ethyl acetate-methanol-water (81: 11 : 8)
system
Detection Kedde reagent (No. 23, p. 302) vis. Fig. 1
Chloramine-trichloroacetic acid reagent
(CTA No. 7, p. 300) UV-365 nm Fig.2
Chromato- Kedde reagent (vis.)

gram Immediately after spraying, cardiac glycosides 1-13 form blue to red-violet colours.
1 With the exception of peruvoside, these colours are fairly stable.
Digoxin and lanatoside C, red-violet.
Gitoxin and Ianatoside B, blue-violet.
Digitoxin and lanatoside A, blue.
The colour range is indicative of the structural type.
2 CTA reagent (UV-365 nm)

All cardiac gIycosides give light blue, blue-green or yellow-green fluorescent zones.
Blue-green fluorescence is given by the Strophanthus, Convallaria and Thevetia glyco-
sides: g- and k-strophanthin (for TLC analysis ofthis gIycoside mixture, see Fig. 14,
p. 216), cymarin, cymaroI, convallatoxin and peruvoside.
Intense, light blue fluorescence is given by the Digitalis and Oleander glycosides,
with the exception of digitoxin, which fluoresces yellow-green.
After CT A treatment, chromatograms of some standard substances show additional zones

in UV-365 nm, due to degradation products and impurities.
204
FRONT
Rf
-0.5
Fig.l
2 3 4 5 6 7 8 9 10 11 12 13
Fig.2
2 3 4 567 8 9 10 111213
205
TLC Synopsis of Cardiac Glycosides U 1
Tracks 1 = g-strophanthin 8=digoxin
2 =" k -strophanthin" 9=gitoxin
3 = convallatoxin (Rf ca. 0.30) lO=digitoxin
4=cymarin l1=cymarol
5 = lanatoside A 12 = peruvoside
6 = lanatoside B 13 = oleandrin (Rf ca. 0.9)
7 = lanatoside C 14 = hellebrin (Rf ca. 0.15)
15 = proscillaridin
Solvent H -1: ethyl acetate-methanol-water (81 : 11 : 8)
system
Detection Sulphuric acid reagent (conc. H 2 S0 4 No. 34, p. 303) vis. Fig.3
UV-365 nm Fig.4
Chromato- 1 g-Strophanthin is immediately recognizable in UV-365 nm as a distinct, yellow-brown

gram fluorescent zone. In visible light a weak brown appears if the TLC plate is exposed
3, 4 for 15 min to the air.
2,4 Strophanthus glycosides, and convallatoxin (3) show only weak, brown in vis., but
intense, blue or yellow-green fluorescent zones in UV-365 nm.
5-10 The Digitalis glycosides give violet or brown in vis., and light to dark blue fluores-
cence in UV-365 nm (see also Fig. 2).
12 Peruvoside gives a fading brown in vis. and blue fluorescence in UV-365 nm.
13 Oleandrin shows brown in vis., and a radiant blue fluorescence in UV-365 nm.
14 Hellehrin gives brown in vis., and a green-brown fluorescence in UV-365 nm.
15 Proscillaridin shows violet in vis., and a yellow fluorescence in UV-365 nm.
Remarks: Treatment with chloramine-trichloroacetic acid reagent (eTA No. 7, p. 300) pro-
duces similar fluorescent zones in UV-365 nm, but only weak colours in visible light.
After treatment with conc. H 2 S0 4 , additional zones appear in UV-365 nm, due to impurities
and degradation products.
206
-FRONT
Rf
, ....
.. .-
-0.5
n -START
Fig.3
2345678 91011 12131415
FRONT
Rf
START
Fig.4
2 3 4 56 7 8 9 10 11 12 13 1415
207
Digitalis Folium
Tracks 1-3 = Digitalis lanatae folium 4-6 = Digitalis purpureae folium
(various commercial patterns) (various commercial patterns)
Tests Tl =lanatoside A T4=digoxin T = mixture of gitoxin
T2 = lanatoside B T5 = gitoxin and digitoxin
T3 = lanatoside C T6 = digitoxin
Solvent H-l: ethyl acetate-methanol-water (81: 11: 8)
system
Detection Chloramine-trichloroacetic acid reagent
(CTA No. 7, p. 300) UV-365 nm Fig.5A
Kedde reagent (No. 23, p. 302) vis. Fig. 5B, 6A, C
Antimony(III) chloride reagent (SbC1 3 No. 3, p. 299) vis. Fig.6B
Chromato- 1 Digitalis lanatae folium

gram After CTA treatment, ab out 9 predominantly blue and blue-green fluorescent zones
5A (UV-365 nm) are seen in the Rf range 0.25-0.75. Weak, blue fluorescent zones of
gitoxin (T5) and digitoxin (T6), and stronger, blue or blue-green zones of lanatosides
A, B, C (cf. Tl-T3) are present. Lanatoside A forms a major zone, while lanatosides B
and C show lower concentrations.
In addition, a green fluorescent zone is present at the origin, and two fluorescent
zones, one green and one orange, are seen at the solvent front.
5B 1 With Kedde reagent, these cardenolides form blue or red-violet (vis.) zones. Lanatosi-
de A is again a major zone.
The orange zones in the region ofthe solvent front are flavones or anthraquinones
(e.g. digitolutein); to some extent, they overlap the genins (red-violet zone).
5A 4 Digitalis purpureae folium
After CTA treatment the chromatogram is similar to that of Digitalis lanatae folium
in the Rf range 0.45 to 0.75, except that gitoxin and digitoxin (T4 and T5) are
more prominent. Purpurea-glycosides A and Bare present at Rf 0.2-0.25, the first
migrating as a main zone at about the same Rf as the lanatoside C test (T3).
5B 4 With Kedde reagent, Purpurea-glycoside A appears as a distinct, blue-violet (vis.)
main zone.
6A,C Digitalis extracts 2 and 5 originate from commercial drugs with relatively high con-
tents of primary glycosides. Digitalis extracts 3 and 6 are from drugs that have
been stored for a rather long time, so that the primary glycosides have become
largely degraded (see Figs. 7 and 8, and accompanying explanations).
6B The primary glycosides, lanatosides A, B, C (cf. Tl, T2, T3) mi grate at Rf 0.2-0.25,
while the secondary glycosides, gitoxin, digoxin and digitoxin are found at Rf
0.5-0.7.
After treatment with SbC1 3 reagent, they form grey-blue (vis.) zones (cf. Figs. 7
and 8, p. 210).
208
-FRONT
Rf
-0 .5
START
Fig.5
Tl-2-3 4 T T6 Tl-2-3 4 T5 T6
A
-FRONT
Rf
-0 .5
-START
Fig.6
T1·2-3 2 T5 3 Tl-2-3 T4 T5 T6 5 T6 6
209
Digitalis lanatae Folium Digitalis purpureae Folium
TLC comparison
Tracks 1 = Digitalis lanatae folium (commercial drug, DAB 8 quality)
2= Digitalis lanatae folium (commercial drug, stored, fermented)
3= Digitalis purpureae folium (commercial drug, DAB 8 quality)
4 = Digitalis purpureae folium (commercial drug, stored, fermen ted)
Tests Tl-3 = lanatosides A, B, C
T4=digoxin
T5=gitoxin
Solvent H-l: ethyl acetate-methanol-water (81: 11: 8)
system
Detection Antimony (III) chloride reagent (SbCI 3 No. 3, p. 299) UV-365 nm Fig.7
vis. Fig.8
For description of drugs see p. 198. Formulae pp. 200-201
Chromato- As seen in Figs. 5 and 6, commercial drugs show very variable contents of primary
gram and secondary glycosides. This is due to different rates of c1eavage of primary into
7,8 secondary glycosides (e.g. Purpurea-glycoside A into digitoxin).
During storage of the drugs, plant enzymes (digipurpidase and digilanidase) preferentially
remove the terminal glucose residues of Purpurea-glycosides. The lanatosides are more stable,
due to the presence of the acetyl group.
TLC comparison of
1, 2 Digitalis lanatae folium and
3,4 Digitalis purpureae folium
After SbCl 3 treatment and inspection in UV-365 nm or vis., the differences between
standardized official drugs (1 and 3) and stored and/or fermented drugs (2 and
4) are very apparent. In Digitalis purpurea, the primary glycosides are less stable
than in Digitalis lanata, as evidenced by the strong zones of digitoxin and gitoxin
(cf. T5).
After 2 hours maceration of Digitalis purpureae folium powder in water, the
primary glycosides are c1eaved into the secondary glycosides. If this mixture is ex-
tracted with DCM (see p. 195), a glycoside mixture is obtained, which gives the
TLC pattern shown on track 4.
210
Fig.7
Tl-2 - 3 2 3 4 T4 T5
-FRONT
Rf
-0.5
-START
Fig.8
Tl - 2- 3 2 3 4 T4 T5
211
Nerii (oleandri) Folium
Tracks 1 = Nerii oleandri folium
Tests Tl = oleaside E
T2 = gentiobiosyloleandrin (partially purified reference substance)1
T3 = glucosylnerigoside (partially purified reference substance)1
T4 = glucosyloleandrin (Rf ca. 0.4)1
T5 = odoroside H
T6 = odoroside A
T7 = oleaside A
T8 = nerigoside (Rf ca. 0.7) and adynerin (Rf ca. 0.8)
T9 = oleandrin
Tl 0 = mixture of Oleander glycosides (from Roth Co.)
Tll = adynerin
Solvent H-l: ethyl acetate-methanol-water (81 : 11: 8)
system
Detection Sulphuric acid reagent (conc. H Z S0 4 No. 34, p. 303) vis. Fig.9
Kedde reagent (No. 23, p. 302) VlS. Fig. IOA
(CTA No. 7, p. 300) UV-365 nm Fig. lOB
For description of drug see p. 198. Formulae p. 202
Chromato- 1 Nerii/olium extracts produce 13-15 zones ofcardenolides in the Rfrange 0.1-0.95.
gram Oleander extracts normally show a high cardenolide concentration. A large
9,10 number ofindividual compounds have been found which occur in varying concentra-
tions, depending on the origin ofthe drug. Oleandrin and adynerin are major constitu-
ents. Freshly harvested drug also contains high concentrations of primary glycosides
(e.g. glucosyloleandrin).
Kedde reagent produces very stable, intense, blue to red-violet (vis.) zones. With
erAreagent, the same compounds form mostly light blue, with some yellow-green,
fluorescent zones in UV-365 nm; and with sulphuric acid reagent, they form zones
with colour gradations of red or brown-green (vis.) (see list below, and chromato-
gram in Fig. 9).
9 Treatment with sulphuric acid reagent pro duces the following coloured (vis.) zones:
Glycosides %leandrigenin (red):
oleandrin Rf ca. 0.9 (T9)} .
nerigoside Rf ca. 0.7 (T8) monoglycosldes
glucosyloleandrin Rf ca. 0.1 (T2)
glucosylnerigoside Rf ca. 0.4 (T3) } d'19IYCOSI'des
Glycosides 0/ digoxigenin (brown):
odoroside A Rf ca. 0.8 (T6)
odoroside H Rf ca. 0.55 (T5)
Glycosides %leagenin (green-brown):
oleaside E Rf ca. 0.1 (Tl)
oleaside A Rf ca. 0.75 (T7)
Glycosides 0/ adynerigenin (red-violet):
adynerin Rfca.0.8 (Tl 1)
1 Some of the test compounds were isolated from the drug and only partially purified.
212
-FRONT
Rf
-0.5
-START
Fig.9
Tl T2 T3 T4 T5 T6 T7 T8
FRONT
Rf
START
Fig. 10
T9 T 10 T 11 T 10 T9
213
Strophanthi Semen
Adonidis and Convallariae Herba TLC Comparison
Tracks 1 = Strophanthi grati semen
2 = Strophanthi kombe semen
3 = Adonidis herba
4 = Convallariae herba
Tests Tl = g-strophanthin
T2 = "k-strophanthin"
T3 = convallatoxin
Solvent H -1: ethyl acetate-methanol-water (81 : 11 : 8)
system
Detection Sulphuric acid reagent (cone. H 2 S0 4 No. 34, p. 303) VIS. Fig.11
UV-365 nm Fig.12
Chromato- The fOUf drug extracts can be quickly differentiated by chromatography in solvent
gram system H-1, treatment with sulphuric acid reagent, and inspection in UV-365 nm
11, 12 or vis:
1 Strophanthi grati semen is characterized by g-strophanthin (cf. Ti) at Rf ca. 0.05
(pale fluorescent zone in UV-365 nm; brown zone in vis.).
2 Strophanthi kombe semen shows the three characteristic, blue-green fluorescent (UV-
365 nm) zones of" k-strophantin" mixture (cf. T2) in the lower Rf range. The glyco-
sides, cymarin and helveticoside, migrate ahead of "k-strophantin" at intermediate
Rfvalues (see also Figs. 13 and 14, p. 216).
These major glycosides form brown zones in vis.
3 Adonidis herba is characterized in UV-365 nm by at least 10 predominantly blue
fluorescent zones between Rf 0.3 and the solvent front. The same zones appear
blue, red-violet and brown in vis.
4 Convallariae herba shows green-blue fluorescent zones in the Rf range 0.2-0.35;
these inc1ude the major zone of convallatoxin (cf. T3; Rf ca. 0.3), which also appears
brown in vis.
Apreeise chromatographie identijication and characterization of the individual drugs
is given in Figs. 13 and 14, p. 216 (Strophanthi semen), and in Figs. 15 and 16,
p. 218 (Adonidis and Convallariae herba);
214
, r · n,..."0.., T
0.5
-START
Fig.11
Tl 2
e ....... -
.-
.
••
-
... _....
,....
I - --
•
•-
....
.....
~
..
Fig.12
215
Strophanthi Semen
Tracks 1 = Strophanthi grati semen
2 = Strophanthi kombi: semen
Tests Tl = g-strophanthin
T2 =" k-strophanthin"
T3 = k-strophanthoside
T4 = k-strophanthin-ß
T5 = erysimoside
T6 = helveticoside
T7=cymarin
Solvent H-l: ethyl acetate-methanol-water (81 : 11 : 8)
system
Detection Kedde reagent (No. 23, p. 302) VIS. Fig. 13A
(CTA No. 7, p. 300) UV-365 nm Fig.13B
Sulphuric acid reagent (cone. H 2 S0 4 No. 34, p. 303) vis. Fig. 14A
SbCI 3 re agent (No. 3, p. 299) UV-365 nm Fig.14B
Chromato- 1 Strophanthi grati semen

gram Kedde reagent produces a powerful violet (vis.) zone at Rf ca. 0.1, due to g-strophan-
13A thin (cf. Tl). Ahead of g-strophanthin, up to Rf ca. 0.45, are 4-5 weaker zones,
due partly to sarmentosides.
13B After CTA treatment, the only distinct blue-green fluorescent zone in UV-365 nm
is that of g-strophanthin. A blue fluorescent zone and a broad, greenish fluorescent
zone (lipids) are present below the solvent front.
14B SbCl 3 treatment pro duces two powerfully yellow fluorescent (UV-365 nm) zones,
identical with those from standard g-strophanthin (Tl), and ab out 5 other yellow
zones up to Rf ca. 0.4 (cf. Fig. 13A).
13 A 2 Strophanthi komM semen
Treatment with Kedde reagent reveals k-strophanthoside direct1y above the start,
and the closely juxtaposed zones of k-strophanthin-p and erysimoside at Rf ca. 0.2.
The intermediate Rf range contains four weaker zones, the upper two being
helveticoside and cymarin (cf. Fig. 14B).
14A Treatment with conc. H 2 S0 4 reveals a similar pattern (vis.) to that obtained with
Kedde reagent.
13 B After treatment with CTAreagent, the glycosides are revealed as light green or
yellowish fluorescent zones in UV-365 nm. The zones ne ar the solvent front are
due to the lipid fraction of the seeds.
14B With SbCl 3 reagent, cardiac glycosides are revealed as intense, yellow fluorescent
zones in UV-365 nm (cf. standards T3-T7).
Remarks: The presence of Strophanthus sarmentosus is recognizable by the detection of very
high concentrations of sarmentosides at Rf 0.25--0.4, sarmentoside A at Rf ca. 0.4, and of
sarmentocymarin and saveroside at RfO.7-0.9.
216
FRONT
Rf
0 .5
Fig.13
Tl 2 T2 Tl 2 T2
FRONT
Rf
START
Fig.14
2 Tl 2 T3 T4 T5 Tb T7 12
217
Adonidis Herba Convallariae Herba
Tracks l=Adonidis herba (DCM extract) Fig.15A, B; 16A, B
(MeOH extract; Flavonoids, p. 163) Fig.16C
2=Convallariae herba (DCM extract) Fig. 15C; 16A, B
(MeOH extract; Flavonoids, p. 163) Fig. 15D
Tests TS =" k-strophanthin" T3 = hyperoside
Tl =cymarin T4 = rutin
T2 = convallatoxin (Rf ca. 0.35) T5 = adonivernith
Solvent H-l: ethyl acetate-methanol-water (81: 11: 8) Fig. 15A-C; 16A, B
system F -1: ethyl acetate-formic acid-glacial acetic acid-water
(100: 11: 11: 27) Fig. 15D; 16C
Detection Kedde reagent (No. 23, p. 302) vis. Fig. 15A, C
(CTA No. 7, p. 300) vis. Fig.15B
SbCl 3 reagent (No. 3, p. 299) vis. Fig. 16A
UV-365 nm Fig. 16B
(NP/PEG No. 28, p. 303) UV-365 nm Fig. 15D; 16C
Chromato- 1 Adonidis herba

gram Kedde reagent gives 4-5 weak, violet zones in the Rf range 0.25-0.6, which corre-
15A spond partly with the glycoside mixture "k-strophanthin" (cf. TS). The upper zone
is cymarin (cf. Tl). Adonitoxin is seen at a similar Rf to that of the convallatoxin
test (cf. T2).
16B CTA reagent produces blue or blue-green fluorescent zones (in UV-365 nm) between
Rf 0.2 and the solvent front. Cymarin (cf. Tl) and adonitoxin (Rf ca. 0.3) fluoresce
green-blue.
16A, B SbCl3 reagent gives blue (vis.) zones at Rf 0.25-0.35 and at Rf 0.75-0.85 (16A),
which are characteristic of the drug extract, but are not due to cardiac glycosides.
Inspection in UV-365 nm (16B) shows conspicuous light blue fluorescent zones,
mainly in the intermediate Rf range.
15C 2 Convallariae herba
Kedde reagent gives a distinct violet (vis.) main zone of convallatoxin. In addition,
there are two weak, violet zones (also present in the standard convallatoxin test;
cf. T2): one above (Rf ca. 0.65) and the other be10w the main zone.
16A, B After treatment with SbCl 3 reagent, only weak, uncharacteristic zones are seen in
vis. (16A), but inspection in UV-365 nm (16B) reveals numerous, predominantly
yellow-brown and light blue fluorescent zones. Convalloside, convallatoxoloside and
glucoconvallatoxoloside (in order of decreasing mobility) mi grate below convalla-
toxin (Rf ca. 0.3; T2). Deglucocheirotoxin migrates directly ahead of convallatoxin.
15 D, 16 C Flavonoids
1 Adonidis herba is characterized by the yellow fluorescent (UV-365 nm) flavone-C-
glycoside, adonivernith (cf. T5), together with three other intensely yellow fluorescent
flavonoid zones at Rf 0.2-0.6.
2 Convallariae herba. Using the same sampie size, this drug shows only weak green
or orange fluorescent zones (in UV-365 nm) of kaempferol and quercetin triglyco-
sides at Rf 0.2-0.25.
218
Fig.15
TS Tl Tl 2 T2 T3 2 T4
FRONT
Rf
Fig.16
2 2 T2 T5
219
U zarae (Xysmalobii) Radix
Tracks 1 = Uzarone extract
Tests Tl = xysmalorin T4 = 3-0-acetyl-xysmalogenin
T2=uzarin T5 = lanatoside B (with traces of lanatosides A and C)
T3 = uzarigenin
Solvent H-l: ethyl acetate-methanol-water (81 : 11: 8)
system
Detection Chloramine-trichloroacetic acid reagent
(CTA No. 7, p. 300) UV-365 nm Fig. 17 A
SbCl 3 reagent (No. 3, p. 299) VIS. Fig. 17B

One dragee of the proprietary pharmaceutical Uzara® contains 50 mg of a dried
extract of Xysmalobium undulatum with ab out 30% total glycosides ("Uzarone
extract ").
Chromato- After treatment with CTAreagent, the chromatogram shows at least 7 light yellow
gram to blue-green fluorescent zones in UV-365 nm. The major zone at Rf ca. 0.1 contains
17A, B the diglucosides, uzarin (T2) and xysmalorin (Tl), while the prominent zone at Rf
ca. 0.3 is due to the corresponding monoglucosides, uzarigenin monoglucoside and
xysmalogenin monoglucoside. The latter migrate at about the same Rf value as for
the lanatoside test (cf. T5), which are not present in this drug. The upper part
of the chromatogram shows uzarigenin and 3-0-acetylxysmalogenin (cf. T3 and T4).
Treatment with SbC/3 re agent reveals mainly the blue-violet (vis.) zone of uzarin
and xysmalorin.
Hellebori Radix
Tracks 2 = Hellebori radix (H. viridis)
3 = Hellebori radix (commercial drug of unknown botanical source)
Tests T6 = hellebrin
T7 = g-strophanthin
Solvent H-l: ethyl acetate-methanol-water (81: 11 : 8) Fig. 18A, B
system H-4: chloroform-methanol-water (64:35:10) (lower phase) Fig.18C
Detection SbCl 3 reagent (No. 3), p. 299) UV-365 nm Fig.18A
VIS. Fig.18B
Anisaldehyde-sulphuric acid reagent (AS No. 2, p. 299) VIS. Fig.18C
Chromato- Chromatography in solvent system H-l, followed by treatment with SbC/3 reagent
gram and inspection in UV -365 nm, reveals at least 13 light yellow fluorescent zones be-
18A, B tween the start and the solvent front (18A). The zone of hellebrin (Rf ca. 0.1;
cf. T6) fluoresces intense light yellow; with AS reagent it produces a dark blue
(vis.) colour (18B).
18C In solvent system H-4, hellebdn migrates at Rf ca. 0.35. g-Strophanthin, which
is chromatographed alongside for comparison, migrates somewhat higher.
Remarks: Hellebrin, the main glycoside of Helleborus viridis and H. odorus may be absent
from drugs derived from other Helleborus species.
220
r- FRONT
Rf
START
Fig.17
Tl T2 T3 T4 T5
a
FRONT
•I
Rf
.. 0 .5
.....
~
Fig.18
- I· .~
T6 2 T6 2 3 T7
START
221
Scillae Bulbus
Tracks 1 = Scillae bulb. var. rubra
2 = Scillae bulb. var. rubra (standarized extract)
3 = Scillae bulb. var. alba (commercial pattern)
4 = Scillae bulb. var. alba (commercial pattern)
5 = Scillae bulb. var. alba (standardized extract)
Tests T = proscillaridin
Solvent H-1: ethyl acetate-methanol-water (81 : 11 : 8)
system
Detection SbCI 3 reagent (No. 3, p. 299) vis. Fig. 19
UV-365 nm Fig.20
Chromato-
gram 1-5 After SbCl3 treatment, chromatograms of Scilla extracts produce predominantly
19,20 blue (vis.) zones, or intense, light yellow, yellow-brown or light blue fluorescent
zones in UV-365 nm. The standardized extracts (2, 5) contain higher concentrations
of primary glycosides.
1-2 Scillae bulbus var. rubra
The drug extract produces only a few weak blue (vis.) zones in the middle and
upper Rf range. The test extract (2) gives more blue zones in the middle and lower
Rfrange.
In UV-365 nm, at least 10 intense, yellow-green or blue fluorescent zones appear,
especially at Rf 0.4 and in the Rf range 0.8-0.9 (aglycones).
Scillirosidin migrates at Rf ca. 0.8, its monoglycoside, scilliroside, at Rf ca. 0.4;
these zones fluoresce light green in UV-365 nm (almost white in the reproduced
chromatogram, Fig. 20), and they appear green-yellow in vis. The corresponding
diglycoside, glucoscilliroside, is detectable only in the test extract (light green fluores-
cence at Rf ca. 0.2). Proscillaridin (cf. T5) migrates at Rf ca. 0.65, and it is present
in especially high concentration in the test extract. Scillaren A is found directly
above scilliroside (see Sc. var. alba 3, 4, 5) as a blue zone in vis.
3,4,5 Scillae bulbus var. alba
The TLC of the test extract (5) is characterized in particular by the blue (vis.)
or light yellow fluorescent (UV-365 nm) zones of proscillaridin (Rf ca. 0.65), scilla-
ren A (Rf ca. 0.4) and glucoscillaren (Rf ca. 0.2). The main glycoside in this extract
is scillaren A. Commercial drugs 3 and 4 have high contents of proscillaridin, and
contain only small quantities of scillaren A; the zones in the upper part of the
chromatogram are also more intense.
222
FRONT
Rf
0 .5
-START
Fig.19
T 2
Fig.20
223
Saponin Drugs
The saponins in official saponin drugs are mainly triterpene derivatives, with a smaller
number of steroids.
Sugar residues may be linked via a single OH-group (usually C-3-0H) of the
aglycone (monodesmoside saponins) or more rarely via two OH-groups or a single
OH-group and a carboxyl group (bis-desmoside saponins).
Triterpene saponins
These saponins possess the oleanane ring system, or more rarely ursane or dammarane
systems. Many are acidic, due to the presence of one or two carboxyl groups in
the aglycone and/or sugar moiety. Other oxygen-containing groups mayaiso be
present in the sapogenin, i.e. -OH, -CH 2 0H or -CHO.
The carbohydrate group usually contains 1-6 mono saccharide units, the most
common of these being glucose, galactose, rhamnose, ara bi no se, fucose, xylose,
glucuronic acid and galacturonic acid. The horse chestnut saponins also contain
various esterified aliphatic acids.
All triterpene saponins possess haemolytic activity, which varies from strong
to weak, depending on the type of substitution.
Steroid saponins
The sapogenins ofthe steroid saponins are mostly spirostanols. Furostanol derivatives
are usually converted into spirostanols during isolation procedures: these sapogenins
do not carry carboxyl groups. Steroid saponins possess fewer sugar units than the
tri terpene saponins. In contrast to the monodesmosides, the bis-desmoside furostanol
glycosides have no haemolytic activity.
Powdered drug (2 g) is extracted by heating for 10 min under reflux with 10 ml

of 70% ethanol. The clear filtrate is evaporated to ca. 5 ml, and 25-40 111 of this
soln. are applied for chromatography.
Exceptions:
Ginseng radix is extracted under similar conditions with 90% ethanol.
Liquiritiae radix, according to Ph. Eur. 11.
Powdered drug (1 g) is shaken for 15 min with 20 ml chloroform, then filtered.
The filtrate is evaporated to dryness, and the residue is dissolved in 2.0 ml chloro-
form/methanol (1: 1) (CHCI 3 extract I).
The extracted drug is then heated under reflux for 1 h with 30 ml of 0.5 M
sulphuric acid. After cooling, the unfiltered mixture is shaken twice with 20 ml
quantities of chloroform. The combined chloroform extracts are dried over anhy-
drous sodium sulphate, filtered and evaporated to dryness. The residue is dissolved
in 2.0 ml chloroform/methanol (1: 1) (CHCI 3 extract 11 after hydrolysis). 10111 of
this soln. are applied for chromatography.
225
Each test compound is prepared as a 0.1 % solution in methanol, and 10111 are
applied for chromatography.
2. Adsorbent
Drug extracts 25-40 111.
Test solutions 10 111.
SP-l Chloroform-methanol-water (64: 50: 10)
SP-1 is suitable for the separation of all saponin mixtures from drugs. How-
ever, the mixture must be prepared exactly: analytical grade CHCI 3 must
be used (technical grade contains ethanol) and chromatography must be per-
formed at 20° C after 30 min chamber saturation. At higher temperatures,
all zones are shifted to the upper Rf range of the chromatogram.
SP-2 n-Butanol-glacial acetic acid-water (50: 10:40); upper phase. This system (spe-
cified by DAB 8) is less temperature-sensitive than SP-1. The disadvantages
are that it separates the main saponins of official drugs in the low Rf range,
and the developing time is 5-6 h on TLC plates.
SP-3 Chloroform-methanol-water (70: 30: 4)
SP-3 is especially suitable for the separation of ginsenosides from Ginseng
radix, and the eleutherosides of Eleutherococci radix.
SP-4 Chloroform-methanol (95: 5)
The solvent system is specified by Ph. Eur. II far separation of glycyrrhetic
acid.
III. Detection
1. Without chemie al treatment

With the exception of glycyrrhetic acid (from Liquiritiae radix), no sapomns are
detectable by exposure to UV-254 nm or UV-365 nm.
2. Spray reagents
a) Blood reagent (BL No. 6, p. 299)
Haemolytic saponins are detected as white zones on a reddish background. Haemoly-
sis may occur immediately, or after allowing the TLC plate to stand, or after drying
the plate in warm air.
b) Vanillin-sulphuric acid reagent (VS No. 38, p. 304)
With this reagent, saponins form mainly blue or blue-violet and sometimes yellowish
zones (vis.).
c) Anisaldehyde-sulphuric acid reagent (AS No. 2, p. 299)
Colours are similar to those with VS re agent.
d) Antimony( III) chloride reagent (SbCI 3 No. 3, p. 299)
Red-violet colours in vis. Mostly red-violet, blue and green fluorescence in UV-
365 nm.
226
e) Vanillin-phosphoric acid reagent (VPA No. 36A, p. 304)
Ginsenosides give red-violet colours in vis., and reddish or blue fluorescence in UV-
365 nm.
The main constituents of Eleutherococcus root form weak, violet zones in vis.,
and intense, yellow, pale blue and orange fluorescent zones in UV-365 nm.
f) Komarowsky reagent (KOM No. 24, p. 302)
The sprayed TLC plate is heated for 5-10 min at 100° C in a drying cabinet with
constant observation. Saponins form blue, yellow and red (vis.) zones.
IV. List of Saponin Drugs


F amily/Pharmacopoeia Haemolytic index (HI)
1,2 TLC synopsis

5, 6 Ginseng Radix 2-3% of a saponin mixture, contammg at
Ginseng root least 10 glycosides, known as "ginsenosides"
Panax ginseng MEYER R x (x = 0, a, b 1 , b z, C, d, e, f, gl' h). These
and other Panax spp. are present in the form of neutral bis-desmo-
Araliaceae sides. The tri terpene sapogenins, 20-S-proto-
panaxadiol and 20-S-proto-panaxatriol have
a dammarane ring system. The aglycone,
oleanolic acid, is found only in ginsenoside
R o . The glycosides contain glucose, arabinose,
rhamnose and glucuronic acid.
HI (drug) < 100
6 Eleutherococci Radix Oleanolic acid glycosides (eleutherosides I-

Siberian ginseng M), related to Hedera saponins.
Acanthopanax senticosus Lignane compounds (eleutheroside E, sy-
(Rupp. et MAXIM. ex MAXIM.) ringaresinol).
Harms Coumarins (e1eutheroside B1 =isofraxidin glu-
Araliaceae coside) and chlorogenic acid.
9 Hederae Folium 5% of a tri terpene glycoside mixture ; The

Ivy leaves main saponin is hederacoside H, accompanied
Hedera helix L. by the monodesmosides, ß-hederin, hederaco-
Araliaceae side C (derivatives of oleanolic acid) and Ot:-he-
derin (derived from hederagenin).
2. AB-DDR
HI (drug) 1,000-1,500, HI (ß-hederin) 15,000.
1,2 TLC synopsis At least 3% of an ester saponin mixture,

10 Hippocastani Semen known as "aescin" (DAB 8), containing de-
Horse chestnut seeds rivatives of protoaescigenin and barringto-
genol C. Both sapogenins are linked to 1 mol
Aesculus hippocastanum L. glucuronic acid and 2 mol glucose, and esteri-
Hippocastanaceae fied with angelica, tiglic, a-butyric or isobu-
DAB 8, 2. AB-DDR tyric, and acetic acids.
HI (drug) ca. 6,000
HI (aescin) 9,500-12,500
227
Fig. Drug(Plant source Main constituents
F amily (Pharmacopoeia Haemolytic index (HI)
1, 2 TLC synopsis
7,8 Liquiritiae Radix Saponins: 8-12% glycyrrhizin, which is pres-
Licorice root ent as the calcium and(or potassium salt of
(peeled(unpeeled) glycyrrhizic acid. It does not show haemolytic
Glycyrrhiza glabra L. activity. The aglycone, glycyrrhetic acid, does
Fabaceae possess haemolytic activity.
DAB 8, Ph. Eur. H, 2. AB-DDR Flavonoids: 1-1.5% of a flavonoid mixture.
(3-5.5% glycyrrhizin), Helv. VI, The main constituent is liquiritin (4',7-dihy-
ÖAB droxyflavanone-7-0-glucoside), accompanied
by liquiritoside (liquiritigenin-gluc-rham) and
the corresponding chalcones.
HI (drug) 250-300.
1,2,11,12 TLC synopsis

3 Primulae Radix 5-10% of a saponin mixture saponin is Pri-
Primrose root mula acid A, which is derived from protopri-
Primula veris L. mulagenin.
Primula elatior (L.) HILL. Primula veris has a higher saponin content
Primulaceae than P. elatior, and contains more individual
DAB 8,2. AB-DDR (HI at not less saponins. P. elatior saponins consist of 90%
than 2,500), ÖAB (HI at not less Primula acid; those from P. veris also com-
than 3,000) prise glycosides derived from the genins, pri-
mulagenin A, dehydroprimulagenin A and
primverogenins A and B.
Adulterant: Vincetoxicum hirundinaria Med.
(=Cynanchum vincetoxicum (L.) Persoon),
containing the steroid glycoside mixture,
" vincetoxin " .
1,2,11, 12 TLC synopsis

Quillajae Cortex 10% saponin mixture "Quillaja saponin",
Quillaja bark with Quillaja acid as the aglycone, and glucu-
Soap bark ronic or galacturonic acid as the sugar moiety.
Quillaja saponaria MOLINA HI 3,500-4,500
Rosaceae
Helv. VI, ÖAB (HI not less than
3,000), DAC
1, 2 TLC synopsis
4 Saponariae Radix
S. rubrae Radix S. rubrae radix: 2-5% saponin mixture, main
Red soapwort root constituents saponasides A and D. Gypso-
Saponaria officinalis L. genin is the aglycone of these acidic bis-des-
Caryophyllaceae mosides.
2.AB-DDR(HI 1,200-1,800),DAC HI 1,200-2,000
S. albae Radix S. albae radix: up to 20% saponin with Gyp-
White soapwort root soside A (a bis-desmoside glycoside) as a main
Gypsophila spp. (G. paniculata L., compound. This saponin mixture serves as
G. arrostii Guss.) standard saponin for the determination of hae-
Caryophyllaceae molytic activity.
HI 2,600-3,900
228
F amily jPharmacopoeia Haemolytic index (HI)
1, 2, 11, 12 TLC synopses

Sarsaparillae Radix 1.8-2.4% steroid saponins, comprising paril-
Sarsaparilla lin, pariglin and others.
Smilax spp. (S. regelü KILIP et The aglycones are sarsasapogenin (= pari-
MORTON, Honduras drug) (S. med- genin) and its isomer, smilagenin.
ica SCHL. et CHAM, Veracruz drug) HI 3,500-4,200
Liliaceae
Senegae Radix 8-10% mixed saponins, comprising at least
Polygalae radix 8 glycosides. Main saponin is senegin. The ag-
Milkwort root lycone is presenegenin, with glucose, galac-
Polygala senega L. and other tose, rhamnose and fucose as sugar residues.
Polygala spp. The fucose is esterified with 3,4-dimethoxy-
Polygalaceae cinnamic acid.
Helv. VI, ÖAB HI 2,500-4,500
9 A venae sativae Herba Steroid saponins: avenacosides A and B. The

(Fructus) aglycone is nuatigenin, with glucose and
Oats rhamnose (ratio 3: 1 or 4: 1) as the sugar resi-
Avena sativa L. dues.
Avena orientalis Schreb. Triterpene saponins: e.g. avenacin.
Poaceae
Saponin drugs that possess little or no haemolytic activity andjor are better identified
on the basis of components other than saponins (relevant chromatograms are repro-
duced in the sections on Flavonoid and Coumarin Drugs).
Betulae Folium Saponin (ca. 3%): betulin (derived from betu-
Birch leaves linie acid).
Betula pendula ROTH. Flavonoids (1.5-3%): main components are
Betula pubescens ERH. hyperoside and myricetin digalactoside (see
Betulaceae Flavonoid drugs, Figs. 11 and 12, p. 182).
2. AB-DDR, ÖAB, Helv. VI,
DAB8
Eqniseti Herba Triterpene saponin: equisetonin.
Common horsetail Flavonoids: isoquercitrin, galuteolin (see Fla-
Equisetum arvense L. vonoid Drugs, Fig. 16, p. 186).
Equisetaceae
2. AB-DDR, DAB 8, Helv. VI,
ÖAB
Herniariae Herba Up to 3% saponin mixture (Herniaria saponins
Rupture-wort land 11), derived from medicagenic acid.
Herniaria glabra L. Flavonoids: glycosides of quercetin and iso-
Herniaria hirsuta L. rhamnetin (rutin, narcissin) (see Flavonoid
Caryophyllaceae Drugs, Fig. 6, p. 177).
ÖAB Coumarins: herniarin (see Coumarin Drugs,
Figs. 5 and 6, p. 156).
Verbasci Flos Saponins without haemolytic activity.
Mullein flowers Flavonoids (up to 3.8%): main compounds ru-
Verbascum densiflorum tin and hesperidin (see Flavonoid Drugs, Fig. 7,
BERTOLONI p. 178).
Scrophulariaceae
Helv. VI, ÖAB
229
V. Formulae of Constituents of Saponin Drugs
HO
Primulagenin A
Oleanolic acid: Rl = H; R2 = CH 3
Quillaja acid: Rl = OH; R2 = C~
HO
Gypsogenin: Rl = H; R2 = C~~
Hederagenin: Rl = H; R2 = CH 20H
HO
HOOe
Presenegenin: R = CH 2 0H
Medicagenic acid: R = H
Hippocastani semen:
COOH
~
o
OH
Gluc-O ~--------~v~----------~
O-Gluc
Protoaescigenin: Rl = R2 = R3 = H; R4 = OH
Barringtogenol: Rl = R2 = R3 = R4 = H
Aescin: R, = Tiglyl, Angelyl, Isobutyryl, cr-Methylbutyryl residues

R2 = acetyl; R3 = H; R4 = H or OH
Kryptoaescin: R, = Tiglyl, Angelyl, cr-Methylbutyryl residues
R2 =H; R3 =acetyl; R4 =H or OH
230
Ginseng radix:
R R'
(20 S-Protopanaxadiol) H H
Ginsenoside Rb, I~-D-Gq 1 ~ 21 ~-D-Gq I~-D-GI11 ~ 61 ~-D-GII
Gi nsenoside Rb 2 1~-D-G111 ~ 21 ~-D-GII la-L-Ar 11 --. 61 i3-D-G q
Ginsenoside Re 1~-D-Gq 1 ~ 21 ~-D-Gq Ia-L-ArfI1 --. 61 i3-D-G1 1
Ginsenoside Rd 1i3-D-G!J 1 ~ 21 ~-D-Gq Ii3-D-G11
RO
R R'
(20 S-Protopanaxatrioll H H
Ginsenoside Re la-L-Rh 11 ~ 21 ~-D-Gq 1i3-D-GI!
Ginsenoside Rg, 1~-D-GII !i3-D-G1 !
Ginsenoside R9 2 la-L-RhI1 ~ 21 i3-D-Gq H
HO
OR
Eleutherococci radix:
MeO
MeO
RO
R0-9-CH=CH-CH20H
OR
MeO
OMe
Eleutheroside 8 R=Glucosyl Eleutheroside E R = Glucosyl Eleutheroside 8,
Sinapyl alcohol R = H Syringaresinol R = H
231
Liquiritiae radix: OH OH
HO HO
Liquiritin ..
Liquiritigenin Isoliquiritigenin
Glr 1-2Glr-O
Glycyrrhizic acid
Primulae radix:
la-L-Rha 11 '-.,.
....... mlIß-O-Gl:r:J 1-3IProto-Primu1agenin AI
) B-0-GlI 1-31 ß-O-Ga 11
Primula acid A
Senegae radix:
\ß-0-G111_3)Presenegeni n128-11 0-FuI2_1) L-Rha 14-1) D-Xy 14+-1)B-D-Gal
4
t
Dimethoxycinnamic acid
Senegin
Hederae folium:
la-L-RhaI1-2Ia-L-ArI1-310Ieanolic acid 128-11 8-0-GlI6+-1IB-0-GlI4+-1Ia-L-Rhal
Hederacoside B
232
STEROID-SAPOGENINE
HO
H H
Sarsasapogenin: R1 = H; R2 = H; 5~. 25~

Smilagenin: R1 = H; R2 = H; 5~. 25a Hecogenin: 5a. 20~. 25a
Sarsaparillae radix:
O-gluc
Gluc~
Rha 14 Gluc-O
H Sarsaparilloside
G!uc~
Avenae herba:
HO Nuatigenin
Ar = Arabinose
Fu = Fucose
Ga = Galactose
GI =Glucose
Glr = Glucuronic acid
Rh = Rhamnose
Xy =Xylose
233
Saponin Drugs TLC Synopsis
Tracks 1 = Senegae radix 5 = Primulae radix
2 = Sarsaparillae radix 6 = Saponariae albae radix
3 = Ginseng radix 7 = Liquiritiae radix
4 = Hippocastani semen 8 = Quillajae cortex
Tests T = mixture of saponins from Senegae radix
Solvent SP-1: chloroform-methanol-water (65: 50: 10)
system
Detection Vanillin-sulphuric acid reagent (VS No. 38, p. 304) vis. Fig. 1
Blood reagent (No. 6, p. 299) vis. Fig.2
Chromato- 1 Senegae radix. Immediately after spraying with VS reagent, strong red zones are
gram formed in the Rfrange 0.3-0.55; the colour fades on heating (cf. T, "Senega saponin
1,2 mixture "). After heating, 7-8 additional violet zones appear in the upper part of
the chromatogram; only some of these are due to saponins. The main components
of Senega saponin mixt ure have strong haemolytic activity.
2 Sarsaparillae radix. After treatment with VS reagent, the chromatogram is character-
ized by at least 8 yellow or yellow-brown zones in the Rf range 0.2-0.75. The zones
between Rf 0.55 and 0.75, and directly below the solvent front, show haemolytic
activity.
3 Ginseng radix produces at least 10 intense, dark violet, main zones (ginsenosides)
in the Rf range 0.35-0.75. The main haemolysis zones are in the intermediate Rf
range (for identification, see Fig. 5, p. 238).
4 Hippocastani semen is characterized by the broad violet zone of "aescin" at Rf
0.5-0.6, which shows distinct haemolytic activity (for identification, see Fig. 10,
p.242).
5 Primulae radix shows the brown zone of " Primula acid" (a mixture of three compo-
nents) at Rf 0.3-0.4. Like the other zones in the upper Rf region (0.6--0.75 and
0.95), Primula acid shows only weak haemolytic activity. (See Fig. 3, p. 236 for
further details).
6 Saponariae radix. After treatment with VS reagent, the chromatogram shows several
intense, dark brown zones in the same region (Rf 0.1-0.3) as the saponin test. The
same zones are also strongly haemolytic. There are also several weaker, violet zones
between Rf 0.7 and the solvent front (the most intensive one of these is at Rf
0.75), which also possess haemolytic activity (for identification, see Fig. 4, p. 236).
7 Liquiritiae radix is especially characterized by intense, yellow-brown zones in the
Rfrange 0.6--0.75 (flavonoids). Haemolytic activity is found only in the region above
Rf 0.75. The triterpene saponin, glycyrrhizin, is detected as glycyrrhetic acid after
hydrolysis (for furt her details and identification of zones, see Figs. 7 and 8, p. 240).
8 Quillajae cortex produces about 6 dark brown zones in the Rfrange 0.15-0.4, which
represent the saponin mixture of this drug. Strongly haemolytic zones are found
between RfO.1 and 0.7 (see also Fig. llA, p. 244).
Remarks: All these drug extracts show strongly haemolytic zones directly below the solvent
front (sapogenins and some sterols).
234
Fig.l
T 2 3 4 5 6 7 8
F
Rf
5
Fig.2
T 2 3 4 5 6 7 8
235
Primulae Radix Saponariae Radix
Tracks 1 = Prim ulae radix (P. veris)
2 = Primulae radix (P. elatior)
3 = Saponariae albae radix
4 = Saponariae rubrae radix
Tests Tl =Primula acid
T2 = standard saponin from Gypsophila spp.
Solvent SP-l: chloroform-methanol-water (64: 50: 10)
system
Detection Blood reagent (No. 6, p. 299) vis. Fig. 3A; 4A
Antimony(III) chloride reagent (SbCI 3 No. 3, p. 299) vis. Fig. 3B; 4B
UV-365 nm Fig. 3C; 4C
For description of drugs see p. 228. Formulae pp. 230-233.
Chromato- Primulae radix

gram 1,2 After treatment with ShCl3 reagent, the standard "Primula acid" (cf. Tl) gives
3 three violet zones in vis., and light brown fluorescence in UV-365 nm.
In Primula veris, "Primula acid" appears as two main zones with a minor zone.
In Primula elatior, the saponins are present in lower concentration. Extracts of
Primula veris also show a violet (vis.) zone at Rf ca. 0.75, which gives a greenish
fluorescence in UV-365 nm; this zone is absent from P. elatior (see also Fig. 1,
p. 234; VS reagent). Only one zone of Primula acid gives an immediate haemolytic
response with Blood reagent; the other zones show positive, but slower haemolysis.
Remarks: Primulaverin is present in high concentration in Primula veris, and is absent from
P. elatior. This phenol glycoside can therefore be used to differentiate between the two roots.
By enzymic c1eavage, it yields methyl methoxysalicylate, which has a characteristic odour.
The root of Cynanchum vincetoxicum, which may be encountered as an adulterant, contains
steroid glycosides. These can be extracted with toluene. If the toluene extract is completely
evaporated, redissolved in ethanol and treated with conc. H 2 S0 4 and Feel 3 , a violet coloura-
tion is produced due to the presence of vincetoxin.
4 3, 4 Saponariae radix
After ShCl3 treatment, both Saponaria extracts show a large number of overlapping
zones in the Rf range 0.05-0.5; these are violet or black-violet in vis., and fluoresce
green-blue in UV-365 nm (cf. also Fig. 1, track 6, TLC synopsis p. 234).
The saponin test mixture shows two main zones (cf. T2; RfO.25-0.4). The immedi-
ate appearance of powerful haemolysis zones in the Rf range 0.2-0.45 is typical
for Saponariae radix.
It can be seen from the chromatogram that S. albae radix has a markedly higher
saponin content than S. rubrae radix (S. alba 6-30%; S. rubra 2-5%).
236
......
c
-F
.......
~'
....,.........r.
-
Rf
...
\ .",
~ y.l~'--~'
~
....,Vr... 1
11 -
s
Fig.3
Tl 2 Tl 2
Fig.4
3 3 T2 4 3 T2 4
237
Ginseng, Eleutherococci Radix
Tracks 1 = Ginseng radix
2 = Eleutherococci radix
Tests ginsenosides: Re, Rb 2 , Rb 1 , Re, Rd, Rg', Rh'.
Solvent SP-1: chloroform-methanol-water (64: 50: 10) Fig. 5A, B, C
system SP-3: chloroform-methanol-water (70: 30: 4) Fig. 5D; 6A, B
Detection Antimony(III) chloride (SbCI 3 No. 3, p. 299) UV-365 nm Fig.5A
VIS. Fig.5B
Blood re agent (No. 6, p. 299) VIS. Fig.5C
Vanillin-phosphoric acid (VPA No. 36A, p. 304) VIS. Fig. 5D; 6A
UV -365 nm Fig. 6B
Chromato- 1 Ginseng radix. In both solvent systems, in vis. and in UV-365 nm, the chromatograms
gram show about 10 zones between the origin and the solvent front.
5,6
5A, B Treatment with SbCl3 and inspection in UV-365 nm reveals pale green fluorescent
zones (ginsenosides), which are especially conspicuous in the Rf range 0.25-0.6.
Weaker fluorescent zones occur in the upper Rf range. In vis. the ginsenoside zones
appear violet-blue. The prominent zone at Rf ca. 0.2 is due to sugars.
5C Treatment with Blood reagent reveals weak haemolytic zones at Rf 0.6-0.65 and
astronger zone at Rf ca. 0.9.
5D VPA reagent gives 8-10 distinct red-violet (vis.) zones in the Rf range 0.05-0.4
(e.g. ginsenosides Re, Rb 2 , Rb 1 , Re, Rd, Rg'), and a few weaker zones in the upper
Rf range. In UV-365 nm the ginsenosides give a distinct red-violet fluorescence
(Fig.6B).
Remarks: The ginsenosides show higher Rf values in SP-I than in SP-3; solvent system SP-l
is more suitable for the TLC comparison of Ginseng and Eleutherococcus extracts.
6A, B 2 Eleutheroeoeeus root extraet. After treatment with VPA reagent, chromatograms
are characterized by three violet-red (vis.), main zones at Rf 0.3-0.5. In UV-365 nm
the two upper zones give characteristic yellow and orange fluorescence, while the
lower zone gives a dull, dark green fluorescence (= E). An additional pale yellow
fluorescent zone appears above the orange zone. The weak blue fluorescence at
Rf ca. 0.1 is due to ehlorogenie acid.
Eleutheroside E is seen as a dull, dark green zone (E at Rf ca. 0.4) directly
below the yellow fluorescent zone, and at about the same Rf as ginsenoside Rg'.
Ginseng radix, on the other hand, shows the characteristic, red-violet fluorescent
zones of ginsenosides mainly between the start and Rf ca. 0.45.
238
F
Rf
0 .5
5
Fig.5
Re Rb2 Rb' Re Rd Rg' Rh '
-F
Rf
0.5
Fig.6
2 Re Rg ' 2 Re Rg '
239
Liquiritiae Radix
Tracks 1 = Liquiritiae radix (EtOH extract, see p. 225)
2 = Liquiritiae radix (hydrolysate, see p. 225)
3 = Liquiritiae radix (CHCI 3 extract I, see p. 225)
Test Tl = hyperoside (Rf ca. 0.6), rutin (Rf ca. 0.35)
T2 = standard saponin
T3 = glycyrrhetic acid
system (100: 11: 11 :27) Fig. 7A, B, C
SP-l: chloroform-methanol-water (64: 50: 10) Fig. 8A, B, C
SP-4: chloroform-methanol (95: 5) Fig.8D
(NPjPEG No. 28, p. 303) UV-365 nm Fig. 7 A
Sulphuric acid (H 2 S0 4 50%, No. 34B, p. 303) UV-365 nm Fig.7B
vis. Fig.7C
Blood reagent (No. 6, p. 299) VlS. Fig.8A
Antimony(I1I) chloride reag. (SbCI 3 No. 3, p. 299) VlS. Fig.8B
UV-365 nm Fig.8C
Anisaldehyde-sulphuric acid reag. (AS No. 2, p. 299) vis. Fig.8D
Chromato- Unlike other saponin drugs, licorice is better identified and characterized on the
gram basis of its flavonoid fingerprint.
7 A, B, C Flavonoids
Chromatography in F-1, followed by treatment with NP/PEG or sulphuric acid
reagent and inspection in UV -365 nm, reveals at least 12 yellow-green or light blue
fluorescent zones, particularly in the Rf range 0.2-0.8. Two characteristic, strongly
yellow-brown (vis.) flavone zones migrate between the test zones of the rutin and
hyperoside. The upper of these two zones (Rf ca. 0.45) is due to the flavanone
glycoside, liquiritin, and its cha1cone form.
8A-C Saponins
The characteristic saponin of Liquiritiae radix is glycyrrhizin, which is present in
the drug in salt form and only partially extractable with EtOH (see p. 225). Glycyr-
rhizin however is easily detectable in aqueous extracts. In SP-1, there is a distinct
quenching of fluorescence in UV -254 nm at Rf ca. 0.2.
8 BjC After treatment with SbCI3 , two violet (vis.) zones are seen at Rf ca. 0.2-0.3 (glycyr-
rhizin and a sugar), and a red-brown jlavonoid zone appears at Rf 0.6-0.75. In
UV-365 nm, these zones give partly dark, and partly light blue fluorescence.
8D 2, 3 Glycyrrhizin is detected as glycyrrhetic acid following acid hydrolysis (T3). It migrates
at Rf ca. 0.25. A CHCI 3 extract from licorice, prepared for comparison, contains
practically no glycyrrhetic acid (detection according to Ph. Eur. II).
240
-FRONT
Rf
-0.5
-STAIH
Fig.7
Tl Tl Tl
-FRONT
Rf
-0.5
-START
Fig.8
T2 2 T3 3
241
A venae Herba, Hederae Folium, Hippocastani Semen
Tracks 1 = Avenae herba (MeOH - or BuOH extract) Fig. 9A, B
2 = Hederae folium Fig. 9C, D
3 = Hippocastani semen Fig.10A-D
Tests T = vanillin glucoside
Tl =avenacoside B
T2 = ß-hederin
T3=aescin
Solvent SP-l: chloroform-methanol-water (64: 50: 10) Fig. 9A-D; lOA-C
system SP-2: n-butanol-glacial acetic acid-water
(50: 10 :40); upper phase Fig. lOD
Detection Vanillin-sulphuric acid reag. (VS No. 38, p. 304) vis. Fig. 9A, B
Blood reagent (No. 6, p. 299) VlS. Fig. 9C; lOA
Vanillin-phosphoric acid reag. (VPA No. 36, p. 304) VlS. Fig.9D
Antimony(III) chloride reag. (SbCl 3 ) No. 3, p. 299) vis. Fig. lOB
UV-365 nm Fig.10C
Anisaldehyde-sulphuric acid reag. (AS No. 2, p. 299) VlS. Fig. lOD
Chromato- 1 Avenae herba

gram After treatment with VS reagent, methanol extracts show about 16 predominantly
9A, B grey to red-violet (vis.) zones over the whole Rf range (9B). If the plate is heated
for a longer period, the zones become more distinct, but uniformly red-brown (9 A).
In the butanol extract (to the right ofTl) the saponin zones are the most conspicuous.
Avenacoside (cf. T1) appears at Rf ca. 0.2, vanillin glucoside at Rf ca. 0.55 (cf. T).
9C,D 2 Hederae folium
VPA reagent reveals the characteristic violet or intense blue-black (vis.) zone of
the hederacoside mixture in the Rf range ca. 0.4-0.6. This zone and the zone of
ß-hederin (cf. T2) show only weak haemolytic activity.
1OA-D 3 Hippocastani semen
Treatment with SbCl 3 reveals the broad zone of the saponin mixture, "aescin ",
at Rf ca. 0.5. This appears violet in vis., and gives an intense, green-grey fluorescence
in UV-365 nm. Aescin shows pronounced haemolysis (cf. T3).
Three other violet (vis.) or black zones (in UV-365 nm) (Fig. lOB; treated with
SbCl 3 ) in the Rf range 0.05-0.2 are due to sugars; with AS reagent they form
brown (vis.) zones (Fig. 10D). In the solvent system, specified by DAß 8 (SP-2;
detection with AS reagent), aescin migrates with a much lower Rf value (cf.
Fig.l0D).
242
8
-0.5
-5
Fig.9
T Tl T2 2 2 T2
-F
Rf
-0.5
-5
Fig. 10
T3 3 T3 3 T3 3 T3
243
TLC-Analysis of Saponins, Using Different Solvent Systems
and Spray Reagents
Tracks 1 = Quillajae cortex 4 = Primulae radix
2 = Senegae radix 5 = Hippocastani semen
3 = Ginseng radix 6 = Sarsaparillae radix
Tests Tl = Senega saponin mixture T2 = Primula acid T3=aescin
Solvent SP-l: chloroform-methanol-water (64: 50: 10) Fig. 11A, B, D; 12A, D, E
system SP-2: n-butanol-glacial acetic acid-water Fi~11C,E,F;12B,C,F
(50: 10:40); upper phase
Detection Komarowsky reagent (KOM No. 24, p. 302) VIS. Fig. 11B, D; 12A, D, E
Anisaldehyde-sulphuric acid vis. Fig. 11 C, E, F; 12 B, C, F
(AS No. 2, p. 299)
Blood re agent (No. 6, p. 299) VIS. Fig. 11 A
General: The chromatograms shown in Figs. 11 and 12 were developed in solvent systems
SP-1 and SP-2 and treated with KOM and AS reagents.
1. Solvent systems
SP-2 is less temperature-dependent than SP-1, but it has the disadvantage that
the development over 15 cm requires 5-6 h. Furthermore, the main saponins migrate
at low Rf values; for example, the differentiation of aescin and Primula acid is
difficult.
2. Detection
Sulphuric acid reagents with anisaldehyde, vanillin or p-hydroxybenzaldehyde pro-
duce colours not only with saponins, but also with other compounds (e.g. sugars).
Specific detection of saponins is possible with Blood re agent only (Fig. 5).
Chromato- 1 Quillajae cortex (cf. TLC synopsis Fig. 1, p. 234)
gram A: SP-1: the main haemolysis zones lie in the region RfO.l-0.4.
11 B: SP-1: three brown zones are obtained with KOM reagent. The start zone is stained
intense red.
C: SP-2: AS reagent produces main brown zones in lower Rf region.
11 2,3 Senegae radix, Ginseng radix (cf. TLC synopsis Fig. 1, p. 234)
D: SP-1: with KOM reagent, Senegae radix gives stable, intense, red zones; Ginseng
radix gives black-violet colours.
E, F: SP-2: the saponin zones of Senega (cf. Tl) lie at Rf ca. 0.2, while the main zones
of Ginseng radix appear between Rf 0.2 and 0.65.
12 4,5 Primulae radix, Hippocastani semen
A, D: SP-1: with KOM re agent, the saponin mixtures "Primula acid" (T2) and "aescin"
(T3) form distinct violet zones at Rf 0.2-0.3 and 0.5, respectively.
B, C: SP-2: Primula acid and aescin show rather similar Rf values.
12 6 Sarsaparillae radix (cf. TLC synopsis Fig. 1, p. 234)
E: SP-1: treatment with KOM re agent pro duces at least 8 typical yellow or yellow-
brown zones over the Rf range 0.25-0.75.
F: SP-2: the main zones appear mainly at Rf 0.1-0.2.
244
f
Rf
0.5
5
Fig.11
2 3 T1 2 3
f
Rf
0 .5
Fig.12
T2 4 T2 4 5 T3 5 T3 6 6
245
Drugs Containing Pungent Principles
The sharp-tasting constituents of these drugs belong mainly to one of the following
types:
Amides
e.g. piperine from Piperis fructus.
e.g. capsaicin from Capsici fructus.
o-Methoxyphenols and phenylpropanes
e.g. eugenol in Caryophylli flos and Myristicae semen; gingerols in Zingiberis rhi-
zoma; elemicin and asarone in Calami and Asari rhizoma.
Phenolic sesquiterpenes
e.g. xanthorrhizol in Curcumae rhizoma.

Piperis fructus, Cubebae fructus
Powdered drug (1 g) is extracted by heating under reflux for 10 min with 10 ml
methanol. The filtrate is evaporated to 3 ml and this soln. is applied for chromatogra-
phy.
Capsici fructus
CHCI 3 • The filtrate is used for TLC.

5 mg standard compound (capsaicin, piperine or cubebin) are dissolved in 5 ml
MeOH.
2. Adsorbent
Drug extracts 5 111 or 10 Ill.
Piperis fructus (ca. 3 % soln.): 5 111 (~ca. 50 Ilg piperine)
Capsici fructus (ca. 0.4% soln.): 10 111 (~ca. 14 Ilg capsaicin)
Solutions of standards: 5 111 (~ca. 5 Ilg standard substance)
S-l toluene-ethyl acetate (70: 30): Piperis, Cubebae, Capsici fructus.
S-2 toluene-diethyl ether-dioxan (62.5: 21.5: 16): Piperis fructus.
S-3 diethyl ether: Capsici fructus.
247
III. Detection
UV-254 nm: capsaicin shows fluorescence quenching only at high concentrations.
Cubebin and piperine cause distinct fluorescence quenching.
UV-365 nm: piperine gives dark blue, piperyline light blue fluorescence.
2. Spray reagents
a) Vanillin-sulphuric acid (VS No. 38, p. 304)
After spraying, the plate is heated for 10 min at 100° C then inspected in visible
light. Piperine gives alemon yellow (vis.); cubebin gives a red-violet (vis.).
b) Conc. sulphuric acid (No. 34C, p. 303)
Piperine: dark brown (vis.).
Cubebin: red-violet (vis.) dark red fluorescence after ca. 10 min in UV-365 nm (gen-
eral reaction of lignanes)
c) Capsaicin colour reaction (DCC No. 8, p. 300)
Capsaicin and other capsaicinoids give a blue (vis.) zone; detection limit 0.1 Ilg.
IV. List of Drugs Containing Pungent Principles

1, 2 1. Amides
Piperis Fructus 4-7% sharp tasting substances, with 2-5%
Black pepper trans-piperine (pungency index 1: 2,000,000).
Piper nigrum L. The accompanying compounds, piperettin, pi-
Piperaceae peranin, piperaesthin A and piperyline, con-
ÖAB tribute only ca. 5% to the pungency of the
drug.
Cubebae Fructus Up to 0.1 % piperine.
Cubebs Ca. 2.5% cubebin (a non-pungent pepper lig-
Piper cubeba L. nane).
Piperaceae
Capsici Fructus Capsaicinoids 0.1-0.5% (C. annum),
Capsicums 0.6-0.9% (C. frutescens), comprising 70%
Capsicum annum var. longum L. capsaicin (the vanillylamide of an 8-methyl-
(trans)-non-6-enoic acid) and the capsaici-
Capsici acris Fructus noids, homocapsaicin, dihydrocapsaicin, ho-
Cayenne pepper modihydrocapsaicin and nordihydrocapsai-
Chillies ein. Pungency index of capsaicin is 1: 2 mil-
Capsicum frutescens L. lion. 1: 5,000 Dilutions of the drug extract
Solanaceae should be still sharp tasting.
DAB 8 (Cayenne pepper), DAB 8 specifies not less than 0.4% capsaicin.
Helv. VI, ÖAB, 2. AB-DDR The capsaicin content of stored drugs is often
less than 0.4%.
248
Fig. Drug(Plant source Main constituents
Family(Pharmacopoeia
2. o-Methoxyphenols and other compounds

Pungent principles present in the essential oil:
Calami rhizoma: 3-5% essential oil, containing 0-95% asarone.
Caryophylli Oos: 14-20% essential oil, containing ca. 90% eugenol.
Myristicae semen: up to 16% essential oil, containing ca. 8% myristicin and eugenol.
Pungent principles present in the resin: e.g. in Galangae and Zingiberis rr.izoma
(galangoi and gingerol).
Curcumae zanthorrhizae rhizoma: xanthorrhizol
For description of the drugs, their constituents, formulae of constituents and TLC
separations, see Essential Oi! Drugs.
v. Formulae
<O~(CH2)2~CO-NH-iSO-ButYI
o Piperaesthin A
Piperettin
o
H
OH
R: -CO-(CH 2)4-CH=CH-CH(CH 3)2 Capsaicin

R: -CO-(CH2)6-CH(CH3)2 Dihydrocapsaicin
R: -CO-(CH2h- CH(CH3l2 Homodihydrocapsaicin
Cubebin'
o HO H
Ginger
H3CO~(CH2)n/CH3 n:4.6.8
HO Gingerols
(For fonnulae of eugenol, asarone and xanthorrhizol, see p. 19-20)
The lignane derivative, cubebin, does not have a sharp taste, but it serves for the differentia-
tion of Piperis nigri and Piperis cubebae fructus.
249
Piperis, Cubebae, Capsici Fructus
Tracks 1 = Piperis nigri fructus Tests Ti = piperettin
2 = Piperis albi fructus T2 = dipiperine (with traces of piperine)
3 = Capsici fructus T3 = piperine
4 = Capsici acris fructus T 4 = capsaicin
5 = Cubebae fructus T5=cubebin
T6 = piperine and dipiperine
Solvent SC-i: toluene-ethyl acetate (70: 30) Fig. lA; 2A, B
system SC-2: toluene-diethyl ether-dioxan (62.5:21.5:16) Fig.2C
SC-3: diethyl ether Fig.1B
Detection Vanillin-sulphuric acid reagent (VS No. 38, p. 304) Fig. lA; 2A, C
Dichloroquinone-chloroimide re agent (DCC No. 8, p. 300) Fig.1B
Sulphuric acid reagent (H 2 S0 4 conc. No. 34, p. 303) Fig.2B

Chromato-
gram 1,2 Piperis Jructus
1A After treatment with VS reagent, the chromatogram is characterized by 3-4lemon
2A yellow (vis.) zones in the lower and intermediate Rf range (piperine and piperine
derivatives), a blue-violet (vis.) zone at Rf ca. 0.6 and 1-2 further blue-violet zones
below the solvent front.
Zone Approx. Rf in SC-l (Fig. 1 A) Approx. Rf in SC-2 (Fig. 2 C)
Piperyline 0.1 0.15

Piperine } 0.50
Piperettin 0.25 0.55
Piperine isomers 0.55
Dipiperine 0.35 0.63
Piperaesthin A 0.4 0.70
2C Black and white pepper show the same qualitative composition of pungent principles,
but differ especially with respect to the quantities of minor constituents. White
pepper has a somewhat lower content of pungent principles.
2A, B 5 Cuhehae Jructus
After treatment with conc. H 2 S0 4 , the chromatogram shows the red-violet (vis.)
zone of cuhehin (cf. T5), which is characteristic of this drug. The weaker, brown
to red-violet zones in the upper Rf range are components of the essential oil fraction
(e.g. cadinen, cadinol), which are stained strongly violet with VS reagent (Fig. 2A).
Remarks: Cubebae fructus contains only traces of piperine; without further enrichment it
is not detectable on TLC. Piperis fructus contains traces of cubebin.
1B 3,4 CapsiciJructus
Treatment with DCC reagent produces the characteristic blue (vis.) zone of the
capsaicin at Rf ca. 0.4 (cf. T4). Extracts of Capsici acris fructus show a pronounced
carotene zone at the solvent front. An exact differentiation between the two drugs
can only be made on the basis of a quantitative capsaicin determination.
250
• -FRONT
Rf
-0.5
-START
Fig.l
Tl T2 TJ J T4 4
A B c
-FRONT
Rf
-0.5
-START
Fig.2
T6 5 T5 T6 5 T5 T6 2
251
Mustard on Drugs
and Allium (Garlic)
The mustard oils (isothiocyanates) are always present in the drug as glucosinolates
(S-glucosides). These are split by the enzyme myrosinase (ß-thioglucosidase) when
plant tissues are damaged, or by steam distillation.
I. Preparation of the Drug Extract, and TLC Methods

A. TLC investigation of the mustard oil glycosides
1. Drug extraction (Sinapis semen)
Ground seeds (10 g) are added to 50 ml boiling methanol, boiled for 5 min, then
allowed to stand 1 h with occasional shaking. The filtrate is evaporated to 5 ml,
then applied to a column (length ca. 20 cm, diam. ca. 1 cm) containing 5 g cellulose
powder (cellulose MN 100, Machery & Nagel, Düren). The column is eluted with
methanol and the first 20 ml of eluate are discarded. The next 100 ml are collected
and evaporated to ca. 1 ml at 20--30 0 C under reduced pressure. 25 J.l.I of this soln.
are applied for chromatography.
2. Thin layer chromatography, and detection methods
Adsorbent: Silica gel 60F 254 pre-coated TLC plates (Merck, Darmstadt).
Chromatography solvent:
n-butanol-n-propanol-glacial acetic acid-water (30: 10: 10: 10).
The developed TLC plate is dried and sprayed with 25% trichloroacetic acid in
chloroform. After heating for 10 min at 140 0 C, the plate is sprayed with a 1: 1
mixture of 1% aqueous potassium hexacyanoferrate and 5% aqueous FeCl 3 (TPF
No. 35, p. 304).
Mustard oil glycosides give intense blue zones on a yellow background. The detection
limit is about 5 J.l.g glucoside.
B. TLC of mustard oils as their thiourea derivatives

1. Drug extraction (Armoraciae radix)
Ground roots (200 g) are mixed with 1 I water, allowed to stand for 2 h, and finally
submitted to steam distillation.
F or the preparation of crystalline thiourea derivatives from horse radish oil
(the procedure is similar for other mustard oils), 0.3 g oil is dissolved in 1 ml of
95% ethanol, and an equal volume of 25% ammonia soln. is added. The mixture
is heated for about 20 min on the water bath until the beginning of the exothermic
reaction. After standing 12 h, the crystals are removed by filtration and recrystallized
from warm ethanol.
2. Thin layer chromatography, and delection methods
Using the upper phase from ethyl acetate-chloroform (analytical grade)-water
(30: 30: 40), chromatography is performed on Silica gel60F 254 pre-coated TLC plates
(Merck, Darmstadt) over a distance of 15 cm.
Inspection in UV-254 nm, without chemical treatment, reveals thiourea deriva-
tives as violet-black zones on a yellow background.
253
C. TLC investigation of garlic
1. Isolation of the amino acid mixture of garlic
Finely chopped, fresh garlic cloves (25 g) are frozen with solid carbon dioxide,
and shaken twice with separate 100 ml quantities of 80% methanol. The combined,
filtered extracts are completely evaporated. The residue (ca. 2 g) is dissolved in
90 ml water, and about 60 ml ethanol are added drop-wise over aperiod of 10 min.
After allowing the mixture to stand 12 h in the refrigerator, the clear supernatant
is decanted and completely evaporated. The light yellow residue (ca. 1 g; hygro-
scopic) is extracted with ab out 15 ml ice-cold methanol for about 2 hat 0° C. After
filtration, the residue is washed with ice-cold methanol, then with anhydrous diethyl
ether, and finally dried at 150° C for 2 h. The product (ca. 0.2 g), which is no
longer hygroscopic, is dissolved in ca. 1 ml of 40% ethanol, and fractionated on
a column (length 20 cm, diam. 1 cm) of silica gel. Each 5 ml fraction is tested with
ninhydrin for the presence of amino acids. Fractions containing amino acids are
combined and evaporated to dryness (yield ca. 50 mg). Asoln. containing 50 mg
in 1 ml of 40% ethanol is used for chromatography.
2. Reference substances
Sulphoxycysteine derivatives can be prepared as follows:
e.g. S-allyl-L-cysteine sulphoxide
S-allyl-cysteine (2 g) is dissolved in 35 ml glacial acetic acid. After the addition
of 1.6 ml of 30% H 2 0 2 , the solution is allowed to stand for 8 h at 10--12° C. Animal
charcoal is added and the solution is filtered. The filtrate is evaporated to dryness
at about 50° C, and the residue recrystallized from acetone.
3. Thin layer chromatography, and detection methods
Adsorbent: Silica gel 60F 254 pre-coated TLC plates (Merck, Darmstadt)
Chromatography solvent: n-butanol-n-propanol-glacial acetic acid-water
(30: 10: 10: 10).
Delection: modified ninhydrin reagent (NIH No. 29, p. 303)
Sulphoxy derivatives give orange colours (vis.).
Alkylcysteines give violet colours (vis.).
254
11. Drug List
Drug/Plant source Glucosinolates M ustard oils
Family
Sinapis nigrae Semen Sinigrin Allyliso thiocyana te

Black mustard seeds
Brassica nigra (L.) KOCH
Brassicaceae
Sinapis albae Semen Sinalhin p-Hydroxybenylisothiocyanate
(Erucae semen)
White mustard seeds
Sinapis alba L.
Brassicaceae
Armoracia rusticana Gluconasturtiin Phenylethylisothiocyanate
MEY. ex SCHREB. +
Horse raddish Sinigrin Allylisothiocyanate
Brassicaceae
Allii sativi Bulbus Cysteine, cystine, methionine, methionine sulphoxide, cycloalliin,

Garlic S-allyl-cysteine sulphoxide (= alliin) and S-allyl-cysteine (=
Allium sativum L. deoxyalliin) are present, together with other S-containing amino
Liliaceae acids and cysteine sulphoxides.
By oxidation in the air, or under alkaline conditions, alliin is
converted into allicin (allylthiousulphinic acid allyl ester) and
other diallyl polysulphides, mainly diallyl disulphide.
111. Formulae
HO-fi-CH -C" S-Glucose

~ 2 ~N-O-S030
Allicin
Sinalbin
Sinigrin
255
Mustard on Drugs
Tracks 1 = Sinapis albae semen (Erucae semen)
2 = Sinapis nigri semen
Tests Tl = sinalbin test mixt ure
T2 = sinigrin
Solvent SC-4: n-butanol-n-propanol-glacial acetic acid-water (30: 10: 10: 10)
system
Detection Trichloroacetic acid-hexacyanoferrate-FeCI 3 reagent vis. Fig. 1 A, B
(TPF No. 35, p. 304)

Chromato- Sinapis semen
gram 1,2 After treatment with TPF reagent, extracts show at least five blue (vis.) zones.
1A, B
1 Sinapis albae semen is characterized by the two main zones of " sinalbin " (cf. Tl),
and the weaker zone between them.
2 Sinapis nigri semen contains sinigrin (cf. T2, Rf ca. 0.35). The zone directly above
this, which is characteristic of Sinapis alba, is absent.
Remarks: If larger quantities (50 111) of Sinapis albae semen extract are used, the chromatogram
shows a large number of weak zones in the upper Rf range.
Thiourea derivatives (TU Derivatives) - Chromatogram 2A

Tracks 1 = methyl-TU 3 = isopropyl- TU 5 = sec.n-butyl- TU 7= benzyl-TU
2= ethyl-TU 4= allyl-TU 6 = ß-phenyl; TU 8= phenyl-TU
Solvent ethyl acetate-chloroform-water (30: 30: 40, upper phase)
system
Detection Without chemical treatment in UV -254 nm
The naturally occurring mustard oils can also be analysed as their thiourea deriva-
tives.
Allium sativum - Chromatogram 2B

Tracks G=amino acid mixture from Alii bulbus
9-14 = S-alkyl-cysteine sulphoxides: 15-20 = S-alkyl-cysteines:
9 = methyl- 12 = n-propyl- 15 = methyl- 18=iso-propyl-
10= ethyl- J3=n-butyl- 16= ethyl- 19=n-propyl-
11 = iso-propyl- 14 = allyl- 17= allyl- 20=n-butyl-
Solvent n-butanol-n-propanol-glacial acetic acid-water (30: 10: 10: 10)
system
Detection Modified ninhydrin reagent (NIH No. 29, p. 303) vis.
The following compounds can be detected in Allii sativi bulbus:

9-14 Cysteine sulphoxides (orange, vis.)
S-methyl-, S-ethyl-, S-n-butyl-cysteine sulphoxide, and S-allyl-cysteine sulphoxide
(= alliin)
15-20 Cysteines (violet, vis.)
S-ally1cysteine (= deoxyalliin) and S-n-methy1cysteine.
256
A 8
-FRONT
Rf
-0.5
-START
Fig.l
Tl Tl 2 T2
A B
FRONT
-
Rf
•
--
-
- -
,J-
-
-- •
~
~ 0
0 ~
&' 0.5
0 <D
START
Fig.2
1- 8 G 9-14 15-20
257
N arcotic Drugs Cannabis sativa var. indica L., Cannabaceae
Marihuana: the flowering or seed carrying, dried branch tips of the female plant.
Hashish: the resin exuded from the leaves and flower stalks of the female plant.
The constituents of cannabis, the cannabinoids, are benzopyran derivatives. Of the
various cannabinoids, only L19,lO-tetrahydro-cannabinol (THC) has hallucinogenic
activity. The type and quantity ofthe various constituents depends on the geographi-
calorigin of the drug, c1imatic conditions of growth, time of harvesting and storage
conditions.
I. Preparation of Drug Extracts and Reference Solutions

Powdered drug (1 g) is extracted by shaking at room temperature for 10 rnin with
10 ml methanol. The filtrate is evaporated complete1y, and the residue dissolved
in 1 ml toluene. This toluene soln. (5~50 111, depending on the cannabinoid concentra-
tion) is applied to the chromatogram.

1. Adsorbent
Silica gel 60F 25" pre-coated TLC plates (Merck, Darmstadt)
CA-i: n-hexane-diethyl ether (80:20)
CA-2: n-hexane-dioxan (90:10)
CA-3: cyc1ohexane; the plate is impregnated with N,N-dimethylformamide by run-
ning unloaded in a mixture of N,N-dimethylformamide and carbon tetrachloride
(6:4). After evaporation of the carbon tetrachloride (room temp., 2 h), the extract
is applied to the origin, and chromatography performed in cyc1ohexane.
Thymol (0.1 % soln. in toluene), 5111.
THC (synthetic productjl mg in 5 ml CHC1 3 ), 3 ,.11.
111. Detection
In UV-254 nm, the cannabinoids show fluorescence quenching.
2. Fast blue salt reagent (FBS No. 12, p. 301)
Fast blue salt B (0.5 g) is dissolved in 100 ml water. The developed TLC plate is
sprayed, dried in a warm air stream, then immediately sprayed with 0.1 M NaOH.
Cannabinoids form violet-red, orange-red or carmine (vis.); standard thymol gives
orange.
The dried plate mayaiso be sprayed with a solution of fast blue salt (15 mg)
in 0.1 M NaOH (20 ml), but this reagent is not stable, and must be used immediately.
259
Cannabis Herba Hashish
Tracks 1 = Hashish (Turkish, 1980)
2 = Hashish (Irani an, 1980)
3 = Hashish cigarette (1979)
4,5,6= Cannabis herba (drug collection of the Institute)
7, 8 = Hashish (unknown origin)
Tests T=thymol
THC = tetrahydrocannabinol (synthetic)
Solvent CA-1: n-hexane-diethyl ether (80:20) Fig. 1A, B
system CA-2: cyc10hexane (after impregnation, see p. 259) Fig.2A
CA-3: n-hexane-dioxan (90: 10)
(double development: 1 x 10 cm and 1 x 15 cm) Fig. 2B, C
Detection Fast blue salt reagent (FBS No. 12, p. 301),
followed by treatment with KOH VlS. Fig. 1, 2
Chromato- Treatment with FBS-KOB produces intense red-violet to red-orange (vis.) zones.
gram Between the start and Rf 0.2 are three to fOUf red zones, due to cannabidiol acid
1A, B and other polar cannabinoids. Cannabinol (CBN, Rf ca. 0.45) migrates direct1y above
the thymol standard (cf. T1), followed by tetrahydrocannabinol (THC, Rf ca. 0.5)
and cannabidiol (CBD, Rf ca. 0.55).
The intensities of the COIOUfS depend strongly on the quantity of applied material
and on the subsequent KOH treatment. COIOUfS vary from red-violet (Fig. 1 A)
to red-orange (Fig. 1 B).
TBC is c1early evident only in sampie 2.
2A The separation in solvent system CA-2 on the impregnated TLC plate approximates
to that in CA-I, but with a better separation of zones in the lower Rf range. Sampie 7
contains TBC.
2B, C After double development in CA-3, the zones are found in a very narrow Rf range
between Rf 0.4-0.6. The two main zones at the same Rf as the thymol test are
due to CBN and CBD. In this system, TBC migrates above or in the same Rf
range as CBD.
Remarks: The Rfvalues of cannabinoids in solvent systems CA 1-3 are dependent on tempera-
ture and chamber saturation (cf. Figs. 1 AlB and 2B/c).
Cannabidiol acid (CBDA) Cannabidiol (CBD)
Cannabinol (CBN) LI 9, 10-Tetrahydrocannabinol (THC)
260
A
-0.5
5
Fig.l
2 3 4 5 6 T THC T
A 8
-F
Rf
-5
Fig.2
7 8 3 3 2 T THC T
261
Drugs Containing Valepotriates
The main active constituents of these drugs, the valepotriates, can best be detected
infreshly harvested and carefully dried roots.
Valepotriates are triesters of a terpenoid, trihydric alcohol. This alcohol has
the structure of an iridoid cyclopenta-(c)-pyran with an attached epoxide ring. The
following acids are fou~d: isovaleric, acetoxyisovaleric, isovaleroxy-hydroxyiso-
valeric, acetic and isocaproic acids.
Valepotriates with conjugated diene structure (valtrate and acevaltrate) form
blue (vis.) salts with acetic acid-HCl reagent; didrovaltrate which lack this diene
structure, give yellow-brown colour.
I. Preparation of Extract for TLC from Drugs and from Pharmaceuticals

Drug extracts
Powdered drug (0.2 g) is extracted with 5 ml dichloromethane for 5 min at ca. 60° C
with occasional shaking. After filtration, the drug residue is washed with a further
2 ml dichloromethane. The combined extracts are evaporated to dryness, and the
residue is dissolved in 0.2 ml ethyl acetate. 10 111 of this soln. are applied for chroma-
tography.
Pharmaceutical preparations
One or two dragees are extracted by shaking with 5 ml dichloromethane for 10 min.
The clear filtrate (15-20 111) is used directly for chromatography.

Solutions ofvaltrate, isovaltrate, didrovaltrate or acevaltrate are prepared by dissolv-
ing 5 mg in 5 ml methanol. 10111 of each solution are applied to the chromatogram.
2. Adsorbent
For good quality drugs, 10111 of extract is adequate for the chromatographie detec-
tion of the main constituents. Commercial valerian roots, however, often have a
low valtrate content, so that 50111 of extract are recommended for the TLC investiga-
tion.
V-I toluene-ethyl acetate (75: 25); single development over 15 Cll.
V-2 n-hexane-methylethyl ketone (80:20); DAB 8, double development.
263
III. Detection
UV-254 nm: acevaltrate and valtrathydrines.
UV-365 nm: yellow fluorescence, e.g. baldrinal and homobaldrinal.
2. Spray reagents
a) Hydrochloric acid-acetic acid reagent (No. 32, p. 303)
This is for the specific detection of valtrate and acevaltrate, which give blue zones
(vis.) (halazuchrome reaction): didrovaltrate shows brown (vis.).
b) Dinitrophenylhydrazine reagent, DAB 8 (DNP No. 10, p. 300)
After heating, green-grey or blue zones (vis.) appear; but all zones become uniformly
brown-yellow (vis.) if the heating is excessive.
IV. Drug List
Drug/Plant source Main constituents

F amily/Pharmacopoeia Content
Valerianae Radix lridoids (valepotriates):

Valerian rhizome valtrate, isovaltrate, IVHD-valtrate, didroval-
Valeriana officinalis L. trate, acevaltrate; for distribution and con-
tents, see Table below.
Valerianaceae
Essentialoi/: ca. 0.25% in fresh Valerian rhi-
Ph. Eur. III, 2. AB-DDR (not less than 1-2% zome, containing valerenal with some vale-
valepotriates ), ranone and valerenic acid.
Helv. VI, ÖAB
The essential oil represents one third of the
total activity of the drug.
Indian valerian
Valeriana wallichii DEcANDoLLE
Mexican valerian
Valeriana edulis ssp. procera MEYER
Table
Main constituent Valeriana Valeriana Valeriana wallichi

ojJicinalis edulis valtrate/ acevaltrate- didrovaltrate-
(ca. %) (ca. %) race (ca. %) race (ca. %)
Valtrate / isovaltrate 0.4-2 3.5 2 0.7

Didrovaltrate 0.1 1.5-4 0.1 1.5-3
Acevaltrate 0.1 0.1 0.4 0.4
Other valepotriates 0.1 1.0 0.1 0.1
264
v. Formulae
Valtrate: R1 , R2 = Isovalerianyl Didrovaltrate: R1 , R3 = Isovalerianyl

R3 =Acetyl R2 = Acetyl, R4 = H
Isolvatrate: R1, R3 = Isovalerianyl Homodidrovaltrate: R1 = Isocapronyl
R2 =Acetyl R2 = Acetyl
Homovaltrate: R1 = Isovalerianyl R3 = Isovalerianyl, R4 = H
R2 = Isocapronyl IVHD-Valtrate: R1 od. R2 od. R3 = Isovalerianyl
R3 = Acetyl R1 od. R2 od. R3 = Isovaleroxy-hydroxy-
Acevaltrate: R1 od. R2 = Isovalerianyl isovalerianyl
R1 od. R2 = Acetoxy- R1 od. R2 od. R3 = Acetyl, R4 = OH
isovalerianyl
HO
H3 Ch H ,
HO-~X_§ CH20 - Gluc
Isovalerianyl -0 0 I
Baldrinal: R = Acetyl
Valeriosidate Homobaldrinal: R = Isovalerianyl
265
Valerianae Radix
Tracks 1 = Valerianae radix (Mexican Valerian)
2 = V. radix (Indian Valerian; didrovaltrate race)
3,4,5= V. radix (drugs with degraded valepotriates)
6= V. radix (freshly harvested root)
7 = V. radix (tincture of Valerian from a dispensary)
8= V. radix (extract from pharmaceutical prep. I)
9= V. radix (extract from pharmaceutical prep. 11)
Tests Tl = valtrate
T2 = acevaltrate
T3 = didrovaltrate
Solvent V-l: toluene-ethyl acetate (75:25) Fig. lA, B
system V -2: hexane-methyl ethyl ketone (80: 20);
double development over 15 cm Fig; 2A, B
Detection Conc. HCI-glacial acetic acid (2:8) (No. 32, p. 303) vis. Fig. 1 A, B
Dinitrophenylhydrazine reagent (DNPH No. 10, p. 300) vis. Fig. 2A, B
Chromato- Valerianae radix
gram The chromatogram shows the blue (vis.) zone of the valtrate-isolvaltrate mixture
lA, B (cf. Tl), the brown zone of didrovaltrate (cf. T3), the blue zone of IVDH-valtrate
and degradation products of valepotriates (valtrathydrins). The type and quality
of the drug determines which of these zones is predominant. The degradation pro-
ducts of isovaltrate (homobaldrinal) and valtrate (baldrinal) (Rf ca. 0.5 and DA,
respectively) give an intense yellow fluorescence in UV-365 nm. Yellow zones are
also formed in vis. (Fig. 1 B, track 9).
Approx. Rf Compounds UV-254 nm HCI-acetic acid
(solvent (Fig. 1 A, B) reagent * vis.
system V-I)
0.7-0.75 lsovaltratefvaltrate + blue * Colours are blue to
0.65 Didrovaltrate brown black-blue, or light
0.55 Acevaltrate + blue to dark brown, de-
0.4 IVDH-valtrate blue pending on the de-
1 Valtrathydrins + blue gree of heating.
Start Degradation products (+) brown/blue
1 A, B 1 Mexican Valerian: high content of isovaltrate/valtrate; medium content of didroval-

trate; low conte nt of acevaltrate.
2 Indian Valerian: predominantly didrovaltrate, with low concentration of isovaltrate/
valtrate and IVDH-valtrate.
3,4,5 Official Valerian: commercial drugs with little or no valtrate, and containing chiefly
degradation products of valepotriates.
6 Official Valerian root (freshly harvested): ca. 90% valtrate.
7 Tincture of Valerian (commercial product): low concentration of valtrate, with medi-
um concentrations of acevaltrate, IVHD-valtrate and valtrathydrins.
8 Pharmaceutical: high content of valtrate (V. officinalis).
9 Pharmaceutical (5 years oId): degradation products of isovaltrate (homobaldrinal)
and valtrate (baldrinal).
2A In solvent system V-2, the order of separation is the same, but the Rf values are lower (0-0.6),
and separation is less satisfactory. The double development often leads to variable Rf values
(see chromatograms 2A and 2B).
2B Treatment with DNPH re agent guarantees the detection of all the constituents, but if the
plate is heated too strongly a11 zones become uniformly ye11ow-brown (vis.).
266
Fig.l
s
2 3 4 5 6 Tl T2 T3 7 8 9
Fig.2
2 3 4 5 6 Tl T2 T3 7 8 9
267
Drugs Containing Pigments
A. Drugs containing anthocyanins (flavylium derivatives)

Anthocyanins are responsible for the red, violet and blue colours of flowers and
other plant parts. They are present in the plant as glycosides of hydroxylated 2-phe-
nylbenzopyrylium salts (flavylium salts). Cleavage by acid hydrolysis produces the
corresponding free flavylium salt.
B. Crocus

1. Cyan i jlos, Hibisci jlos, Malvae jlos, Paeoniae jlos (anthocyanins)
Powdered drug (1 g) is extracted by shaking for 15 min with 6 ml methanol/HCl
(9 parts methanol: 1 part 25% HCl). 25111 of the filtrate are used for chromatogra-
phy.
2. Croci stigma
Four or five crushed stigmas are moistened with one drop of water. After about
3 min, ca. 1 ml methanol is added and extraction continued for ab out 20 min in
the dark, with occasional shaking. 10111 of the supernatant or filtrate are used
for chromatography.

a) Anthocyanins: 1 mg standard compound dissolved in 1 ml methanol; sampie 5 111.
b) Methylene blue: 5 mg dissolved in 10 ml methanol; sampie 10111.
c) Naphthol yellow/Sudan red: 5 mg naphthol yellow in 5 ml methanol, and 5 mg Sudan
red in 5 ml chloroform. The two solutions are mixed, and 5 111 are used for chroma-
tography.
2. Adsorbents
Cellulose pre-coated TLC plates (Merck, Darmstadt).
Silica gel plates are used for TLC of Croci stigma extracts.
Chromatography of flower pigments (anthocyanins) is performed on both silica
gel and cellulose plates.
a) Anthocyanins
n-Butanol-glacial acetic acid-water (40: 10: 20) (for silica ge1 and cellulose plates)
b) Croci stigma
Ethyl acetate-isopropanol-water (65: 25: 10)
269
111. Detection
1. Withaut chemical treatment
Anthacyanins form red to blue-violet (vis.) zones. The constituents of Craci stigma
are yellow.
2. Anisaldehyde-sulphuric acid reagent (AS No. 2, p. 299)
Craci stigma
After spraying, picrocrocin gives a red-violet (vis.) zone, while crocin gives a blue-
violet (vis.) zone.
IV. Drug List
Fig. Drug/Plant source Main compounds

F amily /Pharmacopoeia Anthocyanins
1,2 Cyani Flos Cyanidin-3,5-diglucoside (cyan in)

Cornflowers Pelargonidin-3, 5-diglucoside (pelargonin)
Centaurea cyanus L.
Asteraceac
1,2 Hibisci Flos Delphinidin-3-xylosyl-glucoside (hibiscin)

3 Hibiscus flowers
Hibiscus sabdariffa L.
Malvaceac
DAB8
1,2 MalvaeFlos Malvidin-3,5-diglucoside (malvin)

Common mallow flowers
Malva sylvestris (L.) MILL.
Malva sylvestris L. ssp. mauritania
(L.) ASCHERSON et GRAEBNER
("Mauretanian mallow, dark violet
mallow")
Malvaceae
Malvae (arboreae) Flos Delphinidin -3-glucoside
Hollyhock Malvidin-3-glucoside
Althaea rosea (L.) CAV. var. nigra (A mixture of these two compounds is known
HORT. as althaein)
Malvaceae
3 Paeoniae Flos Paeonidin-3,5-diglucoside

Peony
Paeonia spp.
Paeoniaceae
4 Croci Stigma 1.9-15% crocin (digentiobiose ester of croce-

Saffron (Crocus) tin)
Crocus sativus L. 2.7-12.9% picrocrocin (ß-hydroxycyclocitral
Iridaceae glucoside)
Ph. Eur. II1, Helv. VI, ÖAB ß-Hydroxycyclocitral and safranal (dehydro-ß-
cyclocitral) are formed from picrocrocin dur-
ing storage or steam distillation. Carotene gly-
cosides are also present.
270
v. Formulae
R, R2 Aglycones
H H Pelargonidin
HO OH H Cyanidin
OH OCH 3 H Peonidin
OH OH Delphinidin
OCH 3 OCH 3 Malvidin
OH OCH 3 OH Petunidin
CH3 CH3
Crocin ____ HOOC AA A A ~ "",,- ~ . . COOH
Pigment , ~ ~ ~ ..." TC "" T + Gentiobiose
H3 CH3
Crocetin
?yC~S
+ Glucose
-;:::::-0
HO~
NC'H ~_HYdö:rOXycYclO~ÖI
Gluc -O~ ~I
C'H
+ Glucose
Picrocrocin
Safranal
Bitter principle
Odour constituents
271
Flower Pigments, Anthocyanins
Tracks 1 = Cyani flos
2 = Malvae sylvestris flos
3 = Malvae arboreae flos
4 = Hibisci flos
Tests Tl = naphthol yellow and Sudan red
T2 = paeonidin mono glucoside
T3 = petunidin-3,5-diglucoside
T4 = delphinidin-3,5-diglucoside and monoglucoside
T5 = malvidin monoglucoside
T6 = cyanidin-3,5-diglucoside
T7 = methylene blue
Adsorbent Silica ge1 60F 254 pre-coated TLC plates Fig.1
Cellulose pre-coated TLC plates Fig.2
Solvent n-butanol-glacial acetic acid-water (40: 10: 20); developed over 10 cm
system
Detection Without chemical treatment in vis. Fig. 1,2
Chromato- On silica gel the zones are less sharply defined, but show more distinct colour
gram differences than on cellulose. On cellulose plates the Rf values are in the lower
1,2 range.
TLC separations 0/ anthocyanins
On silica gel On cellulose

Approx. Rf Approx. Rf
J Cyanijlos main zone 0.3 1 main zone 0.15

secondary zone 0.5 2 secondary zones 0.25-0.3
2 Malvae sylvestris jlos main zone 0.4 1 main zone 0.15
2 secondary zones 2 secondary zones 0.25-0.3
0.45 and 0.7
3 Malvae arhoreae jlos 2 main zones 0.25-0.55 Closely neighbouring zones
3 secondary zones of about equal intensity
0.6-0.8-0.9 0.1-0.25
4 Hihisci jlos 2 main zones 2 main zones
2 secondary zones 2 secondary zones
0.25-0.5 0.15-0.3
Remarks: A better differentiation and identijication of anthocyanins can be achieved by ascend-

ing or descending paper chromatography, using Ion ger migration distances and solvents based
on mixtures of butanol, acetic acid and water.
272
-FRONT
Rf
-0.5
-START
Fig.l
Tl - T 7 2 3 4
-FRONT
-0.5
-START
Fig.2
Tl - T7 2 3 4
273
Flower Pigments, Anthocyanins Croci Stigma
Tracks 1 = Hibisci flos
2 = Paeoniae flos
3 = Croci stigma
Tests T1 = methylene blue
T2 = naphthol yellow (Rf ca. 0.2) and Sudan red (Rf ca. 0.95)
Solvent n-butanol-glacial acetic acid-water (40: 10: 20) Fig. 3A, B
system ethyl acetate-isopropanol-water (65: 25: 10) Fig. 4A, B, C
Adsorbent Silica gel 60F 254 pre-coated TLC plates
Detection Without chemical treatment VIS. Fig. 3A, 4B
Without chemical treatment UV-365 nm Fig.3B
Without chemical treatment UV-254 nm Fig.4A
Anisaldehyde-sulphuric acid reagent (AS No. 2, p. 299) vis. Fig.4C
Chromato- 1 Hihisci jlos

gram The extract is characterized by two main, blue-violet zones in the Rf region of
3A, B the methylene blue standard. In UV-365 nm two zones appear in this region, one
dark and the other light blue fluorescent.
2 Paeoniae jlos
A red-violet (vis.) or orange fluorescent (in UV-365 nm) zone is present in approxi-
mately the same Rf region as the methylene blue test (cf. Tl). A weaker, blue-violet
zone is also present below the main zone.
4A-C 3 Croci stigma
Four zones of strong fluorescence quenching are seen in UV -254 nm. In vis., 3-4 yel-
low zones are seen in the Rf range 0.05-0.45. The main zone above the start is
due to crocin, which shows grey-green with AS reagent.
Picrocrocin (Rf ca. 0.55) shows distinct fluorescence quenching in UV-254 nm, and
violet (vis.) with AS reagent.
The fluorescence-quenching zones of 4-hydroxycyclocitral and safranal, in the Rf
region of the Sudan red test (Rf 0.8-0.95), are not always present.
274
Fig.3
Tl 2 Tl Tl 2 Tl
A
• FRONT
Rf
-0.5
Fig.4 -START
T2 3 T2 3 T2 3
275
Drugs with Miscellaneous Constituents
These drugs cannot be assigned to any of the foregoing drug groups. They contain
a wide variety of constituents, some of which do not occur in other drugs.

1. Salicis cortex
methanol. The filtrate is evaporated to ca. 3 ml, and 20 f.tl are used for chromatogra-
phy. Extracts may be similarly prepared in CHCl 3 or ethyl acetate. Alternatively,
the concentrated methanol extract may be extracted by shaking with two separate
10 ml quantities of CHCl 3 or ethyl acetate; combined extracts are evaporated to
ca. 3 ml, and 20 f.tl are used for chromatography.
2. Pyrethri jlos
Pyrethrins are extracted from the flowers with methanol (1 g drug, 5 ml methanol,
10 min extraction). This extract is used directly for chromatography (15 or 30 f.tl,
depending on the pyrethrin concentration).
3. Filicis rhizoma
Powdered drug (1 g) is mixed with 15 ml saturated barium hydroxide and shaken
for ca. 30 min. The filtrate is adjusted to pH 3-5 with dilute HCI, then extracted
with three separate 10 ml quantities of diethyl ether. The combined ether extracts
are dried over anhydrous sodium sulphate, then completely evaporated. The residue
is dissolved in ca. 1 ml CHCI 3 , and 10 f.tl are used for chromatography.
4. Hamamelidis folium/cortex
methanol. The filtrate is evaporated to ca. 5 ml, and 10 f.tl are used for chromatogra-
phy.
5. Lichen islandicus
Powdered drug (2 g) is heated for 5 min with 5 ml methanol and 10 f.tl of the c1ear
filtrate are used for chromatography.
6. Podophylli resina/rhizoma
Resin (0.5 g) is extracted by heating under reflux for 30 min with 5 ml CHCI 3 ;
20 f.tl of the c1ear filtrate are used for chromatography.
7. Visci albi herba (folium)
of 50% methanol. The c1ear filtrate is evaporated to ca. 5 ml.
For the investigation ofjlavonoids, 20 f.tl are applied to the chromatogram. Alter-
natively, 2.5 ml of the methanol extract can be extracted with 10 ml ethyl acetate,
the ethyl acetate phase evaporated to ca. 2 ml, and 25 f.tl of this soln. used for
chromatography.
For the investigation of amino acids, 30 f.tl of the concentrated methanol extract
are applied to the chromatogram.
277
H. Thin Layer Chromatography
Salicis cortex Salicin, pieein, triandrin: 1 mg dissolved with warming in 1 ml
methanol; sampie volume 15111.
Pyrethri flos Thymol: 5 mg in 5 ml methanol; sam pie volume 5 111.
Filicis rhizoma Resorcinol, phloroglucinol: 5 mg in 5 ml methanol; sam pie vol-
urne 511l.
Hamamelidis folium Hamamelitannin: 10 mg in 5 ml methanol; sampie volume
10 111.
Lichen islandicus Caffeie acid: 5 mg in 5 ml methanol; sampie volume 10111.
Fluoreseein: 5 mg in 10 ml methanol; sampie volume 10111.
Podophylli rhizoma Podophyllotoxin: 5 mg in 5 ml methanol; sam pie volume 10 111.
(resina)
Visci albi herba Chlorogenie acid: 5 mg in 5 ml methanol; sampie volume 10 111.
(folium)
Amino acids: 5 mg of each amino acid in 5 ml methanol; sam-
pIe volume 10111.
2. Adsorbent
3. Chromatography solvents and detection methods

See the corresponding chromatograms (Figs. 1-8, pp. 283-287).
IH. List of Miscellaneous Drugs


Family jPharmacopoeia
Salicis Cortex ca. 4--5% phenol glycosides in varying propor-

Willow bark tions:
Salix spp., e.g. Salicin (salicyl alcohol-ß-D-glucopyranoside),
Salix alba L., 0.5% in Salix alba; 1-3% in Salix fragilis.
White willow Triandrin (3-(-4-hydroxyphenol-2-propen-l-
Salix viminalis L. ol)-1-ß-D-glucopyranoside, up to 6% in Salix
Common osier viminalis L.
Salix cinerea L. Picein (4-hydroxyacetophenone-ß-D-gluco-
Grey willow side), ca. 2% in Salix cinerea L.
Salix fragilis L. Esters of salicyclic acid and salicyl alcohol.
Crack willow Acetylated salicin, salicortin and salireposide
Salicaceae mayaiso OCCUf.
Tannins
2A Hamamelidis Folium Not less than 8% tannins (Helv. VI), with ß-

Witch-hazelleaves hamamelitannin (formed from gallic acid and
Hamamelis virginia L. hamamelose = 2-C-hydroxymethyl-D-ribose)
Hamamelidaceae
2. AB-DDR, Helv. VI
278
2B Filicis Rhizoma Not less than 1.5% crude filicin, and 20%
(Polypodii filicis maris radix) flavaspidic acid (Helv. VI).
Male fern rhizome Main compounds are the hutanone-phloroglu-
Dryopteris filix-mas (L.) SCOTT eides.
Polypodiaceae Dimeric phloroglucides: albaspidin, flavas-
Helv. VI pidic acid and others.
Trimeric phloroglucides: filixie acid, trifla-
vaspidic acid and others.
Tetramerie phloroglucides: methylene-bis-
nor-flavaspidic acid.
2C Pyrethri Flos Not less than 1% total pyrethrins, containing

Insect flowers 50% pyrethrin I (ÖAB).
Chrysanthemum cinerariifolium Main compounds are pyrethrins land II, and
(TREVISAN) VISANI einerins land II. They are converted into per-
Dalmatian insect flowers oxides and lumi-compounds on exposure to
Chry. marschellii ACHERSON air and light.
Chry. occineum WILLD.
Caucasian insect flowers
Asteraceae
ÖAB
3A Lichen Islandicus Main compounds are polysaccharides (ca.
3B Ice1and moss 50%): liehen in and isoliehenin.
3C Cetraria islandica (L.) ACHARIUS Also present are 2-3% bitter tasting depsi-
Parmeliaceae dones (lichen acids): fumarprotocetraric aeid,
Helv. VI (the 2. AB-DDR alsoad- protocetraric aeid and cetraric aeid (1-2%),
mits C. tenuifolia (RETz) HOWE with protolichesteric acid and lichesteric acid.
4A Podophylli Rhizoma Main compounds are lignanes (podophyllo-

4B May-apple root toxin, (X- and ß-peltatin) and flavonoids.
4C Podophyllum peltatum L. Podophylli resina contains ca. 50% podo-
Berberidaceae phyllotoxin.
Podophyllinum
ÖAB, Helv. VI
5, 6 Visci albi Herba (Folium) Main compounds in branches and leaves:

Mistletoe leaves Flavonoids:
Viscum album L. ssp. album Quercetin-3-0-rhamnoside, Q-3-0-arabino-
Deciduous mistletoe (on practically side; in V. alb. ssp. coloratum:
all European deciduous trees, ex- Flavoyadorinin A, Flavoyadorinin B (7,3'-di-
cept beech) O-methyl-luteolin-4' -O-monoglucoside) and
ssp. abies (WIESB.) homoflavoyadorinin B (sugar residue D-glu-
Abromeit cosylapiose).
Silver fir mistletoe (onlyon silver Plant acids: chlorogenie acid, caffeic acid, sin-
fir; not on Scots pine or deciduous apic acid.
trees) Fresh leaves contain high concentration of
ssp. austriacum (WIESB.) amino acids (ca. 0.43%), e.g. arginine, aspara-
VOLLMANN gine, proline, lysine, serine, alanine, threo-
Seots pine mistletoe (on Pinus spp.; nme.
rarelyon spruce) "Viscotoxin": a polypeptide with 46 amino
Loranthaceae acid residues (Mol. Wt. 5000).
7, 8 Remarks: TLC separation of amino aeids; see Figs. 7 and 8 for reference compounds
for Visci albi herba.
279
IV. Formulae
Q
OH
0 CH,OH
CH=CH-CH2-0~
HO
OH
Triandrin
OH
Salicin
CH3
I
Q
C=O
Salicortin
CH,OH
~ OH
HO
OH
Picein
Salireposide
OH
OH
Galloyl -O-~:O-GaIlOYI HO
OH OH OH
Gallic acid
(3,4,5-Trihydroxy- ~-Hamamelitannin Catechin
benzoic acid)
280
CH3
~oc~
o OH
Aspidinol Filixic acid
Chrysanthemum-mono- Pyrethrolone,
and dicarbonic acid Cinerolone
Pyrethrin I: R1 = CH 3 ; R2 = -CH 2 -CH=CH-CH=CH 2 R= -CO-CH=CH-COOH

Pyrethrin 11: R1 = COOCH 3 ; R2 = -CH 2 -CH=CH-CH=CH 2 Fumarprotocetraric acid
Cinerin I: R1 = CH 3 ; R2 = -CH 2 -CH=CH-CH 3 R= -C 2 H.
Cinerin 11: R1 = COOCH 3 ; R2 = -CH 2 -CH=CH-CH 3 Cetraric acid
R=-H
Protocetraric acid
Podophyllotoxin: R1 = H; R2 = OH; R3 = CH 3
a-Peltatin: R1 = OH; R2 = H; R3 = H
~-Peltatin: R1 = OH; R2 = H; R3 = CH 3
281
Salicis Cortex, Hamamelidis Folium
Filicis Rhizoma, Pyrethri Flos
Tracks 1 = Salicis cortex (MeOH total extract)
2 = Salicis cortex (CHC1 3 extract)
3 = Salicis cortex (ethyl acetate extract)
4 = Hamamelidis folium (MeOH extract)
5 = Filicis rhizoma (diethyl ether extract)
6 = Pyrethri flos (MeO H extract)
Tests Tl = salicin T4 = phloroglucinol T7 = resorcinol
T2 = triandrin T5 = catechin T8 = phloroglucinol
T3=picein T6 = hamamelitannin T9 = thymol
Solvent AN-1: ethyl acetate-methanol-water (100: 13.5: 10) Fig. lA, B
system E-1: ethyl formate-formic acid-water (80: 10: 10) Fig.2A
E-2: chloroform-methanol (85: 15) Fig.2B
E-3: hexane-ethyl acetate (90: 10) Fig.2C
Detection Fast blue salt reagent (FBS No. 12, p. 301) Fig.1A
FBS, followed by 5% KOH Fig. lB, 2B
Iron (III) chloride (FeCI 3 No. 14, p. 301) Fig.2A
Phosphomolybdic acid reagent (PMA No. 27, p. 303) Fig.2C
Chromato-
gram 1,2,3 Salicis cortex (commercial drugs, botanically not defined). FBS reagent produces
1A, B 3-4 red or yellow-brown (vis.) zones in the intermediate Rf range. The colours
are intensified by KOH treatment.
The phenol glycosides that are characteristic of individual Salix species mi grate
at Rf 0.4-0.6. In some species, salicin is the main compound. The glycosides are
partially c1eaved during extraction. The main zones in the upper Rf range, which
also stain red with FBSjKOH, are due to catechin, p-coumaric acid, saligenin and
salicylic acid (yellow-brown) and salicyl alcohol. Some Salix species show only a
low content of salicin.
2A 4 Hamamelidisfolium
The zones of catechin (cf. T5), phloroglucinol (cf. T4) and hamamelitannin (cf. T6)
show varying intensities of blue-black (vis.) by FeCI 3 .
2B 5 Filicis rhizoma
FBS treatment reveals 7-8 red (vis.) zones of the butanone phloroglucides. There
are also two zones in the region of the resorcinol test (the lower of these is filixic
acid), one weaker zone in the region of the phloroglucinol test (= jlavaspidic acid)
and major zones at Rf ca. 0.15 and the solvent front (=aspidinol)
2C 6 Pyrethri jlos
The pyrethrins are stained blue (vis.) by PMA reagent. The main compounds, pyreth-
rins land 11, and cinerins land 11 become rapidly converted into lumi-compounds
or peroxides on exposure to light and air.
A satisfactory detection of pyrethrins can only be performed with fresh flowers.
The chromatogram shown, was made with an extract of commercially available
flowers; the main components migrate below the thymol test (cf. T9).
282
A
• -FRONT
Rf
-0.5
-START
Fig.l
Tl 2 3 2 3 T2 Tl T3
FRONT
Rf
-START
Fig.2
T4 T5 4 T6 T7 5 T8 T9 6
283
Miscellaneous Drugs
Tracks 1 = Lichen islandicus
2 = Podophylli resina
Tests Tl = caffeic acid and methyl ester of caffeic acid
T2 = fluorescein
T3 = podophyllotoxin
Solvent E-4: chloroform-glacial acetic acid-methanol (90: 5: 5) Fig.3A-C
system E-5: chloroform-methanol (90: 10), chromatography over 5 cm, Fig.4A-C
followed by 15 cm in chloroform-acetone (65: 35)
Detection Without chemical treatment UV-365 nm Fig. 3A, 4A
Vanillin-sulphuric acid re agent (VS No. 38, p. 304) VlS. Fig.3B
Phenylenediamine reagent (PD No. 31, p. 303) UV-365 nm Fig.3C
Conc. sulphuric acid reagent (No. 34C, p. 303) UV-365 nm Fig.4B
vis. Fig.4C
Chromato- 1 Lichen islandicus

gram 'The untreated chromatogram shows a strong fluorescence-quenching zone (Rf ca.
3 0.25) ofJumarprotocetraric acid in UV-254 nm.
3A In UV-365 nm, the untreated chromatogram shows a black-brown zone (scarcely
visible against the plate background) at about the same Rf value as the caffeic
acid test (Tl).
3B Treatment with VS re agent produces a pronounced blue-violet (vis.) zone of Jumar-
protocetraric acid at about the same Rf value as the caffeic acid test, and weaker
zones at Rf ca. 0.7.
3C After treatment with PD reagent, the chromatogram shows a yellow-green fluores-
cent zone in UV-365 nm. In addition, there are yellow or brown-green fluorescent
zones in the upper Rf range, due to protolichesteric acid, cetraric acid and protoce-
traric acid.
Remarks: In MeOH, caffeic acid is rapidly converted into its methyl ester (cf. T1), which
has a higher Rf value.
4A 2 Podophylli rhizoma (resina)
The main compound, podophyllotoxin, shows fluorescence quenching in UV-254 nm,
but does not fluoresce in UV-365 nm.
4B, C After H 2 S0 4 treatment, it appears as a dark brown zone in UV-365 nm, or red-
brown in vis. (Rf ca. 0.8; cf. T3). There is a weaker, dark brown (UV-365 nm)
or yellow-red (vis.) zone at Rf ca. 0.6 (rz-peltatin) , and another zone directly below
the solvent front (deoxypodophyllotoxin and ß-peltatin). The yellow zones in the
intermediate Rf range are due to flavonoids.
284
FRONT
Rf
START
Fig.3
TI Tl T2 T2
-FRONT
Rf
-0.5
-START
Fig.4
2 2 T3 2 T3
285
Visci albi Herba (Folium)
Tracks 1-3= Visci albi herba (drug patterns I-IH; extracted with 50% methanol)
4= Vi sei albi herba (drug pattern H; ethyl acetate extract)
Test Tl = chlorogenie acid
system (100: 11 : 11 : 27) Fig.5
AA-l: n-butanol-acetone-glacial acetic acid-water
(35:35:10:20) Fig. 6A, B
(NPjPEG No. 28, p. 303) UV-365 nm Fig. 5; 6A
Ninhydrin reagent (NIH No. 29, p. 303) vis. Fig.6B
For description of drugs see p. 279. Preparation of extracts p. 277.
Chromato-
gram 1-3 After treatment with NP/PEG re agent, prominent features of the chromatogram
5A are white-blue fluorescent zones above the start, in the Rf range of Tl (ehlorogenie
acid and other caffeoylquinic acids) and in the region of the solvent front (eaffeie
acid).
The yellow-green fluorescent zones in the upper Rf range are due to jlavonoids
(e.g. Quercetin-glycosides). The flavonoids in the intermediate Rf range are seen
better in the ethyl acetate extract (4).
6A In solvent system AA-l (for separation of amino acids), treatment of the chromato-
gram with NP/PEG reveals blue fluorescent zones in the middle and upper Rf
range, and the flavonoids unresolved at Rf ca 0.9.
6B Treatment with ninhydrin reagent produces about ten red-violet to red-brown zones
of amino acids in the lower and middle Rf range.
The following amino acids can be identified: L-lysine and L-arginine (low Rf
region), proline, L-serine, alanine, threonine, glutamie acid (overlapping in the region
of the brown zone), and tyrosine and leucine (Rf 0.5 and higher) (cf. Figs. 7 and
8, p. 288).
According to the literature, at least 18 free amino acids are present. With few
variations, the TLC amino acid patterns of all Viscum drugs are similar, and may
therefore be used for identification purposes.
286
FRONT
Rf
Fig.5
4 Tl
-FRONT
~O . 5
-START
Fig.6
2 3 2 3
287
Amino Acids Tests
Track 1 = L-lysine 12 = D L-serine
2 = L-arginine 13 = L-threonine
3 = D-asparagine 14 = L-cysteine
4 = L-asparagine 15 = 4-amino butyric acid
5 = glycine 16=D-valine
6 = L-proline 17 = DL-methionine
7 = L-glutamine 18= DL-tyrosine
8= L-serine 19= L-tyrosine
9 = glutamic acid 20 = D-isoleucine
10= DL-alanine 21 = L-leucine
11 = DL-threonine 22 = DL-phenylalanine
Solvent AA-1: n-butanol-acetone-glacial acetic acid-water
system (35:35:10:20) Fig. 7A, 8B
AA-2: n-butanol-glacial acetic acid-water
(50: 10:40); upper phase Fig. 7B, 8A
Detection Ninhydrin reagent (NIH No. 29, p. 303) Fig. 7, 8
Chromato- Most amino acids show violet-red to red-brown (vis.) with ninhydrin: the exception
gram is proline (6), which gives yellow (vis.).
Solvent system AA-1 generally gives higher Rf values than AA-2.
AA-1 Amino acids 1-11 (Fig. 7 A) and 12-17 (Fig. 8 B)
AA-2 In solvent system AA-2, amino acids 2-5 migrate at almost the same Rf (Fig. 7B),
whereas amino acids 12-22 show marked differences in Rf (Fig. 8 A).
For the identification of all the amino acids by TLC, several different solvent
systems must be used.
288
A
• -F
Rf
-0.5
-5
Fig.7
T1-T11 T1- T6
-0.5
-5
Fig.8
T12 -T22 T12 -Tl7
289
TLC Screening
of an Unknown Commercial Drug
Using a special analytical procedure, an unknown commercial drug can be identified

or assigned to a group on the basis of its constituents.
Analyses are performed for the following main active constituents:
1. alkaloids 6. jlavonoids
2. anthra-glycosides 7. saponins
3. arbutin 8. essentialoils
4. cardiac glycosides 9. coumarins and phenol carboxylic acids
5. bitter principles 10. valepotriates
I. Preparation of Extracts
1. For analysis of anthra-glycosides, arbutin, bitter principles andjlavonoids. Powdered
drug (1 g) is extracted by heating on a water bath for 15 min with 5 ml methanol;
20 III and 100 III of the filtrate are applied to the chromatogram.
2. For analysis of alkaloids. Powdered drug (1 g) is moistened with ca. 1 ml of 10%
ammonia soln., then extracted by shaking for 15 min at 60 0 C with 5 ml methanol;
20 III and 100 III of the filtrate are applied to the chromatogram.
3. For analysis of saponins. A methanol extract is prepared as in 1. (above). This
is evaporated to ca. 1 ml, mixed with 0.5 ml water, then extracted with 3 ml butanol.
20 111 and 100 III of the butanol phase are applied to the chromatogram.
4. For analysis of cardiac glycosides. Powdered drug (1 g) is mixed with 5 ml of 50%
methanol and 10 ml of 10% lead (11) acetate solution, then heated for 10 min on
the water bath. The cooled filtrate is extracted with two separate 10 ml quantities
of dichloromethane. The combined DCM extracts are completely evaporated. The
residue is dissolved in DCM-methanol (1: 1), and 100 111 of this soln. are applied
to the chromatogram.
5. For analysis of essential oils, coumarins, phenol carboxylic acids and valepotriates.
a) Dichlo.romethane extract (DCM extract)
DCM. The filtrate is evaporated to dryness, and the residue is dissolved in 1 ml
toluene; 20 111 and 100 111 of this toluene soln. are applied to the chromatogram.
b) Microdistillation of essential oils according to Luckner or T AS method (see Essential
Oil Drugs, p. 6)
Using the TAS method, all those compounds that are volatile at ca. 200 0 C can
be obtained (essential oils, coumarins, etc.).

From each extract, 20 111 and 100 111 (prepared as described above, under I), are
applied to a TLC silica gel plate (60F 254 , 10 cm x 10 cm). Ten such plates are pre-
pared, one for each of the main classes of constituents 1-10 (see above). To each
plate is also applied a different selection of standard substances belonging to the
class of constituents to be analysed (see separation scheme on p. 294). Chromatogra-
phy is performed in one of the following solvent systems:
291
a) Ethyl acetate-methanol-water (100: 13.5: 10)
For the analysis of anthra-glycosides, arbutin, cardiac glycosides, bitter principles,
jlavonoids, alkaloids and saponins.
b) Toluene-ethyl acetate (93: 7)
For the analysis of essential oils, coumarins, valepotriates and plant acids.
Both solvents are allowed to run for a distance of 8 cm. After inspection in UV-
254 nm and UV-365 nm, each chromatogram is analysed for the presence of one
of the groups of drug constituents by spraying with an appropriate reagent (see
separation scheme on p. 294, and list of spray reagents on p. 299).
III. Detection and Classification of Components

The following reactions can be used to determine the types of compounds present:
1. Bornträger reaction (10% ethanolic KOH No. 21, p. 302)

Red in vis.; red fluorescence in UV-365 nm: anthraquinones (e.g. frangulin, rhein,
etc.)
Yellow in vis.; yellow fluorescence in UV-365 nm: an thrones (e.g. aloin, aloinosides,
cascarosides, etc.)
For further identification, see Anthraquinone drugs, Figs. 1-12, pp. 102-114).
2. Kedde reagent (No. 23, p. 302)

Pink and blue-violet (vis.) zones: cardenolides. For further identification, see Cardiac
glycoside Drugs, Figs. 1-8, pp. 204-220).
Remarks: BufadieilOlides are detected with antimony (III) chloride reagent (SbCI 3 No. 3,
p. 299). This produces blue (vis.) zones (scillaren A) or yellow-green fluorescent zones (in
UV-365 nm) (scilliroside) in the intermediate Rfrange.
3. Dragendorffreagent (DRG No. 11, p. 300)

Red-brown (vis.) zones; the colour may be unstab1e: alkaloids. Some types of alka-
loid fluoresce in UV-365 nm without chemical treatment, e.g. Cinchona alkaloids,
emetine, cephaeline and the Rauwolfia alkaloids give blue fluorescence. Alkaloids
of the berberine type fluoresee yellow; boldine gives a violet fluorescence.
Some of the strongly basic alkaloids remain at the start in the screening system.
If at the start a positive DRG reaction is given, a second chromatogram should
be run in solvent system AL-1 (toluene-ethyl acetate-diethylamine, 70: 20: 10). For
further identification, see Alkaloid Drugs, Figs. 1-26, pp. 66-90.
4. Berlin blue re agent

Blue in vis.: arbutin and its derivatives. For further identification, see Arbutin Drugs,
Fig. 1, p. 120.
5. Natural products-polyethyleneglycol re agent (NP/PEG No. 28, p. 303)

Intense yellow, orange and green fluorescent zones in UV -365 nm: flavonoids.
Without chemical treatment, flavonoids must show a distinct quenching of fluores-
cence in UV-254 nm, and yellow, green or weak blue fluorescence in UV-365 nm.
The screening system for flavonoids does not produce such sharp zones as the
solvent system F-1. For positive identification, chromatography should be repeated
with F -1 ethylacetate-formic acid-glacial acetic acid-water (100: 11 : 11 : 27).
Chlorogenie acid, which is frequently present in flavonoid containing extracts,
remains at the start in the screening system, and mi grates at Rf ca. 0.5 in solvent
system F-l.
For further identification, see Flavonoid Drugs, Figs. 1-20, pp. 172-190.
292
6. Vanillin-sulphuric acid reagent (VS No. 38, p. 304)
a) Bitter principles
If the extract tastes distinct1y bitter, and the screening system shows red-brown,
yellow-brown or dark green (vis.) zones in the intermediate Rf range, the drug
may be one ofthe known bitter principle drugs (see Fig. 1, p. 132).
Very lipophilic bitter principles, such as quassin, absinthin, cnicin and marrubiin
migrate unresolved at the solvent front in this screening system. For further identifi-
cation, see Bitter Principle Drugs, Figs. 1-12, pp. 132-142.
Remarks: Extracts containing alkaloids or cardiac glycosides also taste partly bitter.
b) Saponins
Saponins also form coloured (vis.) zones with VS reagent. In the screening system,
however, the known saponins (e.g. aescin, Primula acid and the saponin test mixture)
remain at the start.
For a precise differentiation, chromatography must be performed in the system
SP-l (chloroform-methanol-water, 64: 50: 10). For further identification, see Sa-
ponin Drugs, Figs. 1-12, pp. 234-244.
c) Essential oils
Blue, brown or red zones in vis. In the screening system, essential oils migrate
unresolved at the solvent front.
Classification is possible after chromatography in solvent system A-l (toluene-
ethyl acetate, 93: 7); see Essential Oil Drugs, Figs. 1-28, pp. 22--48.
7. Hydrochloric acid - acetic acid reagent (HCl/AA No. 32, p. 303)

Valepotriates
8. NH3/KOH - reagent (No. 21, p. 302)

Coumarins
293
IV. Scheme of Separation and Identification
Ethyl acetate-methanol-water 100: 13.5: 10 TLC I-TLC 7
TLCl Anthra-glycosides
Extract 20111/100111
Tests -> red (vis.): -> Identification:
a10in 5 111 anthraquinones see Anthraquinone
frangu1in 10 111 yellow (vis.): Drugs, p. 93
Detection anthrones
KOR reagent No. 21
TLC2 Arbutin
Extract 20111/100111
Tests -> blue (vis.) -> Identification:
arbutin 10 111 see Arbutin
hydroquinone 10 111 Drugs, p. 117
Detection
Berlin b1ue No. 5
TLC3 Cardiac glycosides

Extract 20 111/100 111
Tests -> pink/vio1et (vis.) -> Identification:
1anatosides A-C 10 111 see Cardiac
k-strophanthin 10 111 Glycoside Drugs,
proscillaridin 10 111 p. 195
Detection -> blue (vis.) -> Identification:
Kedde reagent No.23 SbC1 3 - see Bufadienolides
(SbC1 3 re agent No. 3) reag.on1y p. 195
TLC4 Bitter principles

Extract 20111/100111
Tests -> red/yellow- -> Identification:
naringin 10 111 brown/bIue-green see Bitter
(rutin 10 111) Principle Drugs,
Detection p. 125)
VS reagent No. 38
TLC5 Alkaloids
Extract 20 111/100 111 Solvent system AL-1
Tests -> orange-brown -> toluene-ethyl
atropine 10 111 (vis.) acetate-diethylamine
reserpine 10 111 (70:20: 10)
emetine 10 111 1
Delection Identification:
Dragendorff see Alkaloid Drugs,
reagent No. 21 p.51
294
TLC6 Flavonoids
Extract 20111/100 111 Solvent system F-1
Tests -+ yellow/green/ -+ ethyl acetate-
rutin 10 111 orange formic acid-glacial
chlorogenic acid 10 111 (UV-365 nm) acetic acid-water
hyperoside 10 111 (100:11:11:27)
Detection !
NP/PEG reagent Identification:
No. 28 see Flavonoid Drugs,
p. 163
TLC7 Saponins
Extract 20111/100111 Solvent system SP-1
Tests -+ blue (vis.) -+ chloroform-methanol-
aescin 10 111 water (64:50:10)
Primula acid 10 111 !
Detection Identification:
VS reagent see Saponin Drugs,
No. 38 p.225
Toluene-ethyl acetate, 93: 7 TLC 8-TLC 10

TLC8 Essential oils
Extract 20111/100111
Test -+ red/yellow / -+ Identification:
Salviathymol ® 5 111 blue/brown see Essential Oil
(p.296) (vis.) Drugs, p. 5
Detection
VS reagent No. 38
TLC9 Valepotriates
Extract 20111/100111
Test -+ blue/brown -+ Identification:
Valtrat or standard (vis.) see Valerianae
Pharmaceuticals Radix, p. 263
Detection
Hydrochloric acid-
acetic acid reagent
No. 32
TLC10 Coumarins
Extract 20111/100111 Solvent system C-1
Tests -+ light blue/ -+ diethyl ether-
scopoletin 5 111 brown toluene (1: 1;
umbelliferone 5 111 (UV-365 nm) saturated with 10%
Detection acetic acid)
UV -365 nm without !
chemical treatment; Identification:
intensified with see Coumarin Drugs,
NH 3 /KOH p. 145
295
TLC Analysis of HerbaI Drug Mixtures
Many herbai preparations contain mixtures of drugs or drug extracts. Chromato-

grams displaya large number of more or less overlapping zones (UV and vis.),
so that identification or c1assification of the compounds present is difficult or only
partly successfu1. In such cases it is necessary to submit the preparation to column
chromatographie fractionation or other special procedures for the separation of
individual c1asses of compounds.
If the different drugs of the herbai formulation contain the same c1asses of
compounds and active principles, identification of the characteristic components
is usually possible.
Salviathymol ® (essential oil components, Fig. 1) and purgative preparations
(anthra-glycoside mixtures, Fig. 2) are described be10w as examples of mixed herbai
preparations.
Fig. 1 Salviathymol ®
Composition
1 g contains: 01. Salviae 2 mg (standardized at not less than 40% thujone); 01.
Eucalypti 2 mg (not less than 75% cineole); 01. Menth. pip. 23 mg (not less than
50% menthol); 01. Cinnamomi 2 mg (not less than 75% cinnamaldehyde); 01. Caryo-
phylli 5 mg (not less than 80% eugenol); 01. Foeniculi 10 mg (not less than 60%
anethole and 10% fenchone); 01. Anisi 5 mg (not less than 90% anethole); Tinct.
Myrrhae (DAB 8) 10 mg; Tinct. Rathanhiae (DAB 8) 4 mg; Tinct. A1chemillae (1 : 5)
20 mg; Menthol 20 mg; Thymol 1 mg; Phenylsalicylate 6 mg, and Guajazulene
0.4 mg.
Chromatography is performed on Silica gel 60F 254 pre-coated TLC plates, in
solvent system A-l (toluene-ethyl acetate, 93: 7). Chromatograms are sprayed with
vanillin-sulphuric acid reagent (VS No. 38, p. 304) or with phosphomolybdic acid
reagent (PMA No. 27, p. 303).
Interpretation of Chromatograms
Identifiable Approx. Standard VS PMA

terpenoids Rf (vis.) (vis.)
Azulene and terpene hydrocarbons 0.98 violet-blue blue

Anethol 0.9 cf. Tl violet-blue blue
Thujone (after PMA) 0.7 red-violet
Thymol 0.5 cf. T2 red-violet blue
Cinnamaldehyde
Eugenol } 0045 brown-orange blue
Cineol/piperitone 004 blue-orange blue
Menthol 0.2 cf. T3 blue blue
Fig. 2 Commercial purgative preparations

Mixed herbai preparations with anthra-glycosides as the main components
296
FRONT
Rf
0 .5
START
Fig.l
S Tl T2 T3 S Tl T2 T3
FRO
Rf
111
IV
START
Fig.2
2 3 4 5 6 7 8 9
297
Preparation 0/ extracts
Three finely powdered dragees are extracted by heating on the water bath for 5 min
with 6 ml methanol and 10 ~l of the c1ear filtrate are used for chromatography.
TLC chromatography, and detection
Adsorbent Silica gel 60F 254 pre-coated TLC plates (Merck, Darmstadt)
Solvent system Ethyl acetate-methanol-water (100: 13.5: 10) (10 cm)
Detection UV-365 nm
Known, commercial purgative formulations were applied to the start of tracks 1-9
of the thin layer chromatogram. They represent mixtures of two to five anthra-
glycoside-containing drug extracts. In some cases, extracts of other drugs are also
present (e.g. Gentianae radix, Bryoniae radix or Curcumae rhizoma).
The identifiable components are labelIed I-VI:
I-VI Approx. Rf Tracks
I Anthraquinone aglycones solvent front 1, 2, 3,4, 5, 6, 7, 8, 9

II A-monoglycosides }
0.8-0.85 2, 4, 5, 6, (7), 9
Frangulins A and B
III Deoxyaloin 0.6 1, 2, (3), (4), (5), (6), (7), 8
IV Aloin } 0.5 1,2,3,4,6, 7, 8, 9
Rhein
V Glucofrangulins }
0.35-0.4 2,3,4,5,8
Aloinosides
VI Cascarosides } 0.05-0.2 (1),2,3,4, (5), (6), (7), 8
A,B,C,D
Sennosides start (UV -254) 3,4,8
For further differentiation, the TLC plates are treated with KOH reagent and NP/
PEG re agent (see Anthraquinone Drugs, p. 93).
For the analysis of sennosides in Sennae folium, the solvent system and detection
method described on p. 110, Fig. 9 should be used.
298
Spray Reagents
For the thin layer chromatography of drug extracts
No. 1 Anisaldehyde-acetic acid reagent (AA)

0.5 ml Anisaldehyde mixed with 10 ml of 98% acetic acid.
The TLC plate is sprayed with 5-10 ml, then heated at 120 0 C for 7-10 min
in a drying cabinet.
Detection 0/ petasin/isopetasin. The plate may be sprayed afterwards with conc.
sulphuric acid and evaluated in vis. or UV-365 nm.
NO.2 Anisaldehyde-sulphuric acid reagent (AS)

0.5 ml Anisaldehyde is mixed with 10 ml glacial acetic acid, followed by 85 ml metha-
nol and 5 ml concetrated sulphuric acid, in that order.
The re agent has only limited stability, and is no longer useable when the colour
has turned to red-violet.
The TLC plate is sprayed with ab out 10 ml, heated at 100° C for 5-10 min,
then evaluated in vis. or UV-365 nm.
Detection 0/ essential oils, pungent principles, bitter principles, saponins, etc.
NO.3 Antimony(lII) chloride reagent (SbCI 3 )

20% solution of antimony(III) chloride in chloroform.
The TLC plate must be sprayed with 15-20 ml reagent, then heated for 5-6 min
at 100° C. Evaluation in vis. or UV-365 nm.
Detection 0/ cardiac glycosides, saponins, visnagin (Ammi visnagae /ructus), etc.
NO.4 Benzidine re agent (BZ)

0.5 g Benzidine is dissolved in 10 ml glacial acetic acid and the volume adjusted
to 100 ml with ethanol. Evaluation in vis.
Detection 0/ aucubin (Plantaginis folium)
NO.5 Berlin blue reagent (BB)

A freshly prepared solution of 10 g iron(III) chloride and 0.5 g potassium hexacyano-
ferrate in 100 ml water. The plate is sprayed with 5-8 ml and evaluated in vis.
Detection 0/ arbutin.
NO.6 Blood reagent (BL)

10 ml of 3.6% sodium citrate are added to 90 ml fresh bovine blood. 2 ml of this
mixture are mixed with 30 ml phosphate buffer pH 7.4*. The plate is sprayed in
a horizontal position.
* Phosphate buffer pH 7.4
25.00 ml potassium dihydrogen phosphate solution (27.281 g potassium dihydrogen phosphate
dissolved in CO 2 -free water (double distilled) and volume adjusted to 1,000 ml) mixed with
39.34 ml of 0.1 M sodium hydroxide, and volume made up to 100 ml with CO 2 -free, double
distilled water.
Detection 0/ saponins; white zones are formed against the reddish background of
the plate. Haemolysis may be immediate, or it may not occur until the plate has
been warmed.
Boroxyl reagent. See Natural products-polyethyleneglycol reagent No. 28.
299
NO.7 Chloramine-trichloroacetic acid reagent (CTA)
10 ml freshly prepared 3% aqueous chloramine T solution (syn. sodium sulphamide
chloride, or sodium tosy1chloramide) is mixed with 40 ml of 25% ethanolic trichloro-
acetic acid.
The plate is sprayed with 10-15 ml, heated at 100° C for 5-10 min and evaluated
in UV-365 nm.
Detection of cardiac glycosides.
NO.8 Dichloroquinonechloroimide reagent (DCC)

1% methanolic solution of 2,6-dichloroquinonechloroimide.
The plate is sprayed with 5-10 ml, then immediately exposed to ammonia vapour.
Detection of arbutin (DAB 8), capsaicin (DAB 8).
NO.9 Dinitrophenylhydrazine reagent (DNPH)

0.1 g of 2,4-dinitrophenylhydrazine is dissolved in 100 ml methanol, followed by
the addition of 1 ml of 36% hydrochloric acid.
After spraying with about 10 ml, the plate is evaluated immediately in vis.
Detection of ketones and aldehydes.
No. 10 DNPH-acetic acid-hydrochloric acid reagent

0.2 g of 2,4-dinitrophenylhydrazine in a solvent mixture consisting of 40 ml 98%
acetic acid, 40 ml 25% hydrochloric acid and 20 ml methanol.
The plate is sprayed with 10 ml and evaluated in vis. It is then heated at 100° C
for 5-10 min and evaluated again in vis. (Ph. Eur. III).
Detection of valepotriates (Valerianae radix). Chromogenic dien es react without
warming.
Remarks: Dienes can also be detected with HCL-AA reagent (No. 32).
No. 11 Dragendorffreagents (DRG)

Detection of alkaloids, heterocyclic nitrogen compounds, quarternary amines.
No. 11A Dragendorffreagent (Ph. Eur. I, p. 139)

0.85 g basic bismuth nitrate is dissolved in 40 ml water and 10 ml glacial acetic
acid, followed by addition of 8 g potassium iodide dissolved in 20 ml water.
No. 11 B Dragendorff reagent Rl (Ph. Eur. III, p. 105)

100 g tartaric acid are dissolved in 400 ml water. 8.5 g basic bismuth nitrate are
added and the solution is shaken for 2 h. 200 ml of 40% potassium iodide are
then added, and the solution is shaken vigorously. After standing 24 h, the solution
is filtered.
Diluted Dragendorff reagent (Ph. Eur. III, p. 105)
50 ml Dragendorff reagent Rl are added to a solution of 100 g tartaric acid in
500 ml water.
No. 11 C Dragendorff reagent with tartaric acid

Solution A: 17 g bismuth subnitrate and 200 g tartaric acid in 800 ml water.
Solution B: 160 g potassium iodide in 400 ml water.
A + B = stock solution
Spray reagent: 50 ml stock solution + 500 ml water + 100 g tartaric acid.
No. 11 D Dragendorff reagent with hydrochloric acid (modified)

Solution A: 0.3 g bismuth sub nitrate, 1 ml of 25% HCl, 5 ml water.
Solution B: 3 g potassium iodide in 5 ml water.
Spray reagent: 5 ml A + 5 ml B + 5 ml of 12.5% HCI + 100 ml water.
300
No. HE Dragendorffreagent according to Vaguifalvi
A mixture of 2.6 g bismuth carbonate, 7 g dried sodium iodide and 25 ml glacial
acetic acid is heated to boiling for a few minutes. After standing overnight, the
precipitated sodium acetate is removed by filtration. 20 ml of the c1ear red filtrate
are mixed with 80 ml ethyl acetate to give the stock solution.
Spray reagent: 2 ml stock solution, 5 ml glacial acetic and and 12 ml ethyl acetate.
Detection limit: approx. 1 Ilg.
After the plate has been dried (i.e. the smell of acetic acid is no longer detectable),
it can be sprayed again with 5% ethanolic sulphuric acid, ihereby giving a ten-fold
increase in sensitivity.
No. HF Dragendorffreagent,Jollowed by sodium nitrite or H Z S0 4

After treatment with any type of Dragendorff reagent, the plate may be additionally
sprayed with 5% aqueous sodium nitrite or with 5% ethanolic sulphuric acid, thereby
intensifying the coloured zones.
No. 12 Fast blue salt reagent (FBS)

0.5 g fast blue salt B is dissolved in 100 ml water. (Fast blue salt B=3,3'-dimethoxy-
biphenyl-4-4' -bisediazonium)-dichloride)
The plate is sprayed with 6-8 ml, dried and inspected in vis. Spraying may be
repeated, using 0.1 M NaOH, followed again by inspection in vis.
Detection of bitter principles /rom hops, and phenolic compounds in general.
No. 13 Fast red salt reagent (FRS)

0.5% aqueous solution of fast red salt B (=diazotized 5-nitro-2-aminoanisole).
The plate is sprayed with 10 ml, followed immediately by either 0.1 M NaOH
or exposure to ammonia vapour.
Detection 0/ amarogentin.
No. 14 Iron(III) chloride reagent (FeCI 3 )

10% aqueous solution.
Spray with 5-10 ml and evaluate in vis.
Detection %leuropein; carnosolic acid (Salviae folium, Rosmarini folium); hop bitter
principles.
No. 15 EP reagent according to Stahl (EP)

0.25 g of 4-dimethylaminobenzaldehyde is dissolved in a mixture of 45 ml of 98%
acetic acid, 5 ml of 85% o-phosphoric acid and 45 ml water, followed by 50 ml
conc. sulphuric acid with cooling.
The sprayed plate is evaluated immediately in vis., or after heating.
Detection 0/ azulenes (Matricariae flos) ; the natural blue of the azulenes is intensified
by EP reagent. After warming at 100° C for 3-5 min, proazulene gives a blue-green
(vis.).
No. 16 Acetic anhydride-sulphuric acid reagent

Liebermann-Burchard reagent (LB)
5 ml acetic anhydride and 5 ml conc. sulphuric acid are added carefully to 50 ml
absolute ethanol, while cooling in ice. The reagent must be freshly prepared.
The sprayed plate is warmed at 100° C for 5-10 min, then inspected in UV-
365 nm.
Detection oftriterpenes, steroids (saponins, bitter principles).
No. 17 Iodine-chloroform reagent (I/CHCI 3 )

0.5% Iodine in chloroform.
301
The sprayed plate is warmed at 60° C for ab out 5 min. It may be evaluated
immediately, or after standing for 10 min at room temperature.
Detection o/Ipecacuanha alkaloids.
No. 18 Iodine re agent
About 10 g solid iodine are spread on the bottom of a chromatography tank. Com-
pounds containing conjugated double bonds give yellow-brown (vis.) zones on expo-
sure to the atmosphere of iodine vapour.
No. 19 Iodoplatinate re agent (IP)
0.3 g hydrogen hexachloroplatinate (IV) hydrate is dissolved in 100 ml water and
mixed with 100 ml of 6% potassium iodide solution.
The plate is sprayed with 10 ml and evaluated in vis.
Detection o/nitrogen-containing compounds, e.g. alkaloids (blue-violet). For the detec-
tion of Cinchona alkaloids, the plate is first sprayed with 5% ethanolic H Z S0 4
(No. 34), and then with IP reagent.
No.20 Iodine-hydrochloric aicd reagent (I/HCl)
1 g potassium iodide and 1 g iodine are dissolved in 100 ml ethanol (soln. I). 25 ml
of 25% HCI are mixed with 25 ml of 96% ethanol (soln. Ir).
The plate is first sprayed with 5 ml soln. 1, followed by 5 ml soln. Ir. Dark brown
(vis.) zones are produced.
Detection o/the purine derivatives, caffeine, theophylline and theobromine.
No.21 Potassium hydroxide (KOH)
5% or 10% Ethanolic potassium hydroxide (Bornträger reaction)
The plate is sprayed with 10 ml and evaluated in vis. or in UV-365 nm, with
or without warming.
Detection 0/ anthraquinones (red), anthrones (yellow, UV-365 nm); coumarins (blue,
UV-365 nm).
No. 22 Potassium permanganate-sulphuric aicd re agent (PPM)

0.5 g Potassium permanganate is dissolved carefully in 15 ml conc. sulphuric acid,
while cooling in ice (Care! Explosive manganese heptoxide is formed).
Detection 0//enchone; the plate is sprayed first with phosphomolybdic acid reagent
(PMA No. 27) (10 min/ll0° C), followed by PPM reagent (5 min/110° C) blue (vis.).
No.23 Kedde reagent (Kedde)
5 ml freshly prepared 3% ethanolic 3,5-dinitrobenzoic acid are mixed with 5 ml
of2 M NaOH.
The plate is sprayed with 5-8 ml of the freshly prepared mixture, then evaluated
in vis.
Detection 0/ cardenolides.
No. 24 Komarowsky re agent (KOM)
1 ml of 50% ethanolic sulphuric acid and 10 ml of 2% methanolic 4-hydroxybenzal-
dehyde are mixed shortly before use.
The sprayed plate is heated at 100° C for 5-10 min under constant observation,
and evaluated in vis.
Detection 0/ essential oils, pungent principles, bitter principles, saponins, etc.
Liebermann-Burchard reagent (LB), see Acetic anhydride-sulphuric acid reagent
No. 16
No. 25 Marquis re agent
3 ml formaldehyde are diluted to 100 ml with conc. sulphuric acid. The plate is
evaluated in vis., immediate1y after spraying.
Detection o/morphine, codeine, thebaine.
302
No. 26 Millons reagent (ML)
3 ml mercury are dissolved in 27 ml fuming nitric acid, and the solution diluted
with an equal volume of water.
Detection 0/ arbutin and phenol glycosides in general.
No. 27 Phosphomolybdic acid reagent (PMA)

20% ethanolic solution of phosphomolybdic acid. The plate is sprayed with 10 ml,
then heated at 100 0 C for 5 min, under observation.
Detection 0/ components 0/ essential oils.
For the detection of rhaponticosides: 4 g phosphomolybdic acid are dissolved with
warming in 40 ml water. 60 ml conc. sulphuric acid are added carefully to the cooled
solution. Rhaponticoside and Deoxyrhaponticoside form strong, blue (vis.) zones.
No. 28 Natural produets-polyethylenglyeol reagent (NP/PEG)

The plate is sprayed with 1% methanolic diphenylboric acid-ß-ethylamino ester ( =
diphenylboryloxyethylamine) (NP), followed by 5% ethanolic polyethyleneglycol-
4000 (PEG) (10 ml and 8 ml, respectively).
Detection 0/flavonoids, aloin. Intense fluorescence is produced immediately or after
15 min in UV-365 nm. PEG increases the sensitivity (from 10 Ilg to 2.5 Ilg). The
fluorescence behaviour is structure-dependent.
No. 29 Ninhydrin reagent (NIH)

30 mg ninhydrin are dissolved in 10 ml n-butanol, followed by 0.3 ml of98% acetic-
acid. After spraying (8~10 ml), the plate is heated for 5~10 min under observation,
and evaluated in vis.
Detection 0/ amino acids, biogenie amines.
No. 30 Nitrosodimethylaniline reagent (NDA)

10 mg Nitrosodimethylaniline are dissolved in 10 ml pyridine, and used immediately
to spray the TLC plate.
Detection 0/ anthrone derivatives (grey-blue zones in vis., e.g. from freshly harvested
Frangulae cortex).
No.31 Phenylenediamine reagent (PD)

0.5% ethanolic solution.
Evaluation in vis. or in UV -365 nm.
Detection o/constituents of Lichen islandicus (e.g. fumarprotocetraric acid).
No.32 Cone. hydroehlorie acid-glacial aeetie acid reagent (HCl/AA)

8 parts conc. hydrochloric acid and 2 parts glacial acetic acid.
After spraying, the plate is heated at 110 0 C for 10 min, and evaluated in vis. or
in UV-365 nm.
Detection o/valepotriates with diene structure (halazuchrome reaction).
No. 33 Nitrie acid (HN0 3 conc.)

Detection 0/ ajmaline and brucine, red in vis.
Detection of sennosides: after spraying with HN0 3 conc. and heating for 15 min
at 120 0 C, the plate is sprayed with KOH reagent (No. 21).
Red-brown (vis.) or yellow-brown fluorescent (UV-365 nm) zones are formed.
No. 34 Sulphurie aeid (HZS04)

A) 5% or 10% ethanolic H Z S0 4
B) 50% ethanolic H Z S0 4
C) concentrated H Z S0 4
303
The plate is heated at 100° C for 3~5 min, and evaluated in vis. or in UV-365 nm.
With conc. H 2 S0 4 coloured (vis.) zones often appear immediately.
Detection of e.g. cardiac glycosides.
No.35 Trichloroacetic acid-potassium hexacyanoferrate-iron(m) chloride reagent (TPF)

Solution 1: 25% trichloroacetic acid in chloroform.
Solution 2: 1% aqueous potassium hexacyanoferrate mixed with an equal volume
of 5% aqueous iron(lII) chloride. The plate is sprayed vigorously with solution 1
and heated at 110° C for 10 min. It is then sprayed with solution 2 and evaluated
in vis.
Detection of sinalhin and sinigrin.
No. 36 Vanillin-phosphoric acid reagent (VPA)

A:l g vanillin dissolved in 100 ml of 50% phosphoric acid.
B: 2 parts 24% phosphoric acid and 8 parts 2% ethanolic vanillic acid.
After spraying with either A or B, the plate is heated for 10 min at 100° C, and
evaluated in vis. or in UV -365 nm.
Detection of e.g. terpenoids, lignanes and cucurbitacins.
No. 37 Vanillin-hydrochloric acid reagent (VHCl)

The plate is sprayed with 5 ml of 1% ethanolic vanillin, followed by 3 ml conc.
HCI, then evaluated in vis. Colours are intensified by heating for 5 min at 100° C.
Detection of myrrh constituents.
No. 38 Vanillin-sulphuric acid reagent (VS)

5% Ethanolic sulphuric acid (solution I)
1% Ethanolic vanillin (solution II)
The plate is sprayed vigorously with 10 ml solution I, followed immediately by
5~10 ml solution II. After heating at 110° C for 5~10 min under observation, the
plate is evaluated in vis.
Detection of e.g. components of essential oUs (terpenoids, phenylpropane derivatives,
phenols, etc.).
No.39 Van URK reagent

0.2 g of 4-dimethylaminobenzaldehyde is dissolved in 100 ml of 25% HCI with the
addition of 1 drop of 10% iron(III) chloride solution.
Detection of Secale alkaloids.
304
Abbreviations and Definitions
1. Ahhreviations
TLC: thin layer chromatography
Silica gel 60: specific surface area 500 cm 2 jg; pore volume 0.75 cm 3 jg; pore diameter
60 A.
UV-254 nm: shorter wavelength ultraviolet light, used to detect substances that
quench fluorescence (Silica gel 60F 254 pre-coated TLC plates from Merck, Darm-
stadt). Zones appear dark blue against a yellow-green fluorescent background.
UV-365 nm: for the detection of substances that fluoresce in long wave ultraviolet
light. UV-Iamps: commercially available lamps or tubes from Philips, Osram, Syl-
vania and others.
vis.: visible light or daylight.
2. General concepts
Without chamber saturation: the chromatography solvent is poured into the chroma-
tography tank, and swirled around vigorously for a few seconds. The TLC plate
is then placed in position, and chromatography allowed to proceed.
With chamber saturation: the solvent is allowed to remain in the c10sed tank for
Ijr1 h before chromatography. The inside of the tank should be lined with filter
paper.
Volume of chromatography solvent: about 100 ml are normally used. Chromatogra-
phy tank dimensions: 20 x 9 x 20 cm.
3. Extraction procedures
Powdered drugs are used for extraction. According to DAB 7, "medium fine
powder" corresponds to sieve No. 5 (0.315 mm mesh). According to Ph. Eur. I,
medium fine corresponds to sieve No. 300 (mesh size 300).
Sampie weights quoted for drug extraction refer to the dried drug.
4. Sampie volume
The volumes quoted are recommended averages. Depending on the quality of the
drug, larger and smaller volumes should also be used. Exact volumes can be applied
with the aid of commercially available, standardized capillaries. If melting point
capillaries are used, it can be assumed that 1 cm is roughly equivalent to 4-5 j..ll.
As a rule, the sampie should be applied to the start as a line about 1 cm wide.
Small sampie volumes (1-3 j..ll), however, are applied as a spot.
305
References
Books on TLC Techniques and on the Constituents of Plant Drugs

Randerath, K.: Dünnschicht-Chromatographie, 2. Aufl., Verlag Chemie, 1965
Stahl, E.: Thin-Layer Chromatography 2nd. edtn., Springer Verlag, Berlin-Heidelberg-New
York,1969
Stahl, E., Schild, W.: Pharmazeutische Biologie, 4. Drogenanalyse 1I, Inhaltsstoffe und Isolie-
rungen. Fischer Verlag Stuttgart-New York, 1981
Wicht!, M.: Die Pharmakognostisch-Chemische Analyse. Untersuchung und Wertbestimmung
von Drogen und galenischen Präparaten. Akademische Verlagsgesellschaft, Frankfurt/M.,
1971
Wagner, H.: Pharmazeutische Biologie, 2. Drogen und ihre Inhaltsstoffe, 2. Aufl. Fischer Ver-
lag Stuttgart-New York, 1982
Wagner, H., Bladt, S.: Pharmazeutische Biologie, Praktikumshandbuch, 2. Ausg., Institut für
Pharmazeutische Biologie der Universität München, 1979
Pharmacopoeias
Deutsches Arzneibuch, 8. Ausgabe 1978. Deutscher Apotheker Verlag, Stuttgart.
Govi Verlag, Frankfurt/Main. (German Pharmacopoeia, 8th. edition 1978) DAB 8
Deutsches Arzneibuch, 7. Ausgabe 1968. 1. Nachtrag 1974, 2. Nachtrag 1975.
Deutscher Apotheker Verlag, Stuttgart. Govi Verlag, Frankfurt/Main. (German
Pharmacopoeia, 7th. edition 1968, 1st. supplement 1974, 2nd. supplement 1975) DAB 7
Ergänzungsbuch zum Deutschen Arzneibuch. Sechste Ausgabe (Arzneimittel, die
im Deutschen Arzneibuch, 6. Ausgabe, nicht enthalten sind). (Supplement to
the 6th. edition of the German Pharmacopoeia, containing material that was
not inc1uded in the 6th. edition) Erg.-B.6
Europäisches Arzneibuch, Band I-IlI, 1974, 1975, 1978,. Deutscher Apotheker
Verlag, Stuttgart. Govi-Verlag, Frankfurt/Main. (Ph. Eur. I: pp. 79-82 Thin
Layer Chromatography) (European Pharmacopoeia, Volumes I-IH, 1974, 1975,
1978 Ph. Eur.
Arzneibuch der Deutschen Demokratischen Republik. 2. Ausgabe 1979, Akade-
mie Verlag, Berlin. (Physical analysis: 12.01 Thin layer chromatography) (Phar-
macopoeia of the German Democratic Republic) 2. AB-DDR
Deutscher Arzneimittel-Codex, 1979 (Ergänzung zum Arzneibuch). Govi-Verlag,
Frankfurt/Main. Deutscher Apotheker Verlag, Stuttgart. (Appendix E: Azeo-
tropic solvent mixtures for TLC and paper chromatography). (German pharma-
ceutical codex, 1979 (supplement to the Pharmacopoeia)) DAC
Österreichisches Arzneibuch, Ausgabe 1981. Österreichische Staatsdruckerei,
Wien (Austrian Pharmacopoeia) ÖAB
Pharmacopoea Helvetica, Editio sexta, 1971 und Supplement. Eidgenössische
Drucksachen- und Materialzentral, Bem (Swiss Pharmacopoeia, 6th. edition,
1971 and supplement) Helv. VI
Kommentar zum Helv. VI pp. 98-105. Erläuterungen zur Dünnschichtchromato-
graphischen Prüfung. Selbstverlag des Schweizerischen Apotheker-Vereins, Bem
1975 (Notes on Helv. VI, with guide to thin layer chromatography)
The Uni ted States Pharmacopeia, 1980. The National Formulary. The United
States Pharmacopeial Convention, Rockville, MD ("Chromatography" pp. 938- USPXX
946 NFXV
307
Subject Index
Page numbers referring to the description of chromatograms are printed in bold type.
Abies species 16, 46 - Macis 10, 28

Abrotani herba 149, 156 - Matricariae 11, 32
Absinthii herba 128, 134 - Melissae 14,40
absinthin 128,131,134 - Menthae 12,36
acacetin rutinoside 180 - Myristicae 10,28
acacia flowers 165, 180 - Petroselini 10,28
Acaciae flos 165, 180 - Pi ni 15, 16,46
Acanthopanax senticosus 227 - Rosmarini 11, 30
acevaltrate 264, 265, 266 - Salviae 13, 38
acid amides 55, 62 - Sassafras 10, 26
aconite root 57 - Serpylli 14, 40
Aconiti herba/tuber 57, 84 - Terebinthinae 16,46
aconitine 57, 64, 84 - Thymi 13, 40
Aconitum napellus 57 ajmaline 55, 62, 68,70
Acorus calamus 9 ajowan fruits 14,40
Adonidis herba 198,214,218 Ajowani fructus 14,40
adonis 198 albopetasin 192
Adonis vernalis 198 alder buckthorn bark 98, 106
adonitoxin 198, 202, 218 al der buckthorn fruits 98, 106
adonivernith 218 Alexandrian sennapods 99
adynerin 198,212 alkaloid drugs 51
aesculetin 150 allicin 255
aesculin 227,230,234,242 alliin 255
Aesculus hippocastanum 227 Allium sativum 255, 256
Aetherolea 5 allyl thiourea derivatives 256
Aetheroleum allylisothiocyanate 255
- Anisi 9,26 allyltetramethoxybenzene 10, 20, 28
- Anisi stellati 9, 26 Aloe species 97, 102
- Anthemidis 11,32 aloe-emodin 97, 102
- Aurantii 15,44 aloes 97, 102
- Basilici 10, 26 aloesine A, B 97, 100, 102
- Calami 9, 24 aloin A, B 97, 100, 102
- Cardamomi 11, 30 aloinoside A, B 97, 100, 102
- Carvi 10,30 Althaea rosea 270
- Caryophylli 10,28 amarogentin 127, 130, 136
- Chamomillae 11, 32 amaropanin 136
- Cinnamomi 9,24 amaroswerin 136
- Ci tri 15,44 amentoflavone 119,122,170
- Citronellae 14,40 amide pungent principles 248
- Cinae 12, 34 amino acids 255, 288
- Curcumae 14, 42 Ammi (Amme os) fructus 148, 160
- Eucalypti 13, 38 Ammi majus, A. visnaga 148
- Foeniculi 9, 26 anabsinthin 128, 134
- Juniperi 11,30 anethole 9, 20, 26
- Lavandulae 12,34 Angelica archangelica 147
309
angelica root 147, 154 bearberry leaves 119
Angelica sylvestris 147 Belladonnae folium 59,88,90
anise 9 belladonna leaves 59
Angelicae radix 147, 154 belladonna root 59
anisaldehyde 9, 20, 26 Belladonnae radix 59, 90
Anisi fructus 9,26 Belladonnae semen 90
Anisi stellati fructus 9, 26 benzo-o:-pyrones 145
Anserinae herba 168, 186 benzoic acid 16, 48
Anthemidis flos 11, 32, 165, 182, 184 benzoins 16, 48
Anthemis nobilis 11 benzyl benzoate 48
anthocyanins 269,272, 274 Berberidis radix 57, 80
anthracene drugs 93 berberine 57, 63, 80
anthracene glycoside drugs 97 Berberis vulgaris 57
anthraquinones 93 bergamot oil 15,44
anthrone, anthranol 93 bergapten 154
apigenin 170 bergenia 119
apiol 10, 20, 28 Bergenia crassifolia 119
arbutin 119,120 Betula species 167
arbutin drugs 117, 120 Betulae folium 167, 182, 229
Arctostaphylos uva-ursi 119 birch leaves 167
Armoracia rusticana 253, 255 bisabolol 11, 21, 32
Arnica chamissonis 165 bisabolol oxide A, B 11,21,32
arnica flowers 165 bitter principle drugs 125
Arnica montana 165 blackthorn flowers 167
Arnicae flos 165,174,176 blessed thistle 128
Artemisia abrotanum 149 boldine 57,61,84
Artemisia absinthium 128 Boldo folium 57,84
Artemisia cina 12 bornyl acetate 15, 19,46
artichoke 129 bilberry leaves 119
Asa foetida 148, 158 Brassica nigra 255
asafetida 148 broom 58, 168
asaresitannol 148 brucine 56, 62, 72
asarone 9, 20, 24 bryomarid 138
ash bark 149 Bryoniae radix 129, 138
Asperulae herba 149, 156 bryony root 129
aspidosperma bark 55 buckbean 128
Atropa belladonna 59 buckthorn berries 98
atropine 59, 63, 88, 90 bufadienolides 195
Aucuba japonica 128 Bulbus scillae 199,222
aucubine 128, 130, 136 burnet root 148
Aurantii flos 15, 44 butanone phloroglucides 279, 282
Aurantii pericarpium 15, 44, 129, 132,
169, 188 cacao seeds 58
Avena sativa 229 Cacao semen 58,86
avenacoside 242 Cacti flos 165, 176
Avenae herba 229, 233, 242 cactin 165
azulene 32 caffeic acid 152, 171
caffeine 58, 59, 65, 86
baldrinal 265 caffeine drugs 86
balsams 16, 48 Calami rhizoma 9,24
Balsamum peruvianum 17,48 Calendula officinalis 166
Balsamum tolutanum 17, 48 Calendulae flos 166, 174, 176
barberry bark 57 Calluna vulgaris 119
basil 10,26 Camellia sinensis 59
Basilici herba 10, 26 cannabidiol (CBD) 260
310
cannabidiol acid (CBDA) 260 Chelidonium majus 57
cannabinol (CBN) 260 Chinae cortex 56, 74
Cannabis herba 260 chlorogenic acid 171, 172
Cannabis sativa 259 Chrysanthemum species 279
cape aloes 97, 102 chrysophanol 98, 100, 108
capsaicin 249,250 Cinae flos 12,34
Capsici fructus 248, 250 Cinchona alkaloids 56
Capsicum annum var. longum 248 Cinchona bark 56
Capsicum frutescens 248 Cinchona ledgeriana 56, 74
capsicums 248 Cinchona succirubra 56, 74
caraway 10 Chinchonae cortex 56,74
Cardamomi fructus 11, 30 cinchonidine 56, 61, 74
cardamoms 11 cinchonine 56, 61, 74
cardenolides 195 cineole 19,22,38
cardiac glycoside drugs 195 cinerin I, Ir 279, 281, 282
Cardui benedicti herba 128, 134 cinnamaldehyde 9, 20, 24
Cardui mariae fructus 169, 190 cinnamein 17, 20, 48
carnosol 13, 142 Cinnamomi cortex 9,24
carnosolic acid 142 Cinnamomum species 9
Carum carvi 10 cinnamon bark 9
carvacrol 13, 19,40 cinnamoyl benzoate 16
Carvi fructus 10, 30 cinnamoyl pigments 42
carvone 10, 19,30 circular TLC 95, 112, 114
caryophyllene 28 citral 14, 19,40
Caryophylli flos 10,28 Citri pericarpium 15,44, 169, 188
Cascarae cortex 97, 104 Citronellae aetheroleum 40
cascarosides 97, 100, 104 citronellal 14,19,40
Cassia angustifolia 99 Citrus species 15
cassia cinnamon 24 Claviceps purpurea 55
Cassia senna 99 clove oil 10, 28
catechin 122 cloves 10
catharticin 98, 106 Cnici herba 128, 134
cat's foot flowers 167 cnicin 128, 131, 134
cayenne pepper 248 Cnicus benedictus 128
Centaurea cyanus 270 codeine 56,61,78
Centaurii herba 127, 132 Coffea arabica 58
Centaurium minus 127 Coffeae semen 58, 86
centaury 127 coffee beans 58
cephaeline 56, 61,76 cola seeds 59
Cephaelis acuminata 56, 76 Colae semen 58
Cephaelis ipecacuanha 56,76 Colchici semen 57, 82
Cereus grandiflorus 165 colchicine 57, 64, 82
Cetraria islandica 279 Colchicum autumnale 57
Cetraria tenuifolia 279 Colchicum seeds 57
cetraric acid 281, 284 Colombo radix 57,80
Ceylon cinnamon 9,24 Colombo root 57
Chamaemelum nobile 11 coltsfoot flowers, leaves 166
chamazulene 11, 21, 32 columbamine 57,80
chamomile flowers 11, 166 Commiphora molmol 16
chamomile flowers (Roman) 11, 166 common osier 278
Chamomilla recutita 11 condurangins 129, 131, 132
Chamomillae flos 11,32, 166, 184 Condurango bark 129
chelerythrin 57, 63, 82 Condurango cortex 129, 132
Chelidonii herba 57, 82 coniferyl benzoate 16
chelidonine 57, 63,82 coniferyl cinnamate 16
311
Convallaria majalis 198 Cynarae herba 129, 142
Convallariae herba 198,214, 218 cynarin 129,142,171
convallatoxin 198,202,218
coriander 11 Daphne mezereum 149
Coriandri fructus 11, 30 daphnoretin 150, 156
Coriandri semen 30 Datura stramomium 60
Coriandrum sativum 11 delphinidin 270, 271
cornflowers 270 demethoxycurcumin 42
cornmint oil 12, 36 deoxyaloin 97, 104
Cortex dianthrones 110
- Cascarae 97, 104 dicaffeoyl quinic acids 171
- Chinae 56, 74 didrovaltrate 265, 266
- Cinchonae 56, 74 Digitalis folium 198,208,210
- Cinnamomi 9,24 Digitalis glycosides 197, 200
- Condurango 129, 132 Digitalis lanata 198
- Frangulae 98, 104, 106 Digitalis purpurea 198
- Fraxini 149, 158 digitoxin 201
- Mezerei 149, 158 Dryopteris filix-mas 279
- Oreoherzogiae 98, 106
- Quebracho 55, 68 eIder flowers 167
- Quillajae 228, 234, 244 e1emicin 20
- Rhamni cathartici 98 Eleutherococci radix 227, 238
- Rhamni purshiani 97, 104 e1eutheroside 227,231,238
- Salicis 278, 282 Elletaria cardamomum 11
- Viburni 119, 122 emetine 56,61,76
- Yohimbe 55, 68 ene-ine-dicycloether 11, 21, 32
coumarin 24, 145, 149 Ephedra 57
coumarin drugs 145 Ephedra species 57
coumarins 150,151,152 Ephedrae herba 57,86
cowberry leaves 119 ephedrine 64
Crataegi flos, C. folium, C. fructus 166, epoxydihydrocaryophyllene 34
178 Equiseti herba 168, 186
Crataegus species 166 Equisetum arven se 168
crocetin 271 ergobasine 55
Croci stigma 270, 274 ergocristine 62, 72
crocin 270,271,274 ergometrine 55,72
crocus 270 ergot 55
Crocus sativus 270 ergotamine 55, 62,72
Cubebae fructus 248, 250 ergotoxin 55
cubebin 249, 250 Ericae herba 119
cubebs 248, 250 eriocitrin 169, 188
Cucurbita pepo 129 Eriodictyonis herba 169, 188
cucurbitacins 129,131,138 Erucae semen 255,256
Cucurbitae semen 129, 138 essential oil drugs 5
cucurbitine 129 Eucalypti folium 13, 38
Curacao aloes 97, 102 eucalyptol 13
Curcuma species 14, 42 eucalyptus leaves 13,38
Curcumae rhizoma 14,42 Eucalyptus species 13
curcumins 14,42 eugenol 29,22,28
Cyani flos 270, 272
cyanidine glycosides 270 Farfarae flos 166, 176
cymarin 206 Farfarae folium 166, 192
Cymbopogon species 14 fenchone 9, 19,26
Cynanchum vincetoxicum 228, fennel 9,26
236 Ferula assa-foetida 148
312
filicin 279 - Nicotianae 58, 86
Filicis rhizoma 279,282 - Oleae 128, 136
Filipendula ulmaria 167 - Oleandri 198,212
filixic acid 281,282 - Orthosiphonis 169, 188
flavone, flavonol, flavanone 170, 171 - Plantaginis 128, 136
flavonoid drugs 163 - Pyri 119
Flos - Rosmarini 11, 30, 128, 142
- Acaciae 165, 180 - Salviae 13,38, 128, 142
- Anthemidis 11,32, 165, 182, 184 - Sennae 99, 110, 112
- Arnicae 165,174,176 - Stramonii 60, 88, 90
- Aurantii 15,44 - Theae 59,86
- Cacti 165,176 - Trifolii fibrini 128
- Calendulae 166,174,176 - Uvae ursi 119, 120
- Caryophylli 10, 28 - Vitis idaeae 119,120
- Chamomillae 11, 32, 166, 184 foxglove leaves 198
- Cinae 12, 34 Frangula-emodin 100, 106
- Crataegi 166, 178 Frangulae cortex 98, 104, 106
- Cyani 270, 272 Frangulae fructus 106
- Farfarae 166, 176 frangulin A, B 98, 100, 106
- Helichrysi 167, 174, 178 fraxidin 149, 158
- Hibisci 270, 272, 274 Fraxini cortex 149, 158
- Lavandulae 12,34 fraxinol 149, 150
- Malvae 270, 272 Fraxinus species 149
- Matricariae 11, 32, 166, 184 Fructus
- Primulae 166, 176 - Ajowani 14,40
- Pruni spinosae 167, 180 - Ammeos 148,160
- Pyrethri 279, 282 - Ammi 148, 160
- Robiniae 180 - Anisi 9,26
- Sambuci 167, 174, 178 - Anisi stellati 9, 26
- Spiraeae 167, 180 - Capsici 248,250
- Stoechados 167,174, 178 - Cardamomi 11, 30
- Tiliae 167, 180 - Cardui mariae 169, 190
- Verbasci 167, 178, 229 - Carvi 10,30
Foeniculi fructus 9,26 - Coriandri 11, 30
Foeniculum vulgare 9 - Crataegi 166, 178
foliamenthin 128, 132 - Cubebae 248, 250
Folium - F oeniculi 9, 26
- Belladonnae 59,88 - Frangulae 106
- Betulae 167,182,229 - Juniperi 11,30
- Boldo 57, 84 - Oleae 136
- Crataegi 166, 178 - Petroselini 10, 28
- Digitalis 198,208,210 - Piperis 248, 250
- Eucalypti 13, 38 - Rhamni cathartici 98, 106
- Farfarae 166, 192 - Sennae 99, 110, 112
- Hamamelidis 278, 282 fumarprotocetraric acid 179, 281, 284
- Hederae 227, 242 furanochromones 148,151
- Hyoscyami 59, 88, 90 furanocoumarins 145,151
- Jaborandi 57,84
- Juglandis 168, 182 Galangae rhizoma 249
- Mate 59,86 Galium odoratum 149
- Melissae 14,40 gallic acid 280
- Menthae 12,36 garlic 255
- Menyanthidis 128, 132 gentian root 127
- Myrtilli 119, 120 Gentiana species 127, 136
- Nerii oleandri 198, 212 Gentianae radix 127, 132, 136
313
gentiopicrin (gentiopicroside) 127, - Centaurii 127, 132
130,132 - Chelidonii 57, 82
geraniol 19,22 - Cnici 128, 134
ginger 249 - Convallariae 198,214,218
gingerols 249 - Cynarae 129, 142
Ginseng radix 227, 234, 238 - Ephedrae 57,86
ginseng root 227 - Equiseti 168, 186
ginsenosides 227, 238 - Ericae 119
glucofrangulin A, B 98, 100, 106 - Eriodictyonis 169, 188
glutamic acid 288 - Herniariae 149, 156, 176
glycine 288 - Hyperici 99, 110
glycyrrhetic acid 228, 240 - Leonuri 168, 186
Glycyrrhiza glabra 228 - Lobeliae 58,84
glycyrrhizic acid 228 - Marrubii 128, 134
glycyrrhizin 228, 240 - Meliloti 149, 156
golden rod 168 - Pulegii 12, 36
grapple plant 127 - Rutae 149, 156, 186
greater celandine 57 - Sarothamni 58,86, 168, 186
Gypsophila species 228 - Scrophulariae 127, 132
- Serpylli 14, 40
Hamamelidis folium 278, 282 - Solidaginis 168
hamamelitannin 280, 282 - Spartii 58, 86, 168, 186
Harpagophyti radix 127, 132 - Thymi 13,40
Harpagophytum procumbens 127 - Veronicae 168, 186
harpagoside 127,130,132 - Violae 168, 184, 186
hashish 259, 260 - Virgaureae 168, 184
hawthorn flowers, leaves, fruits 166, - Visci 279, 286
178 herbaI drug mixtures 296
heather 119 Herniaria glabra 149
Hedera helix 227 Herniariae herba 149, 156, 176
hederacoside 227,232,242 herniarin 149, 150, 152, 156
Hederae folium 227,242 hesperidin 167, 170
hederin, 0:-, ß- 227,242 Hibisci flos 270, 272, 274
Helichrysi flos s. Stoechados flos 167, hibiscus flowers 270
174 Hippocastani semen 227,234, 242,
Helichrysum arenarium 167 244
hellebore rhizome, white 58 hogweed root 148
hellebore root, black 199 hollyhock 270
Hellebori radix 199,220 hop bitter principles 129
Helleborus species 199 hops 129
hellebrin 199, 203, 220 horse chestnut seeds 227
henbane 59 horse radish 255
Heraclei radix 148, 152 horsetail 168
Heracleum spondylium 148 Humuli lupuli strobulus 129, 140
Herba humulone 129,131,140
- Abrotani 149, 156 Humulus lupulus 129
- Absinthii 128, 134 hydrastine 57,80
- Aconiti 57, 84 Hydrastis rhizoma 57, 80
- Adonidis 198, 214, 218 hydroquinone 119, 120
- Anserinae 168, 186 Hyoscyami folium 59, 88, 90
- Asperulae 149, 156 hyoscyamine 59, 63, 88, 90
- Avenae 229,233,242 Hyoscyamus muticus 59, 90
- Basilici 10, 26 Hyoscyamus niger 59
- Cannabis 260 Hyperici herba 99, 110
- Cardui benedicti 128, 134 hypericins 99,101,110
314
Hypericum perforaturn 99 Lignum Sassafras 10,26
hyperoside 170, 172 lime flowers 167
linalool 19,22
lceland moss 279 linalyl acetate 19,22
Ignatii semen 56, 72 ligustilide 147, 154
Ignatius beans 56 lily of the valley 198
Ilex paraguariensis 59 Liquiritiae radix 228, 234, 240
Illicium species 9 liquiritin 228, 240
irnidazole alkaloids 58 Lobelia inflata 58
Imperatoriae radix 148, 154 Lobeliae herba 58,84
imperatorin 151,154 lobeline 58, 64, 84
indole alkaloids 55 lovage 147, 154
insect flowers 279 lupulone 129, 131, 140
ipecacuanha root 56 luteolin 170
Ipecacuanhae radix 56,76 luteolin glycosides 170, 172
isobarbaloin 97 lysergic acid 55
isopetasin 171
isoquercitrin 170, 172 ma-huang 57
isoquinoline alkaloids 56 mace 10,28
isorhamnetin 170 male fern 279
isorhamnetin glycosides 176 mallow flowers 270
IVDH valtrate 265, 266 Malva species 270
ivy leaves 227 Malvae flos 270, 272
marigold florets 166
Jaborandi folium 58, 84 marihuana 259
jaborandi leaves 58 Marquis reaction 78
Jateorhiza palmata 57 Marrubii herba 128, 134
jateorhizin 57, 63, 80 marrubiin 128, 131, 134
Java citronella oil (lernon grass oil) 14, Marrubium vulgare 128
40 Marsdenia condurango 129
Juglandis folium 168, 182 master-wort 147
Juglans regia 168 Mate folium 59, 86
juniper berries 11 mate leaves 59
Juniperi fructus 11, 30 Matricaria chamomilla 11
Juniperus communis 11 Matricariae flos 11, 32, 166, 184
matricin 11
kaempferol 170 may-apple root 279
kaempferol glycosides 172 meadow-sweet flowers 167
khellin 148,151,160 meconic acid 61
Mei radix 154
lanatosides 198,208,210 Meliloti herba 149, 156
lavander flowers 12 Melilotus officinalis 149
lavandin oil 12, 34 melissa leaves 14
Lavandula species 12 Melissa officinalis 14
Lavandulae flos 12,34 melissa oil substitutes 40
lemon balm leaves 14 Melissae folium 14, 40
lemon grass oil 14, 40 melon pumpkin seeds 129
lemon peel 15 Mentha species 12
Leonuri herba 168, 186 Menthae folium 12,36
Leonurus species 168 menthiafolin 128, 132
Levistici radix 148, 154 menthofuran 12, 21,36
Levisticum officinale 148 menthol 12,21,36
Lichen islandicus 279, 284 menthone 12, 21, 36
licorice root 228 menthyl acetate 12, 21, 36
Lignum Quassiae 129, 134 Menyanthes trifoliata 128
315
Menyanthidis folium 128, 132 opium 56,78
methyl chavicol 9, 20, 26 opium alkaloids 56,61,78
Meum species 148, 154 orange flowers 15
Mezerei cortex 149, 158 Oreoherzogiae cortex 98, 106
mezereon bark 149 orthosiphon leaves 169
milk-thistle fruits 169 Orthosiphon spicatus 169
milkwort root 229 Orthosiphonis folium 169, 188
mistletoe leaves 279 osier 278
morphine 56,61,78 oxypeucedanin, -hydrate 151,152
mother cloves 10
motherwort 168 Paeoniae flos 270, 274
mountain pi ne oil 15 paeonidin 271
mullein flowers 167 palmatine 57, 63, 80
mustard 255 Panax ginseng 227
mustard oil drugs 253 Papaver somniferum 56
mustard oil glycosides 253 papaverine 56, 61, 78
Myristica fragrans 10 parsley fruits 10, 28
Myristicae arillus 10, 28 parsley species 10, 28
Myristicae semen 10,28 Pastinacae radix 148
myristicin 10, 20, 28 Pausinystalia yohimba 55
Myroxylon species 17 pear leaves 119
myrrh 16,46 pelargonidin 271
Myrrha 16, 46 peony 270
Myrtilli folium 119, 120 pepper 248
peppermint leaves 12
narcissin 165,176 peptide alkaloids 55, 62
narcotic drugs 259 Pericarpium Aurantii 15, 44, 129, 132,
narcotine 56, 61, 78 188
naringin 131, 132, 188 Pericarpium Citri 15,44,169,188
nerigoside 212 Peru balsam 17,48
Nerii folium 198,212 Peruvianum balsamum 17,48
Nerium oleander 198 peruvoside 202, 204
neroli oil 15, 44 petasin 171,192
nerolidol 48 Petasites species 166, 192
Nicotiana tabacum 58 petit grain oil 15, 34, 44
Nicotianae folium 58, 86 Petroselini fructus 10, 28
nicotine 58, 64, 86 Petrose1ini radix 10
noscapine 56,61,78 Peucedanum ostruthium 148
nutmeg 10 Peumus boldus 57
Nux vomica seeds 56 phenol carboxylic acids 171
phenylpropane derivatives 10
oats 229 phthalides 147
Ocimum basilicum 10 physcione 98, 100, 108
odoroside 198,212 physostigmine 66
Olea europaea 128 Picea species 16, 46
Oleae folium 128, 136 picein 278, 282
Oleae fructus 136 Picrasma exce1sa 129
oleander lea ves 198 picrosalvin 13
Oleandri folium 198, 212 pigment drugs 269
oleandrin 198, 202, 212 pilocarpine 58, 65, 84
oleanolic acid 230 Pilocarpus species 58
oleaside 198,212 Pimpinella anisum 9
oleoresins 16, 48 Pimpinella species 148
oleuropein 128, 130, 136 Pimpinellae radix 148, 152
olives 128 pimpinellin 151, 152
316
pine oils 46 - Berberidis 57,80
Pinus species 15, 16 - Bryoniae 129, 138
Piper cubeba 248 - Colombo 57, 80
Piper nigrum 248 - Eleutherococci 227,238
piperine 249,250 - Gentianae 127, 132, 136
Piperis fructus 248,250 - Ginseng 227, 234, 238
piperitone 19,22 - Harpagophyti 127, 132
Plantaginis folium 128, 136 - Hellebori 199,220
Plantago lanceolata 128 - Herac1ei 148, 152
Podophylli resina/rhizoma 279, 284 - Iperatoriae 148, 154
podophyllotoxin 281,284 - Ipecacuanhae 56, 76
Podophyllum peltatum 279 - Levistici 148, 154
Polygala senega 229 - Liquiritiae 228, 234, 240
Polygalae radix 229 - Mei 154
polyines 11,21,32 - Pastinacae 148
Polypodii filicis maris radix 279 - Petroselini 10
Potentilla anserina 168 - Pimpinellae 148, 152
primrose root 228 - Polygalae 229
primula acid 228, 232, 236 - Polypodii filicis maris 279
Primula species 166, 228 - Primulae 228,234,236
Primulae flos 166, 176 - Rauwolfiae 55, 70
Primulae radix 228,234, 236 - Rhei 98, 108, 114
proscillaridin 199, 203, 222 - Saponariae 228, 234, 236
protropine 82 - Sarsaparillae 229,234,244
protoveratrine A, B 64 - Scopoliae 59,90, 149, 154
Pruni spinosae flos 167, 180 - Senegae 229,234,244
Prunus spinosa 167 - Uzarae 199,220
psychotrine 61, 76 - Valerianae 264, 266
Pulegii herba 12, 36 - Xysmalobii 199,220
pulegone 12, 36 rau ba si ne 62, 70
pungent principle drugs 247 raupine 52, 53
purine drugs 58,86 Rauwolfia alkaloids 68
Purpurea glycosides 198, 200, 208 Rauwolfiae radix 55, 70
pyranocoumarins 151 Rauwolfia root 55, 70
Pyrethri flos 279, 282 Rauwolfia species 55
pyrethrins 281 rauwolscine 70
Pyri folium 119 rescinnamine 55, 62, 68, 70
reserpine 55, 62, 68, 70
Quassia amara 129 Rhamni cathartici cortex 98
quassia wood 129 Rhamni cathartici fructus 98, 106
Quassiae lignum 129, 134 Rhamni purshiani cortex 97, 104
quassin 129, 131, 134 Rhamnus species 97, 98
Quebracho cortex 55, 68 rhaponticin (rhaponticoside) 98, 101,
quercetin 170 108
quercetin glycosides 170, 172 Rhei radix 98, 108, 114
quillaja bark 228 rhein 100, 108, 114
Quillaja saponaria 228 Rheum species 98
Quillajae cortex 228, 234, 244 Rheum-emodin 100
quinidine 56, 61, 74 Rhizoma
quinine 56, 61, 74 - Calami 9, 24
quinoline alkaloids 56 - Curcumae 14, 42
- Filicis 279, 282
Radix - Galangae 249
- Angelicae 147, 154 - Hydrastis 57, 80
- Belladonnae 59,90 - Podophylli 279,284
317
Rhizoma Scopoliae radix 59, 90, 149, 154
- Veratri 58 scopoline 149
- Zingiberis 249 scots pi ne oil 46
rhubarb rhizome 98 screening (of unknown drugs) 291
ribwort leaf 128 Scrophulariae herba 127, 132
Robinia pseudoacacia 165 Secale alkaloids 55
Robiniae flos 180 Secale cornutum 55, 72
robinin 180 secoiridoids 127, 128
rosemary leaves 11 Semen
Rosmarini folium 11, 30, 128, 142 - Belladonnae 90
rosmarinic acid 13 - Cacao 58, 86
Rosmarinus species 11, 30 - Coffeae 58, 86
round turmeric 14 - Colae 58
rue 149 - Colchici 57, 82
rupture-wort 148 - Coriandri 30
Ruta graveolens 149 - Cucurbitae 129, 138
Rutae herba 149, 156, 186 - Erucae 255, 256
rutamarin 150 - Hippocastani 227, 234, 242, 244
rutin 170,172 - Ignatii 56, 72
- Myristici 10,28
Sabadillae semen 58, 84 - Sabadillae 58,84
safrol 10, 20, 26 - Sinapis 255, 256
sage leaves 13 - Stramonii 90
salicin 278, 280, 282 - Strophanthi 199,214,216
Salicis cortex 278, 282 - Strychni 56, 72
Salix species 278 Senegae radix 229, 234, 244
Salvia officinalis 13 senegin 229, 232
Salvia triloba 13 sen na lea ves 99
Salviae folium 13, 38, 128, 142 sen na pods 99
salvigenin 13 Sennae folium 99, 110, 112
Sambuci flos 167,174,178 Sennae fructus 99, 110, 112
Sambucus nigra 167 sennosides 99,101,110,112
sanguinarin 57, 63, 80, 82 serpentine 55, 62, 68, 70
santonin 12,19,34 Serpylli herba 14,40
Saponaria officinalis 228 Seville orange peel 15, 169
Saponariae radix 228, 234, 236 Siam benzoin 48
saponin 228 si! ver fir bil 46
saponin drugs 225 silverweed 168
sarmentosides 216 si!ybin 170, 190
Sarothamni (Spartii) herba 58, 86, 168, Si!ybum marianum 169
186 sinalbin 255, 256
Sarothamnus scoparius 58, 168 Sinapis alba 255
sarsaparilla 229 Sinapis semen 255, 256
Sarsaparillae radix 229,234,244 sinensetin 171, 188
Sassafras lignum 10, 26 sinigrin 255, 256
sassafras wood 10 Smilax species 229
Schoenocaulon officinale 58 soap bark 228
Scillae bulbus 199, 222 soapwort root 228
scillaren 199, 203, 222 socrotine aloes 97, 102
scilliroside 222 Solanaceae drugs 59,88, 90
scoparin 168, 186 Solidaginis herba 168
scopolamine 59, 63, 88, 90 Solidago species 168
scopoletin 150, 152 sophora buds 169
Scopolia carniolica 59, 149 Sophora japonica 169
scopolia root 59 Sophorae gemma 169, 186
318
southernwood 149 tobacco leaves 58
sparteine 58, 65, 86 tolu balsam 17
spearmint leaves 12 Tolutanum balsamum 17
speedwell 168 Trachyspermum ammi 14
spike oil 12, 34 triandrin 278, 280, 282
Spiraeae flos 167, 180 Trifolii fibrini folium 128, 132
spiraeoside 167 trigonelline 86
spray reagents (list) 299 triterpene saponins 225, 230
spruce oil 46 tropane alkaloids 59
squill 199 turmeric 14
standard saponin 228 turpentine oil 16,46
star anise 9, 26 Tussilago farfara 166
steroid saponins 225, 233
Stigma croci 270, 274 umbelliferone 150, 152
St. John's wort 99 Urginea maritima 199
Stoechados flos 167,174,178 Uvae-ursi folium 119,120
Stramonii folium 60, 88, 90 uzara root 199
Stramonii semen 90 U zarae radix 199, 220
Strobulus humuli lupuli 129, 140 uzarin 203, 220
Strophanthi semen 199,214,216 uzarone 220
strophanthin, g- 199,202,204,206
strophanthin, k- 199,202,204, 206 Vaccinium species 119
Strophanthus seeds 199 valepotriates 263, 265
Strophanthus species 199 valerian rhizome 264
Strychni semen 56, 72 Valeriana species 264
strychnine 56, 62, 66 Valerianae radix 264,266
Strychnos species 56, 72 valine 288
Styrax species 16 valtrate 265,266
Sumatra benzoin 48 valtrathydrins 266
sweet flag 9 vanillin glucoside 242
swertiamarin 127, 132 Veratri rhizoma 58
Syzygium aromaticum 10 veratrin (Veratrinum) 58, 84
Veratrum album 58
tall melilot 148 Verbasci flos 167, 178,229
taxifolin 171, 190 Verbascum species 167
tea 59 Veronica officinalis 168
Terebinthinae aetheroleum 16,46 Veronicae herba 168, 186
terpineol 19,22 Viburni cortex 119,122
terpinyl acetate 19 Viburnum species 119,122
tetrahydrocannabinol (THC) 260 Viola tricolor 168
Theae folium 59, 86 Violae herba 168, 184, 186
thebaine 56, 61, 78 Virgaureae herba 168, 184
Theobroma cacao 58 Visci herba 279,286
theobromine 58, 59, 65, 86 viscotoxin 279
theophylline 59, 65, 86 Viscum album 279
Thevetia glycosides 204 visnagin 148, 151, 160
thiourea derivatives 253, 256 vitexin 170,178
thornapple leaves 60 vitexin-2" -O-rhamnoside 170, 178
thujone 13, 19,38 Vitis idaeae folium 119, 120
thyme 13
Thymi herba 13, 40 walnut leaves 168
thymol 13, 19, 40 white hellebore rhizome 58
Thymus species 13, 40 white willow 278
Tilia species 167, 180 wild pansy 168
Tiliae flos 167, 180 wild parsnip root 148
319
wild thyme 14 Xysmalobii radix 199,220
willow bark 278 Xysmalobium undulatum 199
witch hazelleaves 278 xysmalorin 199,203,220
woodruff 148
wormseed 12 Y ohimbe cortex 55, 68
wormwood 128 yohimbehe bark 55
yohimbine 55, 62, 68
xanthorhamnin 98, 106
xanthorrhizol 14,42 Zingiberis rhizoma 249
320
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Plant Drug Analysis

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H. Wagner S. Bladt E.M.

Translated by Th.A. Scott

With 170 Colored Photographs

Springer-Verlag Berlin Heidelberg GmbH 1984

Translation of the German edition' Drogenanalyse '

ISBN 978-3-662-02400-3 ISBN 978-3-662-02398-3 (eBook)

Library of Congress Cataloging in Publication Data

Essential Oil Drugs (Aetherolea) 5

Extraction and TLC . . . . . . . . 5

Extraction and TLC . . . . . . . . . . . . . 93

Arbutin Drugs 117

Extraction and TLC 117

Bitter Principle Drugs 125

Extraction and TLC . . . 125

Coumarin Drugs 145

Flavonoid Drugs 163

Cardiac Glycoside Drugs 195

Extraction and TLC . . . . 195

Saponin Drugs 225

Extraction and TLC 225

Drugs Containing Pungent Principles 247

Extraction and TLC . . . . . . . . . . 247

Mustard Oil Drugs and Allium 253

Extraction and TLC 253

N arcotic Drugs . 259

Extraction and TLC 259

Extraction and TLC 263

Drugs Containing Pigments 269

Extraction and TLC 269

Drugs with Miscellaneous Constituents 277

Extraction and TLC . . . 277

TLC Screening of an Unknown Commercial Drug 291

TLC Analysis of Herbai Drug Mixtures 296

Subject Index 309

I. Thin Layer Chromatographie-Analysis (TLC) of Drugs

11. Doeumentation of Thin Layer Chromatograms

111. Photo graphie Reeord of Thin Layer Separations of Drug

IV. Compilation of a TLC Drug Atlas

1. Source of the drugs

5. Concentration of substances for TLC

I. Extraction of Essential OUs

2. Abridged official method

Drug Content of Sampie Water Time Rate

Absinthii herba 0.3 50 300 3 2-3

a distilled from 1% NaCI solution.

b) Thermomicrodistillation after Stahl (TAS-method)

c) Extraction with methylene chloride (dichloromethane (DCM)-extract)

d) Extraction with methanol (MeOH-extract)

11. Thin Layer Chromatography

Solvent system Drug or essential oil

A-2 Benzene a Anisi fructus

a Benzene is carcinogenic and should be replaced by toluene.

b) Vanillin-sulphuric acid (VS No. 38, p. 304)

c) Phosphomolybdic acid (PMA No. 27, p. 303)

Fig. Drug/Plant source/Family/Phar- Content of essential oil

3 Cinnamomi Cortex Ceylon cinnamon: 1-1.5% ess. oil.

4 Calami Rhizoma (Radix) Triploid European race: up to 3 % ess. oil with

5,6 Foeniculi Fructus F. vulgare var. dulce (French sweet or Roman

5 Basilici Herba 0.1-0.45% ess. oi!.

5 Sassafras Lignum 1-2% ess. oi!.

7A Petroselini Fructus 3-6% ess. oi!.

9B Coriandri Fructus 0.2% ess. oil (Indian eoriander) 0.8-1 % ess.

10C Rosmarini Folium 1-2% ess. oi!.

14 Cinae Flos 2-3% ess. oil.