Chapter 14 Phytochemical variation within a species

As was indicated in Chapter 3, the species represents the unit of plant classification; it may constitute a homogeneous taxon of plants with little variation from one specimen to another or it may include various varieties or races which each have some distinctive feature(s). Thus with Datura stramonium four varieties are described: var. stramonium—white flowers, thorny capsules; var. inermis—white flowers, bald capsules; var. tatula—lilac flowers, thorny capsules and var. godronii—lilac flowers, bald capsules. Often such varieties represent single gene mutations (in the above case two independent genes are involved) and are morphologically recognizable. In other instances the mutation gives rise to a variant having a different secondary metabolite profile—not necessarily discernible in the morphological form; these are termed chemical races or chemodemes. The mutation may involve the presence or absence of a single component or, if acting at an early stage of the biosynthetic route, may involve a whole series of compounds.

Knowledge of the existence of such chemical races is not new and A. Tschirch (1856–1939) in his Handbuch der Pharmakognosie deals with the various ‘physiological forms’ of Vanilla planifolia, Cinchona spp., fennel, Piper nigrum and the balsam-containing trees. Also, 60 years ago A. R. Penfold and co-workers were describing chemical races of various Australian oil-producing species of Leptospermum, Boronia and Eucalyptus.

In addition to the above which involve submicroscopic point mutations associated with an alteration in the DNA chromosomal material, there are other genetic variations which may affect chemical constituents of the species. These include (a), the existence of polyploids in which chromosome sets become doubled, tripled, quadrupled etc., (b), the addition of one or a few chromosomes above the normal complement (extrachromosomal types), (c), gross structural changes to a chromosome, (d), artificially produced transgenic plants.

Two consequences of the above are, on the positive side, it provides the possibility for the selection and establishment of superior strains with respect to the chemical constituents as described in this chapter. The negative aspect concerns the arrival on the drug market of material which is low in active constituents. This, together with other factors, necessitates the need for strict quality control (Chapter 16) which is being vigorously addressed by various legislative bodies (Chapter 2). Transgenic crops present their own problems.

CHEMICAL RACES, CHEMOTYPES, CHEMODEMES

The plant kingdom has been subjected to an extensive, but not exhaustive, chemical investigation. Thousands of samples have been screened for substances of medicinal value or for suitable precursors of therapeutically active compounds. Many other plants have been studied chemically from the viewpoints of manural treatments, plant resistance and biosynthesis of active constituents. From such observations has emerged evidence for the existence of ‘chemical races’, ‘chemotypes’ or ‘chemodemes’. These are defined as chemically distinct populations within a species and have similar phenotypes but different genotypes and as such are identical in external appearance but differ in their chemical constituents.

Before the existence of a chemical race can be established, certain fundamental observations are necessary. A chemical analysis of a number of random samples of a particular species may show a variation between the samples but would be insufficient to demonstrate any genetical differences, since factors such as age, climate and soil can all exert profound effects on the result of the ultimate analysis. Samples of seed, or clones from different plants, must be raised together under uniform conditions, and to exclude hybrids, which do not breed true, cultivation for a number of generations is desirable. It may then be possible to demonstrate that differences occur in either the nature or quantity of a particular constituent and that these differences are of a hereditary nature.

Such observations necessitate numerous assays and precise horticultural work, to which must be added the difficulties of dealing with plants which may take years to mature. Furthermore, many of the more important vegetable drugs cannot be successfully cultivated in temperate climates, so that it is not surprising that the number of medicinal plants fully investigated under ideal conditions is still limited. However, with the world-wide increase in genetic studies on medicinal plants, progress is being made towards the isolation of those enzymes associated with the existence of specific chemical races; recently, for example, the cloning of an enzyme involved in ginkolide biosynthesis (K. SangMin, Phytochemistry, 2006, 67, 1435).

The clinical significance of chemical races is illustrated by Valerian; the plant normally contains both volatile oil and iridoid compounds, the latter with reported cytotoxic activity. As the sedative properties of the drug are ascribable to the valerenic acid and valerone constituents the cultivation of chemical races lacking the iridoids was introduced.

Fixed oils

Agriculturally, the cultivation of seed oil plants is second only in importance to that of cereals. Most of the fixed oil produced is used by the food industry but there are also important industrial and other, including pharmaceutical, uses. It is not surprising therefore that sustained breeding programmes for the improvement of yields and quality of oil have been in progress over many years. Normal rapeseed oil contains, as an acylglycerol, 20–40% of erucic acid, an acid having an extra long carbon chain (C₂₂) and one double bond. Its presence in quantity renders the oil unsuitable for edible purposes but varieties are now extensively grown which contain no erucic acid. The value of the crop has been further enhanced by coupling low erucic acid content with one giving low glucosinolates in the protein meal thus improving the animal feed properties. However, erucic acid is industrially important for the manufacture of lubricants, artificial fibres and plasticizers, so that varieties of rape developed for their high erucic acid content are also important agricultural crops.

The production of oil from sunflower seed has been improved by varieties that yield linoleic acid-enriched oil and which are more convenient for harvesting by having a large single flower head and no side-shoots. Groundnuts, the source of Arachis Oil BP, exist as various strains with different relative proportions of fatty acids.

Safflower constitutes an important oil-seed crop and its genetic variability has facilitated the breeding of varieties with widely differing oil constitutions. High oleic varieties are used for oil for human consumption and high linoleic varieties are important for oils used as industrial coatings and lubricants.

The above examples involve plants with a short life-span so that breeding by classical methods is a relatively rapid procedure. However, this is not so with plants such as the coconut palm, olive and cocoa so that in these cases modern techniques involving gene transfer would have an obvious advantage for the introduction of new or modified oil characteristics.

Cyanogenetic glycosides

A well-known chemical race in the cyanogenetic series is the almond. There are many varieties of Prunus communis showing different morphological forms, with and without amygdalin, but some varieties have similar characters and differ only in the presence or absence of the glycoside. Also in this group the clovers, especially Trifolium repens, have been extensively studied and Linaria has been shown by Dillemann to produce a chemical race by introgressive hybridization. L. striata contains cyanogenetic glycosides, and, crossed with the non-active L. vulgaris, gives rise to hybrids which, on repeated back-crossing with L. vulgaris, give some plants difficult to distinguish from L. vulgaris but which contain the cyanogenetic principles of L. striata.

Alkaloids

The Duboisia species form an important commercial source of the tropane alkaloids and have been extensively studied by Australian workers. With both D. myoporoides and D. leichhardtii, trees from natural stands in various locations were examined and their progeny were raised side by side in experimental plantations. The trees produce hyoscine, hyoscyamine, norhyoscyamine, tigloidine and valeroidine, and the proportion of any one alkaloid to total alkaloid may vary greatly. It was shown not only that seasonal and environmental factors are involved in this variation, but also that within a species there exists a wide range of alkaloid genotypes. Other varieties containing nicotine and nornicotine were also reported. Interspecific hybrids between the two species were studied and four hybrid clones were selected for possible exploitation as high alkaloid yielding strains. Thus, in this genus we have the possibility of two distinct types of chemical race—different alkaloid types within a species and different alkaloid types among hybrid phenotypes.

An example of the improvement of the morphine content of opium poppies by genealogical selection is furnished by the work of Lecat. The original seed gave capsules having an average morphine content of 0.385%. From this heterogeneous population were selected six individuals whose capsules analysed about 0.7% morphine. The seeds of these plants formed the heads of the lines cultivated in successive years, during which the best plants were collected and all those containing less than 0.7% morphine were rejected. The harvest of 1955 gave capsules with an average morphine content of 0.765%, thus doubling the original morphine content of the population. Such a method of breeding does not produce a race of plants surpassing individual morphine contents from the original heterogeneous population; it merely produces a homogeneous race of the alkaloid-rich plants.

Phillipson and colleagues reported at least three different chemical races of Papaver fugax and P. armeniacum in which either (1) 1-benzyltetrahydroquinoline, proaporphine, aporphine, (2) morphinane or (3) rhoeadine types are the major alkaloids; there are at least two different chemical strains of P. tauricola containing either the first or third types of the above. Three different isoquinoline alkaloid chemotypes of Thalictrum minus have been reported from Bulgaria. Papaver bracteatum is a species exhibiting races with respect to thebaine.

From Claviceps purpurea a number of races have been isolated containing different groups of ergot alkaloids and these have obvious implications for the commercial production of alkaloids.

One fodder crop in which the presence of alkaloids is undesirable is lupin seed. Ordinary wild forms are bitter and contain alkaloids of the lupinane series but over the years a number of sweet forms have been developed for commercial purposes in Europe. The strains depend for their low alkaloid content on the presence of a particular recessive gene. However, as a number of such genes exist, cross-fertilization between two different sweet strains will again give bitter progeny. To avoid this happening, considerable care is necessary in regions where different strains are grown side by side. Other plants for which there is evidence of alkaloid varieties include Ephedra distachya and the Lycopodium species.

Chemical races appear to be lacking in the pharmaceutically important indole alkaloid-containing genera Strychnos, Rauwolfia and Catharanthus.

Anthraquinones

The purgative anthraquinone drugs owe their activity to complex mixtures of the 1,8-dihydroxy derivatives of anthranols, their glycosides and free anthraquinones. The relative proportions of the constituents of the mixture, which greatly influence the pharmacological activity, depend not only on time of collection, age of plant, drying conditions and geographical source, but also on genetical factors. In a programme involving Rheum palmatum, van Os produced races varying in their rhein/chrysophanol ratio and other hereditary strains for high- and low-yielding total anthraquinones. The analysis of individual Cassia angustifolia plants has indicated that selection of individuals for high sennoside B-yielding strains is a possibility.

Cardiac glycosides

With Digitalis purpurea the property of high glycoside content is hereditary. The proportion of glycosides derived from digitoxin and gitoxin is also very different in plants of different origin and remains so during subsequent cultivation under standardized conditions. The strains were distinguished chemically as digipurpurin, strospeside and digitoxin types. It now remains to prove that these characters are independent of the phenotype (i.e. that they are not inseparably associated with other characters of the parent plant). One race, ‘Cambridge’, which is relatively rich in digitoxin, is easy to distinguish; the other digitoxin race found in the Vosges differs little from the other selections. Variation in the proportion and quantity of glycosides in D. lanata has also been noted in mixed populations and, by the selfing of selected individuals, strains rich in a particular glycoside have been produced, the inherited character being strongly developed. Valuable physiological forms could thus be produced and Ligeti has recommended that these strains be designated by such names as ‘D. lanata Ehrh, chemo-varieties A and C’, depending on the respective predominance of lanatosides A and C. It appears that with such in-bred lines continuous selection is still required to prevent reversion to the normal character level of the species.

The great value of the radioimmunoassay (q.v.) for the rapid selection of high-yielding strains of Digitalis lanata has been demonstrated by Weiler and Westekemper. After two selection steps involving the analysis of over 10 000 individual plants, the average digoxin content of the plants could be raised two to threefold and several strains with average digoxin concentrations in the leaf of 0.6% were isolated. Individual plants were found with 0.9–1.0% digoxin content. As is usual with this type of selection, no plants better than the few best of the original selection were obtained.

Following intensive chemical investigation of the genus Strophanthus, Reichstein and his colleagues differentiated four chemical varieties of the polymorphous S. sarmentosus from different geographical sources. They are sarverogenin-, sarmentogenin- and sarmutogenin-producing types with glycosides of these, and a fourth form which has a low glycosidal content (Fig. 14.1). Although the locality of growth may produce quantitative differences in the constituents of the various races, the overall type is genetically controlled. Similar variation may exist among those plants that yield steroidal saponins, several thousand of which, from different localities have been screened for their sapogenin content.

Fig. 14.1 Steroidal constituents of chemical races of Strophanthus and Withania.

Withanolides

The plant Withania somnifera (Solanaceae), in addition to producing alkaloids, contains steroidal lactones. Investigations over the years, carried out on various sources of plant material, and concerning the non-alkaloidal constituents, had given differing results, which were explained by the work of Abraham et al. (1968) on Israeli plants. Three chemotypes were discovered among 24 populations of W. somnifera collected in various parts of the country. Chemotype I contained predominantly withaferin A (0.2% of the dry weight), which is the principle responsible for the plant’s bacteriostatic and antitumor properties. Chemotype II contains a compound of similar structure, and chemotype III a mixture of related compounds comprising a group of steroidal lactones—the withanolides (Fig. 14.1). The only morphological difference observed between the chemotypes was a difference in flowering time (12 days early) for chemotype III. Since then, other chemotypes of W. somnifera have been reported from India and South Africa.

Steroidal alkaloids

Solanum spp. (Solanaceae) contain steroidal glycosidic alkaloids some of which have been investigated as potential intermediates in corticosteroid synthesis. In S. dulcamara (the woody nightshade) Sander has distinguished a west European tomatidenol group and an east European soladulcidine-solasodine group (Table 14.1). Although polyploid forms do occur in the genus, these chemical varieties all had 2n = 24 chromosomes and were genetically stable. Subsequent work demonstrated that the different chemotypes can occur in the same locality. With the commercial species, about 3500 individual 6-month-old Solanum laciniatum and S. aviculare were analysed by radioimmunoassay (q.v.) and found to contain average leaf concentrations of 1.6–1.7% solasodine; from these a few individuals were selected for future breeding work.

Table 14.1 Chemical races of Solanum dulcamara.

Aglycone	Sugars	Glycoside
Soladulicidine (25D)	Galatose (1 mol)	Soladulcidine-tetraoside
	Glucose (2 mol)
	Xylose (1 mol)
Solasodine (25D)	Galactose (1 mol)
	Glucose (1 mol)	Solasonine
	Rhamnose (1 mol)
Δ5-Tomatidenol (25L)	Galactose (1 mol)
	Glucose (1 mol)	α-Solamarine
	Rhamnose (1 mol)

Essential oils

The biochemical group of plants offering evidence of the largest number of chemical races is that containing volatile oils. Here, again, many of the differences within a species which have been reported may be due to factors other than genetic ones. Australia offers unique opportunities for the investigation of this problem as the flora is rich in oil-bearing plants. As an example, the common form of Eucalyptus dives contains piperitone as the chief constituent of the oil, but other races are known which produce principally phellandrene or cineole, while still others produce oils intermediate in composition.

There are three races of Melaleuca bracteata producing volatile oils containing chiefly methyl eugenol, methyl iso-eugenol and elemicin, respectively. They can be transformed one into the other by simple chemical steps, which suggests that one of the compounds (e.g. methyl eugenol) occurs in all the races. With the appropriate enzyme, methyl iso-eugenol could be formed by a simple double-bond shift and elemicin by the addition of a hydroxyl group and subsequent methylation. Because of this a one-gene-one-enzyme hypothesis suggests itself and it would be possible to test this by breeding experiments.

In the American turpentine industry, breeding investigations have shown that oleoresin yields in pines are inherited. Two chemotypes differing in their Δ³-carene content of the oil have been recognized for Pinus sylvestris.

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