WO2017118760A1

WO2017118760A1 - Phytochemical recovery from plants

Info

Publication number: WO2017118760A1
Application number: PCT/EP2017/050333
Authority: WO
Inventors: Elliott James PRICE; Paul David Fraser
Original assignee: Royal Holloway And Bedford New College
Priority date: 2016-01-08
Filing date: 2017-01-09
Publication date: 2017-07-13
Also published as: GB201810601D0; GB201600393D0; ZA201805183B; GB2560853A; GB2560853B; CN108601809A; AU2017205705A1; BR112018013754A2

Abstract

Use of plant parts of the family Dioscoreaceae in the production of shikimate, methods of extracting shikimate therefrom and methods the production of shikimate therefor.

Description

PHYTOCHEMICAL RECOVERY FROM PLANTS

Introduction

The present invention relates to the recovery of chemicals of interest from plants of the genus Dioscorea L . , typically to the recovery of chemicals in plants of the family Dioscoreaceae .

Background to the Invention

Certain plants of the Dioscoreaceae family are used extensively by man. The tubers or rhizomes of certain species may be used in various ways, such as food sources or in herbal medicines. Plants of the Dioscoreaceae are also known as 'yams' and many are cultivated by man for food. Examples of the major cultivated species of the Dioscoreaceae include D. rotundata ( 'white yam' ) , D. cayensis ( 'yellow yam' ) , D. alata (also known as 'purple yam' ) , D. opposita (also known as D. batatas or 'Chinese yam'), D. bulbifera ('air potato'), D. esculenta ( 'lesser yam' ) , D. dumetorum ( 'bitter yam' ) and D. trifida ( 'cush-cush yam' ) .

In Chinese medicine, rhizomes of certain yam species, such as D. opposita, are used for the extraction of chemicals from the tuber. Chemicals that are extracted from tubers include ones that can be used as steroid hormone intermediates, for example, diosgenin and/or its variants, which may then be converted into steroid hormones via synthetic chemical means. The tubers of other yam species such as D.hypoglauca (Chinese name being 'bixie' ) , D. tokoro, and D. septemloba are also used in Chinese herbal medicine for the production of decoctions or extracts which contain compounds of alleged pharmaceutical use.

In African medicine, the rhizomes of D. sylvatica (primarily known as Elephant's Foot Yam) have been harvested extensively for their use in producing diosgenin, and latterly are now grown for sale as ornamental plants. D. sylvatica grows wild over much of South Africa, Zimbabwe and Zambia, and is known by various synonyms such as Testudinaria sylvatica, D. hederifolia, D. junodii, D. sylvatica subs. Lydenbergensis , D. montana , D. brevipes . D. marlothii , Testudinaria multiflora , Testudinaria paniculata, D. rehmannii, Testudinaria rehmannii , ramus sylvestris , Testudinaria glaucescens and various varieties thereof. For the purposes of the present invention, the accepted scientific name, 'D. sylvatica' shall be used throughout from here on. A further member of the Dioscoreaceae, D. elephantipes (also known as 'Turtleback' , 'Tortoise Plant', 'Tortoise Back Plant' and 'Hottentot's Bread' ) is used in African medicine where its rhizomes are also used in producing diosgenin and as a food source (hence the trivial name 'Hottentot's Bread') in Southern Africa. This species is amenable to cultivation and is found in much of southern Africa where it is also known by various synonyms such as D. testudinaria , Rhizemys elephantipes , Tamus elephantipes, Testudinaria elephantipes , D. elephantopus, D. montana, Rhizemys montana , Testudinaria elephantipes f. montana, and Testudinaria montana. For the purposes of the present invention, the accepted scientific name, 'D. elephantipes', shall be used throughout from here on.

Gao et al . 2001 describe the use of tubers of D. bulbifera to treat various conditions such as sore throats, cancers, piles, syphilis, ulcers and diabetes. The authors carried out a chemical analysis of the dried tubers and found, amongst a number of other compounds, shikimic acid was present in unknown quantities.

Hence, it is the tuber of certain Dioscorea species which is used as a food source and/or a source of chemical compounds. It is the tuber of species of interest that may also be used to obtain extracts of chemical compounds which are then either converted into pharmaceuticals through chemical synthesis or are used in the form of decoctions and/or pastes in traditional medicines.

Certain chemical compounds of use in the pharmaceuticals industry have been sourced from plants of other species.

L-shikimate, an intermediate in oseltamivir manufacture, has traditionally been sourced from Illicium verum (also known as 'star anise') in China. Oseltamivir is used in flu remedies and goes under the trade mark of 'tamiflu' (Roche) . Oseltamivir is on the list of the World Health Organisation's List of Essential Medicines and is stockpiled as drug of first resort in the event of an outbreak of H5N1 influenza virus. A problem with the use of star anise as a source of shikimate for oseltamivir production is that I. verum is grown only in southwest China and a small area of Vietnam and so alternative sources for the supply of shikimate are needed if supply is to meet world demand.

JP 2003/012470 describes the extraction of medicinal compounds, including shikimic acid, from the plant Illicium anisatum (also known as 'Japanese star anise' ) . The extracted shikimic acid is said to be useful as a hair-restoring cosmetic.

CN102826994 similarly refers to a shikimic acid extraction method carried out on star anise plants, while WO 2011/159144 describes a method carried out on palm-based materials and US 2007/161818 describes a method carried out on sweetgum, pine and cedar plant tissues .

Nevertheless, Shikimic acid remains almost exclusively extracted from the Illicium species star anise.

While shikimate and ultimately oseltamivir can be manufactured via organic chemical pathways, or indeed, using methods employing biotechnology e.g. production in transgenic organisms such as recombinant E. coli, it remains expensive and/or challenging to render shikimate readily available, for example, for oseltamivir production and stockpiling worldwide.

There thus exists a need to provide further sources of shikimate for pharmaceutical production, particularly wherein shikimate extraction need not compete with another known use of the source.

For the purposes of the present invention, the skilled addressee will appreciate that the term 'shikimate' is used to describe the recovery of 'shikimate', also referred to as 'shikimic acid' from plant sources from the family Dioscorea, as described herein. The terms 'shikimate' and 'shikimic acid' are used interchangeably, and have the same meaning unless context demands otherwise.

Summary of Invention According to one aspect of the invention there is provided use of plant parts of the family Dioscoreaceae in the production of shikimate, wherein the plants are selected from D. elephantipes, D. sylvatica , D. villosa, D. communis, D. caucasia , D. nipponica , D. deltoidea, D. tokoro, D. preussi, D. alata, D. altissima, D. sansibarensis, D. bulbifera, D. antaly, D. praehensilis and D. minutiflora .

Typically, plant parts of species of the Dioscorea of use in the production of shikimate have been found to be aerial parts, preferably selected from stems and leaves, and most preferably are selected from leaves and petioles of certain species of the Dioscorea. Species of the Dioscorea of use in the invention may be selected from D. elephantipes, D. sylvatica , D. villosa, D. communis , D. caucasia , D. bulbifera , D. antaly, D. nipponica , D. deltoidea , D. tokoro, D. preussi and D. alata. It has been found that the level of shikimate obtained from leaves and petioles of the species D. elephantipes, D. sylvatica , D. villosa, D. communis , D. caucasia , D. bulbifera , D. antaly, D. nipponica , D. deltoidea , D. tokoro, D. preussi and D. alata, wherein shikimate is found in at least the leaves and petioles, on a dry weight basis may be in the range of from about 5% w/w to about 9% w/w, depending on species.

For certain species, those that are selected from D. elephantipes, D. sylvatica , D. villosa, and D. communis the level of recovery of shikimate from at least leaves and petioles on a dry weight basis has been found to be from 5% w/w to about 9% w/w. For other species of the Dioscorea, the level of recovery of shikimate has been found to be up to about 5% w/w on a dry weight basis. Examples of species of the Dioscorea from which shikimate has been recovered at a level up to 5% w/w on a dry weight basis include D. bulbifera , D. antaly, D. caucasia, D. nipponica, D. tokoro, D. deltoidea, D. preussi, D. alata, and D. minutiflora.

The skilled addressee will appreciate that preferably the shikimate form that is recovered is L-shikimate.

Additionally, the skilled person will be aware that a small number of Dioscorea species grow over-ground structures that are sometimes referred to as bulbs or "aerial tubers". These species include D. bulbifera and D. sansibarensis . Nevertheless, these structures are generally understood by the skilled person as not being included in the definition of "aerial plant parts".

For the avoidance of doubt, any reference made herein, below or above, to aerial parts of plants excludes tubers of any kind. Aerial plant parts typically include leaves, stems and petioles. Aerial plant parts exclude the bulbs / "aerial tubers" of D. bulbifera and D . sansibarensis . Additionally, in certain embodiments of the invention, shikimate may be extracted from the listed Dioscorea other than D . bulbifera .

A particular advantage of the invention is that a new source of shikimate has been made available, i.e. the aerial parts of certain species of Dioscorea named above and below. Unlike the sources noted in the prior art, Dioscorea is grown all over the world. Currently, the main source of shikimate is star anise which is endangered and only grown in certain parts of China and Vietnam. The yields of shikimate found in Dioscorea aerial parts are generally equivalent to, and in some cases higher than, those found in star anise.

A further advantage of using the aerial parts of Dioscorea is that the plant can be kept alive throughout successive harvests of the aerial parts. The tuber can thus be retained for use as a food crop.

In cases wherein the Dioscorea tuber is annual, the leaves and other aerial plant parts can be harvested numerous times throughout the year, before and after flowering, providing the tuber itself remains. In cases wherein the tuber is perennial, the aerial plant parts can be harvested even after the tuber is harvested.

Traditionally, biomass left behind after tuber harvest of Dioscorea is disposed of. Some sources calculate this waste to be approximately 6 million tonnes a year worldwide. This compares to approximately 400,000 tonnes for star anise production. The invention therefore provides a source of shikimate that is normally considered to be waste material, allowing the plant to be harvested for both its food (tuber) and shikimate (aerial plant parts) , whilst minimising the costs associated with waste disposal. In a further aspect of the invention, there is provided a method of extracting shikimate from plants of the family Dioscoreaceae , the plants being selected from D. elephantipes , D. sylvatica , D. villosa, D. communis , D. caucasia, D. nipponica , D. deltoidea , D. tokoro , D. preussi, D. alata , D. altissima, D. sansibarensis, D. bulbifera, D. antaly, D. praehensilis and D. minutifloraD, wherein the process comprises the steps of:

i) freeze-drying aerial parts of the plants;

ii) extracting shikimate with solvent, wherein the solvent is selected from organic alcohols and hot water;

iii) partitioning the product of ii) in the aqueous phase, preferably using an organic solvent;

iv) purifying shikimate from the aqueous phase.

Preferably, the aerial parts of i) are selected from leaves and petioles.

Preferably, the organic alcohol solvent of ii) is selected from short chain (Ci-Cg) alcohols, such as methanol, ethanol, propan-l-ol, propan- 2-ol, 1-butanol, 2-butanol, 2-methyl-l-propanol, and 2-methyl-2- propanol, pentan-l-ol, pentan-2-ol, pentan-3-ol, 3-methylbutan-l-ol, 2-methylbutan-l-ol, 2 , 2-Dimethylpropan-l-ol , 3-Methylbutan-2-ol , 2- Methylbutan-2-ol, and water. More preferably, the alcohol solvent of ii) is selected from methanol, ethanol, iso-propanol , and iso-butanol, and most preferably is selected from methanol and ethanol or a mixture thereof . Where the solvent of ii) is water, it is preferably warmed water at a temperature of at least 80°C, preferably at least 90°C, and most preferably at 100°C or water at any temperature there between.

In a preferred embodiment, the solvent of ii) is selected from methanol, ethanol, and hot water at 100°C. The solvent of step iii) is selected from short chain organic solvents different to the short chain organic solvents of step ii) . Preferably, the solvent of step iii) is a halogenated short chain polar compound, preferably selected from haloalkanes, and most preferably from haloalkanes possessing one, two or three halogen groups on a methyl base, for example, chloroform and dichloromethane . Most preferably, the solvent of step iii) is chloroform.

Shikimate is purified from the resultant aqueous phase of step iii) in a C18 reversed phase column, preferably a C18-reversed phase column run at 30°C, running an isocratic gradient of water at lml/min.

In a further aspect of the invention there is provided a method for the production of shikimate comprising extracting shikimate from the Dioscorea of the invention, preferably from their aerial plant parts as elsewhere defined herein. Suitably the method comprises the steps of

i) harvesting the leaves and stems of plants of the family Dioscoreaceae, wherein the plants are selected from D. elephantipes, D. sylvatica , D. villosa, D. communis, D. caucasia , D. nipponica , D. deltoidea , D. tokoro, D. preussi , D. alata, D. altissima, D. sansibarensis, D. bulbifera, D. antaly, D. praehensilis and D. minutiflora; and

ii) extracting shikimate from the harvested leaves and stems.

Plants of the family Dioscoreaceae that are considered useful for shikimate production include crop plants, that is to say, species amenable to cultivation, and native species of plants wherein the level of shikimate present in the leaves and stems thereof typically lies in the range from at least 1% dry weight to about 10% dry weight, preferably lies within the range of about 2% dry weight to about 9% dry weight, and most preferably lies within the range of about 5% dry weight to about 9% dry weight. Such plants may be selected from D. elephantipes, D. sylvatica , D. villosa, D. communis, D. caucasia , D. nipponica, D. deltoidea , D. tokoro, D. preussi, D. alata, D. altissima, D. sansibarensis, D. bulbifera, D. antaly, D. praehensilis and D. minutiflora.

Preferably, plants of use in a method for the production of shikimate are selected from D. elephantipes, D. sylvatica , D. villosa, D. communis, D. caucasia, D. bulbifera , D. antaly, D. nipponica , D. deltoidea , D. tokoro, D. preussi and D. alata. Preferably still, plants of the family Dioscoreaceae of use in the production of shikimate are selected from crop plant species as defined herein. Most preferably, plants of use in the invention are selected from D. elephantipes and D. sylvatica .

There now follow examples relating to the invention. It is to be understood that the examples are not to be construed as limiting the invention in any way.

Examples

Plant material

Dioscorea material was sourced from the Royal Botanic Gardens (RBG) , Kew Living Collection (http://epic.kew.org/index.htm) (Table 1). Youngest mature leaf and petiole material was sampled. Materials were cut from the vine and quenched in liquid nitrogen immediately before samples were lyophilised, homogenised and stored at -80°C until further processing.

Additional dried leaf material of D. elephantipes (accessions 19900643A & 19280228C) and D. sylvatica (accession 19803437A) were sourced from the RBG, Edinburgh Living Collections (htt : //elmer . rbge . org . uk/bgbase/livcol/bgbaselivcol . php) .

Table 1 . Dioscorea ( ^λϋ. species') accessions from The Kew Living Collection

D . Species Accession Native habitat

elephan tipes (1) 2007· -447 S South Africa

elephantipes (2) 2012- -54 SW South Africa

sylvatica (1) 1963· -26704 South Africa

sylva tica (2) 1963· -26705 South Africa

an taly 1998· -523 Madagascar

bulbifera (1 ) 1998- -533 Africa & Asia

sansibarensis (1) 1998- -525 Tropical Africa

altissima 2005- -1233 South America

rotunda ta 1976· -1475 West Africa

praehensilis I960· -1002 Tropical West Africa & East & South preussi 1968- -57006 Tropical Africa

minutiflora I960- -1001 Tropical Africa / Madagascar

bulbifera (2) 1987- -1993 Africa & Asia

sansibarensis (2) 1598· -543 Tropical Africa South Mexico through Central

composi ta (1) 1969-11715 America

South Mexico through Central

composi ta (2) 1978-1830 America

alata 1982-1316 Asia

elephantipes (3) 2012-54 South West Africa

elephan tipes (4) 2001-2252 South West Africa

Extraction of Shikimic acid (3 , 4 , 5-trihydroxy-l-cyclo exene-l- carboxylic acid) from yam species D. elephantipes

Foliage material from D. elephantipes is freeze-dried following conventional procedures as outlined above. lOmg samples of freeze- dried foliage are placed in separate micro-centrifuge tubes. 400μ1 of HPLC grade methanol is added to each sample before vortexing for 5 seconds, then 400μ1 of HPLC grade H₂0 is added to each sample and the mixture is vortexed for a further 5 seconds . The samples are then rotated (mixed) at 40 rpm for 1 hour. All procedures are carried out at room temperature and under ambient light conditions.

After mixing, 800μ1 of analytical grade chloroform (CHCI3) is added to each sample followed by vortexing for 5 seconds, followed by centrifugation for 5 minutes at 14,000 rpm for partition phasing. Alternatively, after addition of chloroform, partition phasing can be performed naturally by leaving the samples to stand for about 10 minutes. The aqueous polar phase containing shikimate is removed (800μ1) .

Alternatively, a hot water extraction method modified from the method described by Ohira, H., Torii, N., Aida, T. M., Watanabe, . , & Smith, R. L. (2009) . Rapid separation of shikimic acid from Chinese star anise (Illicium verum Hook, f.) with hot water extraction. Separation and Purification Technology, 69(1), 102-108. doi: 10.1016/j . seppur .2009.07.005 may be employed:

In glass tubes, 8 OOmicroliter boiling water is added to lOmg of powdered freeze-dried yam foliage material and vortexed for 5 seconds. The glass tubes are incubated at 100°C for 10 minutes, vortexed for 5 seconds, then cooled on ice. 8 OOmicroliter chloroform is added and the vials vortexed for 5 seconds. Partition phases by centri fugation for 5 minutes at 14,000 rpm (or leave to stand for -10 mins and naturally partition) . Remove 800μ1 aqueous polar phase (upper) which contains shikimic acid.

Quantification via GC-MS :

Aliquot lOOmicroliter of the upper polar phase into a glass vial and add lOmicroliter of internal standard (succinic-D4 acid; dissolved to Img/ml in HPLC grade methanol) .

The sample is dried down via centrifugal evaporation (or under inert nitrogen flow) . To each dry sample is added 30microliter of dissolved methoxyamine hydrochloride [CH₃NHOH . HC1] (MeOx; dissolved to 20 g/mL in 99.9% pyridine] and samples are placed in a heat block at 40°C for 2 hours. 70μΙ_ N-Methyl-N- (trimethylsilyl) trifluoroacetamide [MSTFA] is added to each sample and each sample is incubated for a further 2 hours at 40^CC. Samples are removed from heat and l\i is injected onto GC-MS system to quantify shikimic acid derivative [shikimic acid ( [TMS ) ] relative to a calibration curve made from pure compound standard (described below) . Detection prior to purification:

Aliquot 700microliter of the upper polar phase into a glass vial. The sample is dried down under inert nitrogen or via centrifugal evaporation .

The dried sample is re-suspended in lOOmicroliter of LC-MS grade methanol, internal standard added (10 of Img/mL methanolic 9- anthacenecarboxylic acid) . The sample is sonicated at room temperature for 5 minutes and vortexed.

The sample is filtered through a 2pm nylon filter into a glass vial. Inject 20pL onto LC-MS/MS System. Shikimate is detected via ESI- negative MS against spectra and retention time of pure standard (described below) . Purification

Aliquot 50 L of the upper polar phase into a glass vial. Inject 20μΙ< onto C18-reversed phase column at 30°C, running an isocratic gradient of water at lml/min. Collect fraction which contains solely shikimic acid (as above, approximately 5.6 mins) .

Dry down sample under inert nitrogen or via centrifugal evaporation. GC-MS based metabolite profiling

Samples were re-dried for 30 min under centrifugal evaporation before oxymation and silyation derivatization via addition of methoxyamine hydrochloride [CH₃NHOH . HC1 ] (MeOx; 30 ]iL, 20 g/ L in pyridine) followed by N-methyl-N- (trimethylsilyl) trifluoroacetamide (MSTFA; 70 ]iL) ; incubated (40 °C, 2 h) after addition of each following the methods of Halket, J. M. et al . Chemical derivatization and mass spectral libraries in metabolic profiling by GC/MS and LC/MS/MS. J. Exp. Bot . 56 , 219-43 (2005) .

Samples (1 pL) were injected into the GC-MS with a split/splitless injector at 290°C. The injection of samples was made in splitless mode; with polar samples of the Kew Living Collection also repeated on a 1:10 split. Metabolites were separated on a DB-5MS+DG 30 m (plus 10 m Duraguard) x 250 μπι x 0.25 μπι column (J&W Scientific, Folsom, California, US) . The GC oven was held for 3 min at 70°C before ramping at 4°C/ min to 325°C and held for a min. Helium was the carrier gas at a flowrate of 1.3 mL/ min. The interface with the MS was set at 280°C and MS performed in full scan mode using 70 eV EI+ and scanned from 50 to 1000 m/z. Retention time locking to ribitol was used (modified from Enfissi, E. M. a et al. Integrative transcript and metabolite analysis of nutritionally enhanced DE-ETIOLATED1 downregulated tomato fruit. Plant Cell 22 , 1190-1215 (2010) . A mixture of n-alkanes, ranging from 8 to 32 carbons, was used for retention index external calibration. RBG Kew Living Collection samples sets (6) were run in two batches of three randomised-blocks , two months apart. This approach was used to assess robustness due to the lack of quality control samples. Samples for D. elephantipes of the compound atlas were analysed in three blocks within a single batch.

To identify chromatogram components found in the Dioscorea profiles, a mass spectral (MS) library was constructed from in-house standards, the NIST "11 MS library (National Institute of Standards and Technology, USA) and the Golm Metabolome Database (Kopka, J. et al. GMD@CSB.DB: The Golm metabolome database. Bioinformatics 21, 1635- 1638 (2005)). with additional manual searches of MassBank, Human Metabolome Database (HMDB) , and the Yeast Metabolome Database (YMDB) ( Horai, H. et al . MassBank: A public repository for sharing mass spectral data for life sciences. J. Mass Spectrom. 45, 703-714 (2010), Wishart, D. S. et al . HMDB 3.0-The Human Metabolome Database in 2013. Nucleic Acids Res. 41, 801-807 (2013) and Jewison, T. et al. YMDB: The yeast metabolome database. Nucleic Acids Res. 40, 815-820 (2012), respectively) . Component peak identification and spectral deconvolution was performed using the Automated MS Deconvolution and Identification System (AMDIS v2.71, NIST); using Kovat's retention indices (RI) and MS for identification using the metabolomics reporting guidelines (Fernie, A. R. et al. Recommendations for reporting metabolite data. Plant Cell 23, 2477-82 (2011); Sumner, L. W . et al. Proposed minimum reporting standards for chemical analysis. Metabolomics 3, 211-221 (2007)). Each compound was assigned a representative ion and response areas were integrated and expressed relative to internal standard.

A shikimic acid standard series at 10, 20, 50, 100 and 200 μg from methanolic stock was made on three occasions and analysed as per samples. Ion 255 was chosen to be representative.

GC-MS Spectra

NAME: Shikimic acid (4TMS)

Retention Index=1802.0 RSN: 255

NUM PEAKS : 168

(53 1) (55 1) (57 1) (58 1) (61 2)

(65 4) (66 2) (67 1) (71 1) (72 4)

(73 409) (74 34) (75 34) (76 2) (77 4)

(78 1) (79 1) (81 1) (83 1) (85 1)

(89 1) (91 4) (92 1) (93 5) (94 1)

(95 2) (96 1) (97 1) (99 1) (101 3)

(103 9) (104 1) (105 5) (106 1) (107 1)

(109 2) (111 3) (113 1) (116 3) (119 3)

(120 1) (121 3) (122 1) (123 3) (124 1)

(125 3) (129 2) (131 10) (133 36) (134 5)

(135 5) (136 1) (137 1) (139 2) (141 7)

(147 180) (148 28) (149 20) (150 2) (151 9)

(152 1) (153 2) (154 1) (155 1) (157 1)

(159 1) (161 1) (163 1) (165 3) (166 2)

(167 11) (168 2) (169 3) (177 3) (178 1)

(179 2) (180 5) (181 8) (182 3) (183 2)

(189 27) (190 5) (191 20) (192 4) (193 18)

(194 3) (195 7) (196 1) (197 1) (204 1000)

(205 197) (206 89) (207 15) (208 3) (209 3)

(210 1) (221 10) (222 2) (223 4) (224 1)

(225 3) (227 1) (228 1) (229 2) (237 1)

(239 10) (240 2) (241 3) (243 11) (244 2)

(245 1) (251 1) (253 3) (254 38) (255 67)

(256 17) (257 7) (265 9) (266 2) (267 16)

(268 4) (269 3) (270 1) (271 1) (281 4)

(282 16) (283 12) (284 4) (285 2) (299 1)

(305 6) (306 2) (307 1) (311 1) (312 3)

(313 2) (314 1) (325 1) (327 1) (329 4)

(331 20) (332 6) (341 2) (342 5) (343 5)

(344 2) (345 1) (346 3) (355 6) (356 10)

(357 50) (358 17) (359 10) (360 3) (361 1)

(371 2) (372 36) (373 12) (374 6) (417 2)

(418 1) (447 14) (448 6) (449 3) (450 1)

(462 17) (463 7) (464 4)

The above procedures were repeated for each sample extracted from the foliage of the following species of yam: D. elephantipes, D. sylvatica, D. villosa, D. communis , D. bulbifera, D. antaly, D. caucasia, D. nipponica, D. tokoro, D. deltoidea, D. preussi, D. alata, D. minutiflora, D. praehensilis, D. altissima, and D. sansibarensis .

The percentage dry weight of shikimate in the foliage of the dioscorea plants tested is found to be as follows:

D. elephantipes: from 5% - to about 9% dry weight. D. sylvatica: from 5% to about 8% dry weight.

D. villosa: from 5% to about 8% dry weight.

D. communis: from 5% to about 8% dry weight.

D. bulbifera: up to about 5% dry weight.

D. antaly: up to about 5% dry weight.

D. caucasia: up to about 5% dry weight.

D. nipponica: up to about 5% dry weight.

D. tokoro: up to about 5% dry weight.

D. deltoidea: up to about 5% dry weight.

D. preussi: up to about 5% dry weight.

D. alata: up to about 5% dry weight.

D. minutiflora: up to about 5% dry weight.

D. praehensilis: up to 0.2% dry weight.

D. altissima: from 1% to about 1.8% dry weight.

D. sansibarensis: from 0.2% to about 3.2% dry weight.

LC-MS/MS method

Polar:

Remaining polar phase (700 pL) from metabolite extraction of leaf material is chilled at -20°C overnight and dried via centrifugal evaporation for ~6h. Samples stored at -80°C until analyses.

Samples are re-suspended in 100 L methanol and internal standard is added (10 L of Img/mL methanolic 9-anthacenecarboxylic acid) . Vortex and sonicate at RT for 5 minutes. Re-vortex and pass through 2μπι nylon filters into glass vial with insert.

Inject lOmicroliter onto 3um C18-reversed phase column (100 x 2mm i.d) coupled to a 10 x 2mm C18 guard column at 30°C, running an isocratic gradient of water +0.1% formic acid at 200 uL/min. Electrospray ionisation (ESI) operating in negative mode was employed. Instrument calibration was performed externally prior to each sequence with sodium formate solution prepared by dissolving NaOH in 50% methanol. In addition, automated post-run internal calibration was also performed by injecting the same sodium formate calibrant solution at the end of each sample run via a six port divert valve equipped with a 20 microliter loop.

Shikimate was identified by extracted ion chromatogram [m/z= 173.15 (M-H) ] . Shikimic acid confirmed with standards.

Caffeoylshikimate putatively identified based on MS/MS fragmentation pattern compared with literature.

LC-MS/MS Spectra

^S/MS of Shikimate (m/z = 173 .15 [M-H; 1 )

# m/z I S/N FWHM Res .

1 50. 8557 200 5. 6 0. 0010 50599

2 65. 5550 100 2. 8 0. 0011 57448

3 71. 0126 320 8. 9 0. 0012 59791

4 82. 4235 124 3. 4 0. 0013 64416

5 84. 5639 124 3. 4 0. 0013 65247

6 89. 5126 128 3. 6 0. 0013 67129

7 93. 0366 160 4. 4 0. 0023 41063

8 94. 0873 240 6. 7 0. 0014 68823

9 94. 7419 108 3. 0 0. 0014 69062

10 115 .0029 768 21 .3 0. 0019 60227

11 115 .0495 156 4. 3 0. 0015 76104

12 122 .2581 116 3. 2 0. 0016 78453

13 130 .9696 156 4. 3 0. 0016 81200

14 137 .0038 172 4. 8 0. 0016 83049

15 137 .0249 384 10 .7 0. 0063 21853

16 143 .0361 188 5. 2 0. 0017 84858

17 146 .9646 144 4. 0 0. 0033 44008

18 154 .9500 260 7. 2 0. 0018 88321 155.0406 192 5.3 0..0018 88347

173. 0470 500 13 1.9 0. .0029 60100

175. 0262 256 7. 1 0. .0047 37111

175. 0617 224 6. 2 0. , 0019 93879

176. 0290 104 2. 9 0. .0019 94138

974. 8114 116 3. 2 0. .0044 221531

1072 .6372 104 2. 9 0. .0046 232381

1176 .9762 104 2. 9 0. .0048 243421

1223 .1413 144 4. 0 0. , 0049 248149

1226 .1351 108 3. 0 0. .0049 248453

1354 .9490 108 3. 0 0. 0052 261179

1452 .1822 108 3. 0 0. .0054 270392

1469 .3111 148 4. 1 0. 0054 271982

Claims

1. Use of plant parts of the family Dioscoreaceae in the production of shikimate, wherein the plants are selected from D. elephantipes , D. sylvatica, D. villosa, D. communis , D. caucasia, D. nipponica , D. deltoidea, D. tokoro, D. preussi, D. alata , D. altissima, D. sansibarensis, D. bulbifera, D. antaly, D. praehensilis and D. minutiflora .

2. Use according to claim 1, wherein the plant parts are aerial parts.

3. Use according to claim 1 or claim 2, wherein the plant parts are selected from stems and leaves.

4. Use according to any one of the preceding claims, wherein the plant parts are leaves and/or petioles.

5. Use according to any one of the preceding claims, wherein the plants are selected from D. elephantipes, D. sylvatica , D. villosa, D. communis, D. caucasia , D. bulbifera , D. antaly, D. nipponica , D. deltoidea, D. tokoro, D. preussi and D. alata.

6. Use according to any one of the preceding claims, wherein the plant is selected from D. elephantipes , D. sylvatica , D. villosa , and

D. communis.

7. Use according to any one of the preceding claims, wherein the shikimate is L-shikimate.

8. A method of extracting shikimate from plants of the family Dioscoreaceae, wherein the plants are selected from D. elephantipes,

D. sylvatica , D. villosa, D. communis, D. caucasia , D. nipponica , D. deltoidea , D. tokoro, D. preussi , D. alata, D. altissima, D. sansibarensis, D. bulbifera , D. antaly, D. praehensilis and D. minutifloraD, wherein the process comprises the steps of:

i) freeze-drying aerial parts of the yam plants;

ii) extracting shikimate with solvent selected from organic polar solvents and hot water; iii) partitioning the product of step ii) in the aqueous phase, preferably using a short chain organic solvent different to the short chain organic solvent of step ii) ;

iv) purifying shikimate from the aqueous phase.

9. A method according to claim 8, wherein the aerial parts of i) are selected from leaves and petioles.

10. A method according to claim 8 or claim 9, wherein the solvent of ii) is selected from short chain alcohols and water.

11. A method according to any one of claims 8 to 10, wherein the solvent of ii) is selected from methanol, ethanol, iso-propanol , and iso-butanol .

12. A method according to any one of claims 8 to 11 wherein the solvent of ii) is water at 100°C.

13. A method according to any one of claims 8 to 12, wherein the solvent of ii) is selected from methanol, ethanol and water at 100°C.

14. A method according to any one of claims 8 to 13, wherein the solvent of iii) is selected from halogenated short chain organic compounds .

15. A method according to any one of claims 8 to 14, wherein the solvent is selected from haloalkanes.

16. A method according to any one of claims 8 to 15, wherein the solvent of step iii) is selected from chloroform and dichloromethane, and is preferably chloroform.

17. A method for the production of shikimate comprising extracting shikimate from aerial parts of plants of the family Dioscoreaceae, wherein the plants are selected from D. elephantipes, D. sylvatica, D. villosa , D. communis , D. caucasia , D. nipponica , D. deltoidea , D. tokoro, D. preussi, D. alata, D. altissima, D. sansibarensis, D. bulbifera, D. antaly, D. praehensilis and D. minutiflora .

18. A method according to claim 17, wherein the plants are selected from crop plant species and native plant species.

19. A method according to claim 17 or claim 18, wherein the plants are selected from crop plant species.

20. A method according to any one of claims 17 to 19, wherein the plants are selected from D. elephantipes, D. sylvatica , D. villosa , D. communis, D. caucasia , D. bulbifera, D. antaly, D. nipponica, D. deltoidea , D. tokoro, D. preussi and D. alata.

21. A method according to any one of claims 17 to 20, wherein the plants are selected from D. elephantipes and D. sylvatica .

22. A method according to any one of claims 17 to 21, wherein the aerial parts are stems and leaves.

23. A method according to any one of claims 17 to 21, wherein the aerial parts are stems, leaves and/or petioles.

24. A method according to any one of claims 17 to 21, wherein the aerial parts are leaves .