IGER INNOVATIONS 2007

N a t u r a l p r o d u c t s a n df e e d s t o c k s f r o mp l a n t sPhil Morris 1 and Robert Nash21IGER - Aberys twyth2Vastox Wales Ltd , Plas Gogerddan, Aberys twyth

Novel bioactive compounds from plants 30

Sustainable production of the natural

product galanthamine from Welsh daffodils 31

Utilisation of polymeric fructans from IGER

varieties of high sugar grasses 33

Isoflavonoids as phytoestrogens from IGER

varieties of red clover (Trifolium pratense L.) 35

lants contain a wide variety of both primary andsecondary metabolites which provide us with animportant source of pharmaceuticals andnutraceuticals for the treatment of disease andillnesses in both human and animal health, as well asproviding carbon skeletons for chemicalmanufacture.

In 2006, the global market for plant-derived drugswas estimated to be valued at over £15 billion andaccounted for 50% of prescribed and over-the-counter sales. The anti-cancer drug Taxol® is a goodexample of a major drug derived from plants andnow produced commercially by modification of aprecursor isolated from yew trees.

The global market for food supplements(nutraceuticals), many of which are derived fromplants, is worth ca. £100 billion, exclusive of herbaland traditional medicines sales. Indications are thatthe nutraceutical market will continue to grow.

Novel bioactive compounds from plantsThe pharmaceutical industry has always reliedheavily on leads from natural products to developnew drugs. Despite constant changes in approach inhow best to evaluate natural products, newcommercial preparations continue to come through,including a novel potential anti-cancer drug derivedfrom casuarine which was first discovered at IGER.

Casuarine and derivatives (e.g., VOX 14400 beingdeveloped by Summit plc) is a very water-solublenon-toxic alkaloid that primes the immune system totackle cancer cells in a safe way, potentially withvery wide therapeutic applications. They highlight a

new range of subtle and targeted approaches totherapies and the importance of water-solublenatural products as potential drugs. Despite the factthat most herbal medicines were traditionallyprepared in water, most analyses of such products donot detect the major water-soluble components andindeed many formulations specifically excludethem. This bias in analysis and commercialpreparation may well be the reason that the activecomponents of most major herbal medicines remainunidentified.

Casuarine

The markets for natural remedies, herbal productsand functional foods have been identified as one ofthe largest growth sectors in the world economy.This is in response to the increasing interest anddemand for “natural” remedies and supplements toprotect against disease, treat illness and generallyimprove health.

There is an ever-increasing myriad of productsavailable to satisfy this demand, ranging fromtraditional Chinese remedies to high quality dietarysupplements sold by major global corporations.

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Natural products and feeds tocksfrom plants

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PPhil Morris and Robert Nash

However, with the majority of plant-basedmaterials, claims are limited to association with thetraditional use of the original plant material.Attempts to perform formal clinical and scientifictrials on the efficacy of these products frequentlyfail. This is not surprising for a number of reasons:

• Plants show considerable variation in chemicalcontent due to natural variability within species,climate, soil conditions, microbial infection,season of harvest and the parts of the plant used.

• Even highly standardised production andextraction techniques may not be sufficient tocontrol variability between batches when theactive components are not identified.

• Many extraction methods (such as those usingorganic solvents and supercritical CO2/fluidextraction) commonly exclude the polar (water-soluble) compounds. Potentially, keycomponents of medicinal value may therefore bediscarded.

• Considerable research to identify the activeingredients of herbal preparations has beenlargely fruitless, probably because the methodstypically used focus on analysing thecomponents that are not very water-soluble.

Whilst global corporations are attracted to thismarket, their interest is impeded by this lack ofeffective competencies to isolate, identify andcharacterise the biologically active components.Companies such as Summit plc, based at IGER,(and working in collaboration with IGER) have theknowledge, competencies, know-how andexperience to unlock much of the potential of thismarket and to help secure protection for thoseproprietary components identified as havingsignificant commercial potential.

(Contact: Rob Nash)email: Robert.Nash@vastox.com

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Sustainable production of the naturalproduct galanthamine from Welsh daffodilsAlzheimer’s disease is a common form of dementiawith an estimated 15 million sufferers worldwide,and this number is expected to treble by 2050.Alzheimer sufferers exhibit a gradual decline incognitive faculties with loss of memory, judgmentand the ability to comprehend the externalenvironment. They can now be treated with a class ofdrug called acetylcholinesterase inhibitors whichslow the progression of the disease and alleviatemany of the symptoms. One such compound isgalanthamine, which is an alkaloid found in plantssuch as daffodils and snowdrops.

Fig. 1. Biosynthetic pathway of galanthamine and relatedalkaloids in daffodils.

Galanthamine is one of a range of alkaloids whichform part of a branched biosynthetic pathway inplants (Fig. 1). They are present in the leaves andbulbs of daffodils at varying levels in differentvarieties, where they are considered to protect theplant from herbivores and microbial infection. In thepast, new varieties have been bred mainly for theirfloral characteristics, with the resulting varietiesbeing propagated vegetatively. This has given rise toa very narrow genetic base within the species, and awide, genotypically fixed variation both in thealkaloid profile and in galanthamine content, to the

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point where different varieties can be characterisedby their alkaloid spectrum (Fig. 2).

Barriers to the development and exploitation of anew supply chain for galanthamine are encounteredat both scientific and logistical levels. From ascientific perspective, the levels and spectrum of thealkaloids present in daffodil bulbs vary greatly beingunder genetic, environmental and developmentalcontrol. Total alkaloid levels in bulbs are generally inthe range of 1 to 2% dry weight, but galanthaminelevels can vary from zero in some varieties up to0.2% dry weight in varieties such as Carlton, notablywhen grown under optimum conditions andharvested at the optimum time during the growthseason (Fig. 3).

With funding from Defra, we have been able toquantify galanthamine levels in material from largefield trials of a number of commercial daffodilcultivars (Fig. 4) grown under different cultivationregimens at several sites in Wales. These data havebeen incorporated into a sustainable supply chain

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model which will allow a reliably quantitativeapproach to be taken to the agronomic,environmental and economic feasibility of creating asupply chain for the production of galanthaminefrom daffodils grown in Wales.

(Contact Phil Morris or Mike Theodorou)email: phillip.morris@bbsrc.ac.ukmike.theodorou@bbsrc.ac.uk

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Fig 2. Spectrum of alkaloids in daffodil bulbs var. Carlton.

Fig. 3. Time course of galanthamine accumulation in daffodil bulbs.

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Utilisation of polymeric fructans from IGERvarieties of high sugar grassesFructans act as storage polymers for carbon,performing much the same function in temperateforage grasses as starch does in many other plants.Fructans may be stored in the long or short term, andcan reach very high concentrations (up to ca. 40%dry matter in the case of IGER high sugar grassvarieties).

Chemically, the fructans in plants occur as branchedor linear poly-fructose molecules, linked either (β-2,1) as inulins or (β-2,6) as levens. They aresynthesised de novo from sucrose and are polymersof D-fructose, carrying a D-glucosyl residue at theend, or in the middle, of the chain. They constitute aseries of homologous oligosaccharides and aredefined as any compound where one or morefructosyl-fructose linkages constitute a majority ofthe links between the constituent entities.

Different forage grass species vary in their fructanmolecule size profiles. Some, like cocksfoot andtimothy, contain large fructan polymers, whileryegrasses (Lolium species) accumulate both smallerfructan oligosaccharides and high-molecular weightmolecules (Fig. 5).

Fig. 4. Different daffodil varieties being trialled in the Welsh hills (Photograph courtesy of Alzeim Ltd).

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Fig. 5. Chromatography of fructans of Lolium grass showingresolution of abundant species between DP =c.105-60 as ahomologous series with polymers up to DP c. 90 (14.6kDa).

Perennial ryegrass has been shown to containoligosaccharides from three structural series, theinulin series, the inulin neoseries (Fig. 6) and thelevan series.

At IGER we have an extensive grass breedingprogramme and have developed high sugar grassvarieties of Lolium perenne (e.g., AberDart) thataccumulate high levels of fructan polymers, as well asgaining extensive knowledge in the physiology,biochemistry and molecular biology of fructanmetabolism.

High molecular weight inulin-type fructans are knownto positively affect gastrointestinal performancethrough their probiotic properties. This type of inulincan only be partially digested by humans and, whenconsumed, the undigested portion serves as food for“friendly” bacteria such as Bifidobacteria andLactobacillus species. Clinical studies have shown thatadministering inulin can increase the number of thesefriendly bacteria in the colon, while simultaneouslyreducing the population of harmful bacteria. Otherreported benefits of inulin include an increasedproduction of beneficial short-chain fatty acids such asbutyrate, better absorption of calcium and magnesium,and an enhanced elimination of toxins. Clinical studieshave also shown an anti-carcinogenic effect for thesefructan probiotics.In comparison, the biological activity and probioticperformance of low molecular weight oligomers and

individual polymers with different degrees ofpolymerisation have not been extensively investigated.Such compounds may well act differentially and thelack of documented research on their activities may bedue to the difficulty in obtaining sufficient quantities ofthe purified oligomers from conventional sources offructans, which generally consist of a narrow range ofvery high molecular weights.

With Lolium and timothy, however, there is theopportunity for isolation of a wider range of fructanoligomers, together with polymers of differentstructures, in sufficient quantities for biological testing.This can be achieved by the manipulation of fructanaccumulation in detached leaves, or by isolation andfractionation of fructans from species whichaccumulate predominantly oligomeric forms.Alternatively, the use of endo- or exo-fructan hydrolaseenzymes to shift the molecular weight profile to lowerdegrees of polymerisation is a possibility. Suchenzymes have been isolated from fructan-degradingmicroorganisms which naturally ferment fructans in therumen and in silage, and these are currently beingevaluated for their ability to degrade polymeric fructansto oligomers.

The functionality of difructose anhydride (DFA) makesthis fructose dimer a potentially useful building blockfor biodegradable non-ionic detergents. Acidic catalysisof DFA in the presence of fatty alcohols leads to theformation of alkylfructosides. These compounds areanalogous to the widely used alkylpolyglucoside (APG)surfactants and can be used as washing agents. DFA cancurrently be produced from high molecular weightinulins found in chicory, artichoke or dahlia, but a lowprice production process based on grass fructansfermented with plant or bacterial enzyme systems couldefficiently generate DFA and strongly influence marketpotential.

(Contact Phil Morris, Joe Gallagher orAndy Cairns)email: phillip.morris@bbsrc.ac.ukjoe.gallagher@bbsrc.ac.ukandy.cairns@bbsrc.ac.uk

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Fig. 6. Example of thechemical structure of anoligomeric inulin groupfructan [material thathas mostly or exclusivelythe (2-1) fructosyl-fructose linkage].

Isoflavonoids as phytoestrogens from IGERvarieties of red clover (Trifolium pratense L.)Isoflavonoids are a class of phenolic secondarymetabolites which are particularly prevalent inlegumes, where they are widely distributed andfunction as preformed or inducible antimicrobial orinsecticidal compounds. They are derived fromflavanones such as naringenin (ubiquitously presentin plants) by an unusual ring migration (Fig. 7) andshow a range of biological properties.

The three most important properties are probably theestrogenic activities of simple isoflavones (such asformononetin and genistein) and coumestans, theantifungal and antibacterial properties of theisoflavonoid phytoalexins (e.g., medicarpin andmaackiain) in clovers (Fig. 7) and the insecticidalproperties of the rotenoids. Pterocarpan-typephytoalexins such as medicarpin and constitutiveisoflavone malonyl glycosides are typical of theisoflavonoids from clovers.

Isoflavonoids exhibit estrogenic, antioxidant andanticancer activities, leading to them becomingpopular as dietary supplements (Fig. 8). They mayalso possess other health-promoting activities,including chemoprevention of osteoporosis and

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Fig. 7. Chemical structures and biosynthetic pathway ofcommon isoflavonoids of red clover.

phillip.morris@bbsrc.ac.ukRobert.Nash@vastox.com

prevention of other postmenopausal disorders, aswell as guarding against cardiovascular disease.There are many products derived from red clover onthe market which claim to alleviate the symptoms ofmenopause. Sheep fed red clover diets high inisoflavonoids are known to show decreasedfecundity but increased live weight gain.

Major sources of isoflavonoids for humans arecurrently derived from the seeds of soybean(daidzein and genistein) and chickpea (biochanin A).Red clover leaves, stems and roots contain very highlevels of these isoflavonoids as malonyl glucosides.High isoflavonoid red clover varieties bred at IGER,such as Norseman and Sabtoron, may therefore offeropportunities for exploitation by UK agriculture forincreased economic production of these compounds.

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Fig. 8. Levels of different isoflavonoids in different tissues of twohigh yielding IGER-bred varieties of red clover.

(Contact: Phil Morris or Mike Abberton)email: phillip.morris@bbsrc.ac.ukmichael.abberton@bbsrc.ac.uk