APPLICATIONS .1 General

While phytochemical procedures have today an established role in practi- cally all branches of plant science, this has not always been so. Although these methods are obviously essential in all chemical and biochemical studies, their application in more strictly biological spheres has only come within the last two decades. Even in disciplines so remote from

Applications

³³

the chemical laboratory as systematics, phytogeography, ecology and palaeobotany, phytochemical methods have become important for solv- ing certain types of problems and will undoubtedly be used here with increasing frequency in the future.

There is only room in this book to mention a few of the many applica- tions of phytochemical methods. Some of the obvious applications in agriculture, in nutrition and the food industry and in pharmaceutical research must be taken for granted. The following applications are simply given to illustrate the value of phytochemical techniques in some of the major branches of plant science.

1.6.2 Plant physiology

The major contributions of phytochemical studies to plant physiology are undoubtedly in determining the chemical structures, biosYnthetic origins and modes of action of natural growth hormones. As a result of continuing collaboration over the years between physiologists and phytochemists, five classes of growth regulators are now recognized: the auxins, cytokinins, abscisins, gibberellins and ethylene. Methods of detec- tion, which vary from GLC through TLC to PC, are discussed in later pages as follows: auxins (p. 218), cytokinins (p. 226), abscisins (p. 122) and gibberellins (p. 127). One of the more remarkable aspects of the gibberellin group of hormones is the large number of known structures (over a hundred), all apparently with a similar range of growth properties.

The need for very precise methods of detection and distinction between gibberellins led to the development of combined GC-MS for their analysis. A general book on methods for the isolation of plant growth substances, edited by Hillman (1978), can be consulted for more de- tails. The necessary requirements for accurate hormone analysis are criti- cally considered by Reeve and Crozier (1980). An excellent review of techniques, including the use of radio-immunoassay, is that of Horgan (1981). A sixth class of growth regulator described recently are the brassinosteroids; methods of steroid analysis are given on p. 134.

1.6.3 Plant pathology

Phytochemical techniques are primarily important to the pathologist for the chemical characterization of phytotoxins (products of microbial syn- thesis produced in higher plants when invaded by bacteria of fungi) and of phytoalexins (products of higher plant metabolism formed in response to microbial attack). A range of different chemical structures are involved inboth cases. The most familiar phytotoxins are lycomarasmin and fusaric acid, amino acid derivatives which are wilting agents in the tomato. Other toxins that have been isolated are glycopeptides, naphthaquinones or

34

Methods

of

plant analysis

sesquiterpenoids (Durbin, 1981). Some phytotoxins are chemically labile so that special precautions have to be taken during their isolation and identification.

Phytoalexins also have different structures, according to the plant source (Bailey and Mansfield, 1982). They may be sesquiterpenoid (rishitin from Solanum tuberosum), isoflavanoid (pisatin from Pisum sativum),acetylenic (wyerone acid from Vida [aba)or 'phenolic' (orchinol from Orchis militaris). Identifications of isoflavanoids and acetylenes are described in Chapters 2 and 5 respectively and a procedure for phytoalexin induction is included as a practical experiment in Chapter 2.

'Pre-infective' substances (naturally occurring secondary constituents) are considered by some plant pathologists to be important in imparting disease resistance to plants. Phenolic compounds, such as phloridzin in apple, tannin in raspberry, are mainly implicated here. Methods of identification of such compounds are covered in detail in Chapter 2.

Other examples of plant compounds that have been characterized as phytoalexins or pre-infective substances can be found in the review of Grayer and Harborne (994).

1.6.4 Plant ecology

Two research areas where secondary plant constituents are significant in plant ecology are in plant-animal and plant-plant interactions. The ana- lytical problems in both cases are difficult because of the very limited amounts of biological material at the disposal of the phytochemiSt. For example, in following the fate of secondary compounds in insects feeding on plants, it is necessary to analyse different organs of the insect to see where the compounds are stored; such analyses are often complicated and time-consuming.

Compounds so far known to be involved in plant-animal interactions are primarily alkaloids and cardiac glycosides, mustard oil glycosides, cyanogens, steroids or volatile terpenes. The plant compounds may vari- ously act as feeding attractants or repellents, have hormonal effects on the insects or provide the insects with a useful defence mechanism against predation (Harborne 1993).

Plant-plant interactions involve so-called allelopathic substances which one plant exudes from its roots or leaves in order to prevent the growth of other plant species in its vicinity. The compounds are either volatile terpenes (e.g. cineole) or simple phenolic acids, depending on whether the plant is growing in a semi-tropical or a temperate climate. The phytochemical study of allelopathy can be difficult since it requires determinations on whole leaf extracts, natural leaf leachates and on soil samples too. The possible rapid turnover of active substances in the soil also provides another analytical hazard in this field (Putnam and Tang, 1986).

Applications 35

1.6.5 Plant tissue culture

Phytochemistry has an important role in plant tissue culture in the detec- tion and analysis of the products of secondary metabolism that may be formed in such tissues. During culture, the concentrations of secondary metabolites may vary depending on the plant hormones added, the pres- ence of light and other factors. In some cases, metabolites are produced which are not present in the plant from which the tissue is derived. For example, anthocyanins may be produced in culture of plants which lack cyanic colour. New pharmaceutically active constituents may be pro- duced in cell or callus culture and good phytochemical techniques are necessary for their detection. Production of such structures may be elic- ited in cell culture by adding bacterial or fungal cell extracts (Stafford and Pazoles, 1997).

Some secondary metabolites such as alkaloids may be produced in cell culture in vanishingly small amounts and radioimmunoassay techniques may be required for their analysis (Zenket al.,1997).Anexcellent series of nine volumes has been published, describing experimental conditions for secondary product synthesis in cell culture and methods of phytochemi- cal analysis for most plants that have been taken into cell culture (Bajaj, 1996).

1.6.6 Plant genetics

In the past, phytochemistry has contributed to higher plant genetics in providing the means of identifying anthocyanin, flavone and carotenoid pigments occurring in different colour genotypes of garden plants. The results have shown that the biochemical effects of these genes have a simple basis and have indicated the probable pathway of pigment synthe- sis in these organisms (Alston, 1964). The inheritance in plants of other chemical attributes (alkaloids, terpenes, etc.) has also been successfully mapped out by phytochemical analysis.

A more recent contribution of phytochemistry to genetics is in the identification of hybrid plants andinthe elucidation by chemical means of their parental origin. Phytochemistry has also won increasing recognition as a useful tool, together with cytology, to be used in the analysis of genetic variation within plant populations (d. Harborne and Turner, 1984).

1.6.7 Plant systematics

One of the most rapidly developing fields in phytochemistry at the present time is the hybrid discipline between chemistry and taxonomy, known as biochemical systematics or chemotaxonomy. Basically, it is concerned with the chemical survey of restricted groups of plant, mainly

36

Methods

of

plant analysis

for secondary constituents but also for macromolecules and the applica- tion of the data so obtained to plant classification (Harborne and Turner, 1984).

Perhaps the most useful class of compounds for such study are the flavonoids. Surveys of many other classes of compounds (notably of alka- loids, non-protein amino acids, terpenes and sulphur compounds) have also yielded potentially useful new information for taxonomic purposes.

Accurate methods are essential, both in preliminary screening of plants and in the more detailed analysis of individual components.

Chemical analyses of the amino acid sequences of plant proteins have also been brought to bear on systematic problems, at the higher levels of classification. Results have been obtained with cytochrome c, plastocyanin and ferredoxin; the sequencing of plant nucleic acids has also yielded data of taxonomic interest (Soltis et al., 1992).

1.6.8 Medicinal plant research

The plant kingdom represents an enormous reservoir of biologically ac- tive molecules and so far only a small fraction of plants with medicinal activity have been assayed. Nearly 50% of drugs used in medicine are of plant origin. There is therefore much current research devoted to the phytochemical investigation of higher plants which have ethnobotanical information associated with them. The phytochemicals isolated are then screened for different types of biological activity. Cytotoxicity via the brine shrimp test is studied in order to reveal new anticancer compounds.

Taxol, the new hospital drug from the bark of Taxus brevifolia, was discov- ered in this way. Alternatively, crude plant extracts can be first assayed for particular activities and the active fractions then analysed phyto- chemically. A variety of bioassays are now available for the phytochemist to use in such work (Hostettmann, 1991).

GENERAL REFERENCES

Bailey, J.A. and Mansfield, J.W. (eds) (1982)Phytoalexins, Blackie, Glasgow.

Bajaj, y.P.5. (ed.) (1996)Medicinal and Aromatic Plants, Volume 9, Springer, Berlin.

Bobbitt, J.M. (1963)Thin Layer Chromatography, Reinhold Pub. Co., New York.

Buckingham, J. (ed.) (1994) Dictionary

of

Natural Products, Chapman and Hall, London.

Burchfield, H.P. and Storrs, E.E. (1962)Biochemical Application

of

Gas Chromatogra- phy, Academic Press, New York.

Cross, A.D. and Jones, R.A. (1969)An Introduction to PracticalIR Spectroscopy, 3rd edn, Butterworths, London.

Culberson, C.F. (1969) Chemical and Botanical Guide to Lichen Products, North Carolina Univ. Press, Chapel Hill, USA.

Derome, A.E. (1986) Modern NMR Techniques for Chemistry Research, Pergamon Press, Oxford.

General references 37 Dey, P.M. and Harborne, J.B. (eds) (1989-1997)Methods in Plant Biochemistry,in 10

volumes, Academic Press, London.

Durbin, RD. (ed.) (1981)Toxins in Plant Disease,Academic Press, New York.

Gillam, A.E. and Stern, E.S. (1957) Electronic Absorption Spectroscopy, 2nd edn, Edward Arnold, London.

Hamilton, RJ. and Sewell, P.A. (1982) Introduction to High Performance Liquid Chromatography,2nd edn, Chapman and Hall, London.

Harborne, J.B. (1967) Comparative Biochemistry

of

the Flavonoids, Academic Press, London.

Harborne, J.B. (1993) Introduction to Ecological Biochemistry, 4th edn, Academic Press, London.

Harborne, J.B. and Turner, B.L. (1984) Plant Chemosystematics, Academic Press, London.

Harwood, L.M. and Claridge, T.D.W. (1996) Introduction to Organic Spectroscopy, University Press, Oxford.

Heftmann, F. (1992) Chromatography: Fundamentals and Applications

of

Chromato- graphic and Electrophoretic Techniques,5th edn., Elsevier, Amsterdam.

Hillman, J.R (ed.) (1978) Isolation

of

Plant Growth Substances, Cambridge University Press, Cambridge.

Horgan, R (1981) Modern methods for plant hormone analysis.Prog. Phytochem., 7, 137-70.

Hostettmann, K., Hostettmann, M. and Marston, A. (1986) Preparative Chromatography Techniques,Springer, Berlin.

Jackman, L.M. (1959) Applications

of

NMR Spectroscopy in Organic Chemistry, Pergamon Press, Oxford.

James, T.L. (1975)NMR in Biochemistry: Principles and Applications.Academic Press, New York.

Kirchner, J.G. (1978)Thin Layer Chromatography,2nd edn, John Wiley, New York.

Lederer, E. and Lederer, M. (1957) Paper Chromatography, 2nd edn, Elsevier, Amsterdam.

Linskens, H.P. (1959) Papier Chromatographie in der Botanik, Springer Verlag, Berlin.

Linskens, H.P. and Jackson, J.F. (1985-) Modern Methods

of

Plant Analysis, New Series,Springer, Berlin.

Morton, R.A. (1975)Biochemical Spectroscopy,Adam Hilger, London.

Reeve, D.R and Crozier, A. (1980) Quantitative analysis of plant hormones.

Encycl. Pl. Physiol. New Series,9, 203-80.

Sargent, J.R (1969)Methods in Zone Electrophoresis,2nd edn, BDH Chemicals Ltd., Poole, England.

Scheinmann, F. (ed.) (1970)An Introduction to Spectroscopic Methods for the Identifi- cation

of

Organic Compounds(Vol.I),Pergamon Press, Oxford.

Scheinmann, F. (ed.) (1970)An Introduction to Spectroscopic Methods for the Identifi- cation

of

Organic Compounds(Vol. II), Pergamon Press, Oxford.

Scott, A.I. (1964). Interpretation of the Ultraviolet Spectra of Natural Products, Pergamon Press, Oxford.

Sherma, J. and Zweig, G. (1971)Paper Chromatography,Academic Press, New York.

Simpson, C. (1970).Gas Chromatography,Kogan Page, London.

Southon, I.W. and Buckingham, J. (1989) Dictionary

of

Alkaloids, Chapman and Hall, London.

Stahl, E. (ed.) (1969)Thin Layer Chromatography,2nd edn, George Allen and Unwin, London.

Touchstone, J.e. and Dobbins, M.F. (1978) PracticeofThin Layer Chromatography, John Wiley, Chichester.

38 Methods

of

plant analysis

Truter, E.V. (963)Thin Film Chromatography,Cleaver Hume Press, London.

Turner, W.B. (1971)Fungal Metabolites,Academic Press, London.

Turner, W.B. and Aldridge, D.C. (983) Fungal Metabolites 11, Academic Press, London.

Wagner, H. and Bladt, S. (996) Plant Drug Analysis,2nd edn, Springer, Berlin.

Waller, G.R (ed.) (1972) Biochemical Applications

of

Mass Spectrometry, Wiley Interscience, New York.

Waller, G.R and Dermer, O.c. (980)Biochemical Applications

of

Mass Spectrometry, First Supplement,John Wiley, Chichester.

Williams, D.H. and Fleming,I.(966)Spectroscopic Methods in Organic Chemistry, McGraw-Hill, London.

SUPPLEMENTARY REFERENCES

Alston, RE. (964) inBiochemistry

of

Phenolic Compounds(ed. J.B. Harborne), Academic Press, London, pp. 171-204.

Bate-Smith, E.C. and Westall, RG. (1956)Biochim. Biophys. Acta,4, 427.

Eglinton, G. (1970) inIntroduction to Spectroscopic Methods for the Identification

of

Organic Compounds(Vol.I)(ed. F. Scheinmann), Pergamon Press, Oxford, pp.

123-144.

Grayer, Rand Harborne, J.B. (994)Phytochemistry,37, 19.

Harborne, J,B. (1969)Phytochemistry8, 419.

Harborne, J.B., Lebreton, P., Combier, H., Mabry, T.J. and Hammam, Z. (971) Phytochemistry,10, 883.

Harley, RM. and Bell, M.G. (967)Nature (LondJ213, 1241.

Hershenson, H.M. (956)UVand Visible Absorption Spectra (Vol.I,1930-54).

Academic Press, New York.

Hershenson, H.M. (1959)Infra Red Absorption Spectra (Vol.I,1945-57). Academic Press, New York.

Hershenson, H.M. (1961)UVand Visible Absorption Spectra (Vol. II, 1955-9).

Academic Press, New York.

Hershenson, H.M. (964)Infra Red Absorption Spectra (Vol. II, 1958-62).

Academic Press, New York.

Hershenson, H.M. (1966) UVand Visible Absorption Spectra(Vol. III, 1960-3).

Academic Press, New York.

Hostettmann,K. (ed.) (991)Methods in Plant Biochemistry,Vol. 6,Assays for Bioactivity,Academic Press, London.

Jones, R (980) inAn Introduction to Spectroscopy for Biochemists(ed. S.B. Brown), Academic Press, London.

Knights, B.A. (995)Phytochemistry4, 857.

Lang, L. (1959)Absorption Spectra,Academy of Science Press, Budapest.

Mabry, T.J. (969) inPerspectives in Phytochemistry(eds J.B. Harborne and T.

Swain), Academic Press, London, pp. 1-46.

Orians, C.M. (1995)f. Chem Ecol.,21, 1235.

Paris, R, Durand, M. and Bounet, J.L. (960)Ann. Pharm. Franc.,18, 769.

Peereboom, J.W.C (971) inComprehensive Analytical Chemistry(ed. CL. Wilson and D.W. Wilson) (Vol. IIC), Elsevier, Amsterdam, pp. 1-129.

Phillipson, J.D. (1982)Phytochemistry,21, 2441-56.

Poindexter, E.H. and Carpenter, RD. (962)Phytochemistry,1,215.

Putnam, A.R. and Tang, C.S. (eds) (986)The Science

of

Allelopathy,John Wiley, Chichester.

Sandford, K.J. and Heinz, D.E. (971)Phytochemistry,10, 1245.

Supplementary references 39

Shannon, J.S. and Letham, D.S.(1966)New Zealand]. Sci.,9,833.

Soltis, P.S., Soltis, D.E. and Doyle,

J.J.

(1992)Molecular Systematicsof Plants, Chapman and Hall, New York.

Stafford, A.M. and Pazole, c.J.(1997)inPhytochemical Diversity - A Sourceof New Industrial Products (ed. by S. Wrigley, M. Hayes,R. Thomas and E. Chrystalt Royal Society of Chemistry, Letchworth, Herts.

Tomas-Barberan, P.A.(1995)Phytochemical Analysis6,177.

Van Sumere, c.P. (1969) Revue des Fermentations et des Industries Alimentaires (Brussels),24,91-139.

Zenk, M.H., EI-Shagi, H., Arens, H., Stockigt, J., Weiler, E.W. and Dens, B.(1977) Plant Tissue Culture and its Biotechnological Application,Springer, Berlin.

2

Phenolic com.pounds

2.1 Introduction

2.2 Phenols and phenolic acids 2.3 Phenylpropanoids

2.4 Flavonoid pigments 2.5 Anthocyanins

2.6 Flavonols and flavones

2.7 Minor flavonoids, xanthones and stilbenes 2.8 Tannins

2.9 Quinone pigments 2.1 INTRODUCTION

40 42 49 60 66 74 83 90 96

The term phenolic compound embraces a wide range of plant substances which possess in common an aromatic ring bearing one or more hydroxyl substituents. Phenolic substances tend to be water-soluble, since they most frequently occur combined with sugar as glycosides and they are usually located in the cell vacuole. Among the natural phenolic com- pounds, of which several thousand structures are known, the flavonoids form the largest group but simple monocyclic phenols, phenylpropanoids and phenolic quinones all exist in considerable numbers. Several impor- tant groups of polymeric materials in plants - the lignins, melanins and tannins - are polyphenolic (see Fig. 2.1) and occasional phenolic units are encountered in proteins, alkaloids and among the terpenoids.

While the function of some classes of phenolic compound are well established (e.g. the lignins as structural material of the cell wall; the anthocyanins as flower pigments), the purpose of other classes is still a matter of speculation. Flavonols, for example, appear to be important in regulating control of growth in the pea plant (Galston, 1969) and their adverse effects on insect feeding (Isman and Duffey, 1981) have indicated that they may be natural resistance factors; neither of these functions, however, has yet been firmly established.

Introduction

OH

41

HO

HO

A structural unit of flavolan

HO

OH OH

HO

A structural unit of plant melanin

OH

A structural unit of lignin

Fig. 2.1 Partial structures for the phenolic polymers of plants.

To the plant biochemist, plant phenols can be a considerable nuisance, because of their ability to complex with protein by hydrogen bonding.

When plant cell constituents come together and the membranes are de- stroyed during isolation procedures, the phenols rapidly complex with protein and as a result, there is often inhibition of enzyme activity in crude plant extracts. On the other hand, phenols are themselves very susceptible to enzymic oxidation and phenolic material may be lost during isolation procedures, due to the action of specific 'phenolase' enzymes present in all plants. Extraction of the phenols from plants with boiling alcohol normally prevents enzymic oxidation occurring and this procedure should be adopted routinely.

The classic procedure for detecting simple phenols is by means of the intense green, purple, blue or black colours many of them giveinsolution when 1^% aqueous or alcoholic ferric chloride is added. This procedure,

42 Phenolic compounds

modified by using a fresh aqueous mixture of 1% ferric chloride and 1% potassium ferricyanide, is still used as a general means of detecting phenolic compounds on paper chromatograms. However, the majority of phenolic compounds (and especially the flavonoids) can be detected on chromatograms by their colours or fluorescences in UV light, the colours being intensified or changed by fuming the papers with ammonia vapour.

The phenolic pigments are visibly coloured and they are thus particularly easily monitored during their isolation and purification.

Phenolic compounds are all aromatic, so that they all show intense absorption in the UV region of the spectrum. In addition, phenolic com- pounds characteristically exhibit bathochromic shifts in their spectra in the presence of alkali. Spectral methods are, therefore, especially impor- tant for the identification and quantitative analysis of phenols.

Of the numerous texts devoted to the plant phenols, the reader is especially referred to the simple student treatisePlant Phenolics(Ribereau- Gayon, 1972) and to the more advanced reference volume of Harborne (1989). A critical review of the chromatography of phenolic compounds is that of Harborne (1992). An introduction to the analysis of phenolic plant metabolites has been provided by Waterman and Mole (1994).

2.2 PHENOLS AND PHENOLIC ACIDS