Methods in Polyphenol Chemistry: Proceedings of the Plant Phenolics Group Symposium, Oxford, April 1963

146 Pages·1964·5.39 MB·English

by  J. B. Pridham (Eds.)

METHODS IN
POLYPHENOL CHEMISTRY
Proceedings of the Plant Phenolics Group Symposium
Oxford, April 1963
Edited by
J. B. PRIDHAM
Lecturer in Organic Chemistry
Royal Holloway College,
University of London
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PREFACE
PREVIOUS Symposia in this series have dealt with the advances that have
been made in the chemistry and biochemistry of phenolic compounds.
Delegates at the most recent Plant Phenolics Group Symposium which was
held at the Dyson-Perrins Laboratory, University of Oxford, during 2-4 April
1963, were concerned with the methods and techniques which made many
of these advances possible.
The development of paper chromatographic and ultraviolet spectroscopic
techniques were particularly important and, no doubt, were largely responsible
for the rapid growth in our knowledge of polyphenols which has occurred
during the last 10-15 years. In addition to these two methods other spectro­
scopic techniques were dealt with at the Symposium. Particular mention
should perhaps be made of nuclear magnetic resonance spectroscopy which
is becoming increasingly important in polyphenol chemistry. Only recently*
Professor W. D. Ollis presented structural formulae of complex polyphenols
which were based almost entirely on N.M.R. studies. The full-scale application
of mass spectrometry to structural studies in this general field is also awaited
with interest. Modern chromatographic methods such as thin layer and
gas-liquid techniques will probably be used extensively in the future. These
will enable the analyses of polyphenol mixtures to be carried out more
rapidly and with greater resolution. Polyamide chromatography also has
high powers of resolution, although this technique has not been widely
used owing to difficulties in obtaining supplies of polyamide powder. This
situation has now improved, however.
We are indebted to Springer-Verlag for permission to publish Fig. 6
(p. 34) and Fig. 1 (p 127) appears by courtesy of W. G. Pye & Co. Ltd.
Colleagues in the Department of Chemistry of Royal Holloway College have
given me valuable editorial assistance during the preparation of this book.
Royal Holloway College, 1963 J. B. PRIDHAM
* Plant Phenolics Group Meeting on the "Comparative Biochemistry of the Legu-
minosae", held at the John Innes Horticultural Institution, 10-11 September 1963.
v
LIST OF CONTRIBUTORS
R. J. ABRAHAM, Department of Organic Chemistry, The University,
Liverpool, England.
E. C. BATE-SMITH, Agricultural Research Council, Low Temperature Research
Station, Cambridge, England.
J. W. BRIDGES, Department of Biochemistry, St. Mary's Hospital Medical
School (University of London), London, England.
B. R. BROWN, The Dyson Perrins Laboratory, University of Oxford, Oxford,
England.
J. L. GOLDSTEIN, Agricultural Research Council, Low Temperature Research
Station, Cambridge, England.
J. B. HARBORNE, John Innes Horticultural Institute, Bayfordbury, Hertford,
England.
L. HORHAMMER, Institut fiir Pharmazeutische Arzneimittellehre der Uni-
versitat Munchen, Munchen, Germany.
J. R. LINDSAY SMITH, The Dyson Perrins Laboratory, University of Oxford,
Oxford, England.
R. O. C. NORMAN, The Dyson Perrins Laboratory, University of Oxford,
Oxford, England.
J. B. PRIDHAM, Department of Chemistry, Royal Holloway College
(University of London), Englefield Green, Surrey, England.
G. K. RADDA, The Dyson Perrins Laboratory, University of Oxford, Oxford,
England.
T. SWAIN, Agricultural Research Council, Low Temperature Research
Station, Cambridge, England.
V. THALLER, The Dyson Perrins Laboratory, University of Oxford, Oxford,
England.
H. WAGNER, Institut fiir Pharmazeutische Arzneimittellehre der Universitat
Munchen, Munchen, Germany.
H. WEIGEL, Department of Chemistry, Royal Holloway College (University
of London), Englefield Green, Surrey, England.
ix
GENERAL INTRODUCTION
B. R. BROWN
The Dyson Perrins Laboratory, University of Oxford
ADVANCES in polyphenol chemistry result from the development of new
theoretical approaches and from the application of new practical techniques.
It is remarkable how much knowledge in this field has accumulated over the
past few years as a result of the use of new techniques; for example, much
of the information we now have concerning the polyphenolic constituents of
plants has resulted from the application of paper chromatography, which
was first introduced by Consden, Gordon and Martin in 1944.*1* Nevertheless
it can be said that chemists interested in polyphenols, in common with the
majority of scientists, tackle today's problems with yesterday's tools, i.e.
current problems are attacked with methods which are inadequate and to
that extent are already out of date. Rapid developments in natural product
chemistry often follow the discovery or resuscitation and development of
practical methods. A good example of this was the development of practical
techniques of infrared spectroscopy in the 1940's which has greatly facilitated
structural investigations of complex organic molecules. The discovery and
quick application of new methods or developments and extensions of existing
methods is therefore of first importance.
However, with the rapid expansion of science, another serious problem
arises, viz. that of disseminating knowledge of existing practical methods,
what they are and how they are best applied. For this reason, the subject
of "Methods in Polyphenol Chemistry" was chosen for this Plant Phenolics
Group Symposium. The hope is that many will learn about methods with
which they are unfamiliar and that they will be able to apply such methods
with advantage to their own problems.
It is logical to consider the purposes for which methods are required in
polyphenol chemistry. The first stage in the investigation of a polyphenol
is its detection for which qualitative methods are required. The quantitative
analysis of polyphenols in the material under investigation is important
since it is necessary to know whether one is investigating a substance present
in microgram quantities per litre or in gram quantities per litre. More often
than not, several polyphenols occur together in living matter, e.g. in plant
materials, and this means that methods of separation are required. Following
on separation, and usually part of the same process, is isolation of a single
polyphenol constituent. Before reliable conclusions can be drawn about the
structure, reactivity, and function of a polyphenol, it must be obtained pure.
1
2 B. R. BROWN
Purification is often part of the process which involves separation and iso­
lation. When the polyphenol under investigation is one which has previously
been described, it is necessary to have methods of identification. It is
important to stress here the wisdom of using as many methods and as varied
methods of identification as possible, for many chemical compounds,
including phenols and their derivatives, are very like each other, and identifi­
cation based upon inadequate evidence can be an annoying occurrence.
If the polyphenol under investigation is one not previously reported, it is
necessary to determine its chemical structure and to synthesize it. Finally,
stereochemistry is of primary importance for biological function; clear-cut
methods of determining the fine detail of the stereochemistry of polyphenols
are therefore very important.
It is of interest to classify broadly the methods applied in polyphenol
chemistry and to assess the value of each broad class. First, and oldest,
are the methods of classical organic chemistry. These depend for separation,
isolation, and purification of the compound in question on simple physical
techniques, such as solvent extraction, recrystallization, and distillation.
Thereafter, classical chemical methods of degradation and of synthesis are
applied. It is significant to note that these methods enabled organic chemists
to isolate and characterize simple naturally occurring phenols, e.g. ellagic
acid (A. G. Perkin) and depsides (E. Fischer), but that progress with more
complicated phenols, especially with phenolic polymers, such as tannins
and ligins, was slow and is as yet incomplete. In recent times and at the
present time, the discovery and application of new physical methods has
enabled us to extend our knowledge to polyphenols of greater complexity
and greater molecular weight. The use of the various partition and absorption
techniques for the detection, separation, isolation, and purification of phenols
and the application of ultraviolet and infrared spectroscopic techniques to
structural determination are a few examples. Most of the lectures at this
symposium deal with this aspect. Finally, there are signs that the application
of biochemical methods, e.g. the use of enzymes as chemical reagents, is
becoming very important. Investigation of the biosynthesis and of the bio­
logical function of polyphenols, as well as direct structural investigations,
are amenable to attack by this method. At present this is one of the rapidly
expanding facets in the chemistry of polyphenols.
I shall now describe briefly a few methods (not to be treated in subsequent
chapters) which we are currently using in Oxford for the investigation
of plant polyphenols.
COUNTER-CURRENT EXTRACTION*2)
Principles
It is well known that an organic compound will distribute itself between two
immiscible liquids according to the equation:
General Introduction 3
K (partition coefficient) = Ci/Cn
where Ci is the concentration of the compound in one liquid and Cu the
concentration in the other liquid. If we suppose that we have a mixture of
two substances, A, and B, in equal amounts (say C grams of each per litre
of ether) and that their partition coefficients between say ether and water
are A^A = 1 and KB = 1/9, then if we shake the original mixture with an
equal volume of water, then pass the ether layer on to an equal volume of
water, equilibrate, and pass again, etc., the concentration of A in the ether
at the nth extraction will be C (l/2)w and that of B will be C(l/10)w.
Table 1 shows, for this very favourable but untypical case, that a good
TABLE 1. DISTRIBUTION OF TWO COMPOUNDS, A AND B,
BETWEEN ETHER AND WATER
No. of extractions, n 0 1 2 10
Ratio in ether, [A]/[B] 1 5 25 —107
separation is easily achieved, the ether layer becoming greatly enriched in A.
In more typical but less favourable cases one may have to carry out as
many as 500 partitions to achieve enrichment of practical value. The labour
involved in such a procedure is greatly reduced by the use of a Craig
machine*2* which enables the method to be used as a standard laboratory
technique.
Application to Flavanoids
(i) Catechins. In 1958 Weinges*3* separated a naturally occurring mixture
of (+)-catechin (I), (—)-epicatechin (II) and (+)-taxifolin (from the bark
of Douglas fir) by means of 500 transfers between ether and water.
W-Epicatechin (-)-Catechin
OH) (2[)
4 B. R. BROWN
In 1959 we<4> wished to obtain pure (+)-epicatechin (III) from the mixture
of C+)-epicatechin (III) and (+)-catechin (I) obtained by epimerization of
(+)-catechin (I) in a slightly alkaline solution. Similarly we required
(—)-catechin (IV) which was obtainable from epimerized (—)-epicatechin
(II).<5> All four isomers (I), (II), (III), and (IV) were required for comparative
stereochemical studies.<6) The solvent system ether-1 per cent aqueous
sodium chloride was first used for the separation of (+)-catechin (I) and
(-f)-epicatechin (III). The partition coefficients of these compounds between
these two solvents were measured at 20° using a spectrophotometric method
(A 280 m/x) and found to be:
(+)-catechin, Ki = 0-29
(+)-epicatechin, AT2 = 0-18
Since an optimal balance of speed and efficiency of separation is achieved
when eqn. (1) is satisfied,(2)
V&i + #0 = 1 (1)
(equal volumes of each phase present)
it is clear that these compounds in this solvent system do not constitute
a favourable case W(K\ + K ) = 0-69, cf. eqn. (1), and K\ and K are
2 2
too close together) and in fact 555 transfers were required to obtain an
efficient separation. Since we wished to carry out this separation several
times on a large scale, we investigated alternative solvent systems. First we
found that addition of ethyl acetate to the ether caused an increase in the
partition coefficient (Ki) of (+)-catechin between ethyl acetate-ether
mixtures and 1 per cent aqueous sodium chloride. We chose an ether-ethyl
acetate mixture of composition 9 : 1 v/v as being optimal; the partition
coefficients for this mixture and 1 per cent aqueous sodium chloride were
found to be:
(+)-catechin Ki = 0-73
(+)-epicatechin K% = 0-46
K + K = 119
1 2
V(Ki + K ) = V09
2
It is seen that this should constitute a favourable case (cf. eqn. (1)). Our
experimental distribution curve is shown in Fig. 1 and tubes 85-60 yielded
pure (+)-epicatechin after one recrystallization from water.
(ii) Polyphenolic constituents of needles of Norway spruce (Picea excelsa).
As part of investigations we are carrying out on the relation between phenolic
constituents of leaves and soil formation and composition/7) we have
examined needles of Norway spruce.(8) Application of counter-current extrac­
tion to aqueous acetone extracts of needles enabled us to isolate and to
identify several flavanoid components (see Table 2). The condensed tannin
Generaljtatroduction
(+) -Catechin
120 110 100 90 80 70 60
Tube No.
Fio. 1. Counter-current distribution curve of (4-)-catechin and (+)-epicatechin between
ether-ethyl acetate (9:1 v/v) and 1 per cent aqueous sodium chloride.
TABLE 2. FLAVANOID CONSTITUENTS OF NEEDLES OF NORWAY SPRUCE (Picea excelsa)
Hydroxylation Pattern
Class of
flavanoid
I 5,7,3',4'-Tetrahydroxy 5,7,3',4', 5'-Pentahydroxy
Tannin + +
(+)-Gallocatechin
OH
1
OH
Catechins
HO
OH
OH
HO
Leucodelphinidin
(+)-Leucocyanidin (detected on paper)
Leucoanthocyanidins
HO OH
HO OH
6 B. R. BROWN
which we have isolated yields, on treatment with hot concentrated hydro­
chloric acid, a mixture of cyanidin and delphinidin, the former in greater
quantity. The catechins which we obtained also showed the same hydroxy-
lation patterns, as did the leucoanthocyanidins.
MASS SPECTROMETRYW
Principles
Organic compounds, when subjected to electron impact in a suitable source,
yield positively charged particles which are deflected by passage through a
magnetic field. The amount of deflection is dependent upon and can be used
to determine the masses of the particles. In order to cause ionization, an
organic compound is bombarded with electrons of energy greater than the
ionization potential of the compound (7 to 15 eV). The annexed scheme shows
in outline the general pattern of behaviour observed from an organic compound
under these conditions of electron impact in the source. The occurrence of
rearrangement complicates the pattern, but in practice this is not found to be
a serious disadvantage, since rearrangement is specifically related to the
structures of the molecules in question.
ABCD+e" ► ABCD+ 4-2e-
AB+ +CD*-* ABCD* ^rJ^g_ement
J >,. . / \. -*-AD ++BC
A + B B IA CD* 4B > + BCD- / ^ .
/ \ A+ + D* D+ + A'
C++D# [£+C*
A disadvantage of the method is that a certain volatility (e.g. vapour
pressure ca. 10-2 mm at 150-250°) is required of the organic compound in
order to introduce it into the electron source as a vapour. Thus, as the mole­
cular weight increases, it becomes more important to choose a derivative
of the compound which will fulfil this requirement. At the present time this
puts an upper limit of about 700-800 on the M.W. of compounds amenable
to investigation in a mass spectrometer. Against this, a great advantage of the
method is that only very small amounts (fractions of a milligram) of compounds
are required.
Mass spectrometry allows the determination of molecular weights with
an accuracy of greater than one mass unit. This means that, as an analytical
method, it is as useful as a conventional CH analysis. Even more important
is the fact that, from the pattern of M.W.'s observed after electron impact,
it is possible to obtain valuable information about the structures of molecules.
Applications to Phenols^1®
The values of peak heights for monophenols recorded in Table 3 (work of
Aezel and Lumpkin<10)) show several important features. Firstly, strong