AU2009337191B2 - Plant manipulation - Google Patents

Abstract

Plants, honey derived from these plants, and related methods are described to increase the concentration of phenolic compounds and in particular, methoxylated phenolic compounds in plant nectar and honey derived from the plant nectar. A key driver to increasing the level of phenolic compounds is by imposing a stress on the plant or plants. Methylglyoxyl concentrations may also be increased in the plant nectar and honey derived from the plant nectar.

Description

WO 2010/082844 PCT/NZ2009/000300 PLANT MANIPULATION TECHNICAL FIELD The invention relates to plant manipulation. More specifically, the invention relates to methods of 5 manipulating plants to produce honey from the plants with tailored and/or elevated levels of phenolic compounds. BACKGROUND ART Over the last 40-50 years bacteria have become increasingly resistant to commonly used 10 antibiotics. As a result many infections previously readily cured by antibiotics are now difficult or impossible to treat (Finch, R.G. 19981). Given this, empirical screening of chemical entities for antimicrobial activity represents an important strategy for the development of novel drugs. Natural products in particular have been a rich source of antimicrobial agents, that in general are associated with low levels of toxicity, and in many cases have a fairly broad spectrum of 15 activity (Silver et al 19902). A natural product that has received significant attention due to its anti-bacterial action is honey. Although honey has been used for the treatment of respiratory infections and for the healing of wounds since ancient times (Moellering 19953, Jones 2001 ) it was not until the late 20th century, as a result of the increasing resistance of micro-organisms to antibiotics that research 20 studies began to document the anti-bacterial activity of honey against a number of pathogens (Allen 19915, Willix 1992). While the majority of honeys have been shown to have anti-bacterial activity, manuka honey, a honey produced by bees from the flowers of the manuka bush (Leptospermum scoparium) have been shown to possess the highest levels of anti-bacterial activity (Molan 1992) and to be active against a range of pathogens including Staphylococcus 25 aureus, coagulase-negative Staphylococci, Enterococci and Pseudomonas aeruginosa (Cooper 25 'Finch, R.G. (1998) Antibiotic resistance. Journal of Antimicrobial Chemotherapy 42, 125-128. 2 Silver, L and Bostian, K. (1990) Screening of natural-products for antimicrobial agents. European Journal of Clinical Microbiology & infectious Diseases 9, 455-461. 3 Moellering RC. (1995). Past present and future antimicrobial agents. American J Medicine, 1995; Supp 6A .11S-18S. 4 Jones HR. Honey and healing through the ages. In Honey and Healing. ed Munn PA and Jones HR. 2001; 1-4. Cardiff, IBRA 8 Allen KL Molan PC. Reid GM. (1991) A survey of the antibacterial activity of some New Zealand honeys Journal of Pharmacy & Pharmacology. 43(12):817-22 6 Willix DJ. Molan PC. Harfoot CG. (1992) A comparison of the sensitivity of wound-infecting species of bacteria to the antibacterial activity of manuka honey and other honey. Journal of Applied Bacteriology. 73(5):388-94. Molan PC. The antibacterial activity of honey. 2. (1992). Variation in the potency of the antibacterial activity. Bee World 73: 59-76.

WO 2010/082844 PCT/NZ2009/000300 199',5Cooper 20022, Cooper 2002,French 20054). Indeed today manuka honey is a well accepted and established clinical treatment for infection associated with wounds and burns where it has been shown to,have.both anti-infective and wound healing properties (Cooper. 99 Molan2001 , All 19916). 5 In addition to its use-for the treatment of wounds it has also been shown that manuka honey, has antibacterial activity against the gastric pathogen N. flon, thie causative agent of gastritis and the major predisposing faotorfor'peptic ulcer disease, gastric cancer and B-cell MALT lymphoma (Somal 1994?, Osato 19998, Mitchell 1999). Indeed a number of in vitro studies have shown that concentrations of manuka honey as low as 5-10% (v/v) can inhibit the growth of H. 10 pylori (Somal 1994, Osato 1999, Mitchell 1999). This finding is of particular interest given that over recent years resistance to currently available antimicrobial agents against H. pylor has increased dramatically leading to an increasing number of treatment failures (Fishbach 2007'"). Indeed, in some populations, the level of resistance to clarithromycin, one of the major antibiotics used in the treatment of H. pylori, has been reported to be as high as 30-40% in 15 some countries and is commonly associated with treatment failure (Raymond 200711). Resistance to metronidazole, a second antibiotic commonly used in the treatment of H. pylori infection has also been reported to be high (30%-40% in US and Europe and > 80% some countries of the developing world), although in some cases in vitro resistance does not translate into eradication failure (Raymond 2007, Marvic 2008'). Given this environment, alternative 20 treatment approaches are of interest. 20 -Cooper RA. Molan PC. Harding KG. (1999).Antibacterial activity of honey against strains of Staphylococcus aureus from infected wounds. Journal of the Royal Society of Medicine. 92(6):283-5 -Cooper RA, Halas E, Molan PC. (2002).The efficacy of honey In inhibiting strains of Pseudomonas aeruginosa from Infected bums. J Bum Care Rehabll 23: 366-70. -Cooper RA, Molan PC, Harding KG. (2002). Honey and gram positive cocci of clinical significance in wounds. J Appl Microbiol; 93:857-63. -V. M. French, R. A. Cooper and P. C. Molan. (2005). The antibacterial activity of honey against coagulase-negative staphylococci Journal of Antimicrobial Chemotherapy 56, 228-231 Molan PC. Potential of honey AM J Clin Dermatol 2001;2;13-19 AT Ali, MN Chowdhury, MS al Humayyd. (1991 inhibitory effect of natural honey on Helicobacter pylori. Trop Gastroenterol -N Al Somal KE Coley, PC Molan and B Hancock. (1 994).Susceptibility of helicobacter pylori to the antibacterial activity of manuka honey. Journal of the Royal Society of medicine 1994;87;9-12 -Soto MS. Reddy SG. (1999) Graham DY. Osmotic effect of honey on growth and viability of Helicobacter pylori. Digestive Diseases & Sciences. 44(3):462-4. -Osato MS. Reddy SG. (1999) Graham DY. Osmotic effect of honey on growth and viability of Helicobacter pylori. Digestive Diseases &*Sciences. 44(3):462-4. - L Fischbach; E. L. Evans. (2007) Meta-analysis: The Effect of Antibiotic Resistance Status on the Efficacy of Triple and Quadruple First-line'Therapies for Helicobacter pylori Aliment Pharmacol Ther. ;26(3):343-357. -Josette Raymond , Christophe Burucoa Olivier Pletrini Michel Bergeret Anne Decoster Abdul Wann Christophe Dupont and Nicolas Kalach (2007) Clarlthromycin Resistance in Helicobacter pylori Strains isolated from French Children: Prevalence of.the. Different Mutations and Coexistence of Clones Harboring Two Different Mutations In the Same Biopsy helicobacter.Volume 12 issue 2 Page 157-163. - 12 Elvira Marvic, Silvia Wittmann, Gerold Barth and Thomas Henlel (2008) Identification and quantification of methylglyoxal as the dominant antibacterial constituent of Manuka (Leptospermum scoparium) honeys from New Zealand Mol. Nutr. Food Res. 2008, 52, 000 - 000 2 WO 2010/082844 PCT/NZ2009/000300 While theantirirobiai actiity of honey has been reported to include osmolarity, acidity,: hydrogen peroxide and plarit-derived componentsnmore recent studies have shown that. osmolarity, acidity and hydrogen peroxide actity:cannot accountifor all of the honey activity and that enhanced activity may be due to phytochemicals found in particular honeys, including manuka honey (Molan 1992). For example Cooper et al. (Cooper 1999) in a study of the antibacterial activity of honey against Staphylococcus aureus isolated from infected wounds showed that the antibacterial. action of honey in infected wounds does not depend wholly on its high osmolarity, and suggested that the action of manuka honey stemmed partly from a phytochemical component (Cooper 1999). Until recently the identity of these phytochemicals in manuka honey remained unclear, however in 2008 a study by Marvic et al reported that the pronounced antibacterial activity found in manuka honey directly originated from a chemical -compound, methylglyoxal (MGO). In this study six samples of manuka honey were shown to contain over 70 times higher levels of methylglyoxal (up to 700 mg/kg) than that found in regular honeys (up to 10 mg/kg) (White 19631). Floral Markers As noted above, phytochemicals are thought to have an important role in relation to activity. Honeys have been known for some time to include a variety of phenolic compounds, flavonoids and abscisic acid. A selection of prior art on this point includes the following documents: Ferreres et al 19962 describes tests done on heather honey to find two non-flavonoid components as the main constituents being two isomers of abscisic acid. The corresponding flower nectar from which the honey is derived was also found to contain both isomers as the main constituents. This document notes that the abscisic acid isomers noted were not detected in other monofloral honey samples so Ferreres suggests that abscisic acid may be used as a marker for heather honey. Gheldof et al June 2002' describes tests completed on honeys for antioxidant capacity and phenolic content. Antioxidant content was found to be proportional to phenolic content and darker honeys such as buckwheat were found to have high antioxidant capacities. This application suggests that the phenolic content of honey may be used as an indicator of honey 30 White, J.W., Schepartz, A.. and Subers, M.H. (1963) Identification of Inhibine, Antibacterial Factor in Honey, as Hydrogen Peroxide and Its Origin in a Honey Glucose-Oxidase System. Biochimica Et Blophysica Acta 73, 57-. 2 Ferreres et at Natural occurrence of abscisic acid In heather honey and floral nectar. J. Agric. Food Chem. 1996 44; 2053-2056. 3 Nele Gheldof, Xlao-Hong Wang and Nicki J -Engeseth (2002) Identification and Quantification of Antioxidant Components of Honeys from Various Floral Sources. J. Agric. Food Chem 2002 50, 5870 5877. 3 WO 2010/082844 PCT/NZ2009/000300 origin. Gheldof et al 2002al describes further experimentation completed from the earlier article. I ne aim in this article was to characterise the phenolics and other antioxidants in the honeys tested. In this article the authors found that honeys have similar types of antioxidants but different amounts of phenolic compounds. The author concluded that the phenolics were significant to antioxidant capacity but not solely responsible. Examples of antioxidant materials noted included proteins, gluconic acid, ascorbic acid, hydroxymethylfurfuraldehyde and enzymes such as glucose oxidase, catalase and peroxidase. Barberan et al 19932 describes analysis of flavonoids in honey. The authors of this article found that flavonoids were incorporated into honey from propolis, nectar or pollen and that honeys. from the northern hemisphere tended to show higher degrees of propolis based flavonoids while equatorial and Australian based honeys were largely devoid of propolis based flavonoids. South American and New Zealand honeys contained flavonoids associated with propolis. Yao et al 20039 describes the use of measuring flavonoid, phenolic acid and abscisic acid content in Australian and New Zealand honeys as a method of authenticating honey floral origins. The authors found that Australian jelly bush honey included myricetin, luteolin and tricetin as the main flavonoids. Phenolics were found to be primarily gallic and coumaric acids along with abscisic acid. By contrast New Zealand manuka honey contained quercetin, isorhamnetin, chrysin, luteolin and an unknown flavanin. The main phenolic compound was found to be gallic acid. In addition, almost three times the amount of abscisic acid was found in New Zealand manuka honey as Australian jelly bush honey. Barberan et al 20014 describes how the phenolic profiles of 52 honeys from Europe were analysed. The different honeys were found to have different markers with different characteristics and UV spectra. Different markers however were found to be present in several honeys rather than being specific to one species. For example, abscisic acid was found in heather honey, rapeseed, lime tree and acacia honeys. As should be appreciated from the above, a variety of experiments have been undertaken to determine characterising compounds in honeys. Knowledge exists that honey contains 28 Nele Gheldof and Nicki J Engeseth (2002) Antioxidant Capacity of Honeys from Various Floral Sources. Based on the Determination of Oxygen Radical Absorbance Capacity and Inhibition of in Vitro Upoprotein Oxidation in Human Serum Samples J. Agric. Food Chem 2002 50,3050-3055. 2 Francisco A. Tomas-Barberan, Frederico Ferreres, Cristina Garcia-Viguera, and Francisco Tomas Lorente (1993) Flavonoids In honey of different geographical origin. Z Lebensm Unters Forsch 196:38-44. .1Uhu Yao, Nivedita Datta, Francisco A. Tomas-Barberan, Federico Ferreres, Isabel Martos, Riantong Singanusong (2003) Flavonolds, phenolic acids and abscislc acid in Australian and New Zealand Leptospermum honeys. Food Chemistry 81 (2003) 159-168. 4Francisco A Tomas-Barberan, Isabel Martos, Federico Ferreresi Branka S Radovic and Elke Anklam (2001) HPLC flavonoid profiles as markers for the botanical origin of European unifloral honeys. J Sci Food Agric 81:485-496. 4 WO 2010/082844 PCT/NZ2009/000300 antioxidant activity and'that this:may, be attributable to compounds such as flavonoids, phenolic acids and- abscisic acid.:What should also be apparent-from the above is that different studies have found that these compounds are present in a variety of honeys and that the amount present ard the types of compound present rmay be a misleading measure of the honey origin due to their variation and lack of correlation between: plant and honey. For example, abscisic acid is found in a variety of different honeys from different plant species but the quantities vary substantially even between samples from the same source. The authors of the above documents do not consider whether honey age has any influence on. the composition of the various compounds analysed. Methoxylation Most dietary polyphenols have very poor bioavailability due faster metabolic breakdown of hydroxyl groups as opposed to methoxyl groups. Methoxylated phenolics are highly resistant* to human hepatic metabolism (Wen and Walle.2006a') and also have much improved intestinal transcellular absorption (Wen and Walle 2006b 2 ). The methylated flavones show an approximately 5- to 8-fold higher apparent permeability into cells which makes them much more bio-available. The higher hepatic metabolic stability and intestinal absorption of the methylated polyphenols make them more favourable than the unmethylated polyphenols for use as potential cancer chemo-preventive'agents. The determination of metabolic stability of four methylated and their corresponding unmethylated flavones with various chemical structures all of the tested methylated flavones, showed much higher metabolic stability than their corresponding unmethylated analogues. It should be appreciated from the above that it would be useful to have a means for adjusting the level of medical and/or nutritional potency of honey. Since plants from which honeys are derived contain key compounds with medical and nutritional potency, it should further be appreciated that methods of manipulating plants to enhance key compound levels would be useful. It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice. All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part-of the 33 Wen, X., Walle T. (2006a) Methylation protects dietary flavonoids from rapid hepatic metabolism. Xenobiotica 36: 387-397. .Wen, X., Walle, T (2006b) Methylated flavonoids have greatly Improved intestinal absorption and metabolic stability. DngMetab. Dispos. 34:1786-1792. .5 common general knowledge in the art, in New Zealand or in any other country. It is acknowledged that the term 'comprise' may, under, varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term 'comprise' shall have an inclusive meaning - i.e. that it will be taker to mean an inclusion of not only 5 the listed components it directly references, but also other non-specified components or elements. This rationale will also be used when the term 'comprised' or 'comprising' is used in relation to one or more steps in a method or process. Further aspects and advantages of the present invention will become apparent from the ensuing description that is given by way of example only. 10 DISCLOSURE OF THE INVENTION Embodiments of the invention broadly relate to maintaining and/or maximising the medical and nutritional potency of honey by use of the finding that phenolic compounds in honey are a key driver of honey potency. Since plants are a key source of such phenolic compounds and integral to honey manufacture, manipulating plants to produce greater numbers of phenolic compounds is of interest. 15 Finding the above synergies was surprising as this goes against recent publications that suggest that methylglyoxal (MGO) is the primary compound and those which have found abscisic acid to be a major factor. A further finding by the inventors was the fact that the free phenolic compounds concentrations change over time in the honey and in response to other factors such as heat and dilution. This change in 20 concentration over time was unexpected and may be a reason why earlier trials looking at phenolic compounds were unsuccessful or gave mixed or inconsistent results. Also contrary to the art was the inventors finding that in fact phenolic compounds in plant nectar (as opposed to pollen or other measures) was highly correlated to the levels found in honey sourced from the same plants. As noted above, the art does not teach of a correlation between plant nectar phenolic 25 compound levels and that observed in honey and therefore concludes that phenolics are not a useful measure. The art also does not recognise the influence of phenolic compounds on medical potency. In contrast, particularly when age is taken into consideration, phenolic compounds are highly correlated between plant nectar and honey. This finding has meant that it is possible for the inventors to measure a wide range of factors in honey related to honey properties and value well beyond that speculated in the art 30 of only origin. The exact mechanism behind why the phenolic levels vary is not certain however the inventor understands that these phenolic entities are initially carried by a tannin molecule(s) present in the nectar, and as the honey ages naturally the phenolic molecules are released due to degradation of the tannin body and the matrix associated with a large organic molecule with both hydrophobic and hydrophilic binding 35 sites. The best researched comparison for such aging is the development of flavour and aroma in red 6 wines due to the release of phenolic groups from tannin bodies. The same result of an increase in MGO concentration over time for honeys that include MGO e.g. manuka honey, was also measured by the inventors although other processing steps could be used to adjust MGO concentration and not adjust phenolic concentration hence a different mechanism of release 5 appears to occur for MGO. Prior art suggests that this may be due to conversion of DHA into MGO as a natural reaction process within the honey and that this conversion is sensitive to age like phenolics as well as other influences e.g. heat and acidification. The improved healing effects or potency of some embodiments are in part thought to be due to the phenolic compounds working alone or with other properties in the honey to confer multiple stages of 10 healing. The different stages are an antimicrobial phase, an immune stimulation phase and an anti inflammatory phase. All of these aspects are understood by the inventors to contribute to potency of honey in medical and nutritional applications. Methods developed by the inventors to influence phenolic concentration in plant nectar are now described. 15 For the purposes of this specification the term 'phenolic compounds' and grammatical variations thereof refers to phenolic acids, phenolic salts, phenolic esters and related polyphenols compounds. The term 'free' in the context of phenolics refers to phenolic compounds being in a readily detectable form. The term 'complexed' in the context of phenolics refers to phenolic compounds being carried in a tannin 20 molecule or otherwise not detectable, for example as a result of in vivo phenolic serf condensation or precipitation reactions occurring as a result of honey bees dehydrating nectar. According to a first embodiment there is provided a method of producing honey with levels of tannin derived phenolic compounds elevated above an average concentration for a given honey type by the steps of: 25 a. increasing the concentration of tannin derived phenolic compounds in the nectar of a plant or plants by subjecting the plant or plants to stress; and, b. producing honey from the nectar of the stressed plant or plants; wherein plant stress in step (a) is induced by artificially subjecting the plant or plants to conditions selected from the group consisting of: drought, pests, selective watering, pruning, lack of nutrient(s), UV 30 exposure, heat, externally derived abscisic acid, externally derived salicylic acid, and combinations thereof. In an embodiment, plants may be selected based on their degree of stress in the natural environment. 7 One method of achieving the above stresses envisaged by the inventors may be to grow a hedge of the target plants (competition for light, nutrients and water) and then periodically prune the hedge to stress the plants and increase flowering. An alternative is to grow the plant or plants in a greenhouse. In one embodiment, the above method is also used to increase the amount of methylglyoxal (MGO) in the 5 honey produced from the plant. As may be appreciated, not all plant varieties produce methylglyoxal but selected varieties such as Leptospermum species produce MGO and therefore the above method may also be used to enhance MGO concentration as well if desired. In the above method, phenolic compounds in the plant nectar may be elevated by 5-10,000 mg/kg above a baseline level without stress being subjected to the plant. 10 According to a second embodiment, there is provided a plant for use in honey production that has been subjected to the method substantially as described above. According to a third embodiment, there is provided a honey with elevated levels of phenolic compounds produced by the method substantially as described above. According to a fourth embodiment, there is provided a plant characterised by being stressed and having 15 an increased concentration of phenolic compounds in the plant nectar. According to a fifth embodiment there is provided a plant or plants that have been stressed with an elevated concentration of phenolic compounds in the plant nectar of between 5-10,000 mg/kg in order to produce honey from the plant or plants that have elevated levels of phenolic compounds. The plant or plants of the fifth embodiment may be stressed by artificially subjecting the plant or plants to 20 conditions selected from the group consisting of: drought, pests, selective watering, pruning, lack of nutrient(s), UV exposure, heat, externally derived abscisic acid, externally derived salicylic acid, and combinations thereof. In an alternative embodiment, stress occurs due to natural environmental conditions. In the above embodiment, the plant or plants may be stressed by growing a hedge of the target plants 25 (competition for light, nutrients and water) and then periodically pruning the hedge to stress the plants and increase flowering. An alternative is to grow the plant or plants in a 8 WO 2010/082844 PCT/NZ2009/000300 greenhouse In the fifth, embodiment, the plant or plants may also have an increased concentration of methyiglyoxal (MGO) in the honey produced from the plant. As may be appreciated, not all plant varieties produce methylglyoxal but selected varieties such as Leptospermum species produce MGO and therefore stressed plants may also have enhanced MGO concentration as well-if desired. According to a sixth embodiment there is provided a method of selecting and breeding plants to tailor and/or maximise the amount of phenolic compounds in honey derived from plant nectar by analysing plant nectar phenolic content and selecting and breeding cultivarsof the plant that produce the highest concentration and/or volume of phenolic compounds in the nectar. The sixth embodiment may also include the step of selecting the plant or plants also based on the extent to which they produce elevated levels of phenolic compounds in the plant nectar when stressed. Preferably, the method above is completed where the plants are also selected based on factors selected from the group consisting of: plant growth rate, flower density, timing of flowering, nectar yield, and combinations thereof. It should be appreciated that the timing of flowering is an important characteristic as to ensure monofloral purity, it is preferable to avoid competition with other plant species. It should also be appreciated that nectar yield is also of interest as it is preferable to maximise concentration and volume of phenolic compounds and optionally also MGO content (species dependent). According to a seventh embodiment there is provided a honey produced from the nectar of a plant or plants that have been stressed to cause an increased concentration and/or volume of phenolic compounds in the honey. In the above embodiments, the phenolic compounds may be in a form selected from the group consisting of: a free form, a complexed form and mixtures thereof. Preferably, the phenolic compounds are selected from the group consisting of: phenolic acids, phenolic salts, phenolic esters, related polyphenolic compounds, and combinations thereof. Preferably, the phenolic compounds are derived from tannin compounds. As noted above, a useful correlation is the comparison to wines where aging is associated with the development of flavour and aroma in red wines due to the release of phenolic groups from tannins. 9 WO 2010/082844 PCT/NZ2009/000300 Preferably, the phenolic compounds aremethoxylated. As noted above, the prior art teaches. some useful properties attributable to methoxylated compounds. The inventors have found: that honey which iricluds methoxylated cormpoundsyexhibit useful medical and nutritional effects. By way of examole,the inventors have anaysed the phenolis prominent in manUka (Leptospermum spp.)andlkanulka (Kunsea spp.) and a large number of these phenolics are methoxylated at one or rnore points of their phenol or acid group. Compounds such as gallic or benzoic acid are present mainly in their methoxylated form such as methoxybenzoic acid, nethox ygallic acid, methyl syringate, methoxyphenylactic acid or syringic acid. Methoxylation is therefore a major feature of the phenolics that are prominent in the above species that are acknowledged to have a higher medical and nutritional activity. The inventor's findings in combination with the artr mean that effects envisaged for medical and nutritional applications include: - Greater bioavailability due to the methoxylated compounds be able to enter the cell faster; * Longer bioavailability due to the methoxylated compounds having a much longer half life within cells to scavenge free radicals; - Phase i enzyme induction properties; For honey wound dressing applications, the methoxylated compounds are also likely to have a much longer half life within wound exudate as they are not rapidly degraded. Methoxylation also results in much longer lived molecules once they are in the cell. Also unexpectedly, the inventors have found that methoxylated compounds are well tolerated by the human cells (low toxicity) but not by bacterial and fungal cells that is highly advantageous in treating microbial infections. In a further embodiment, methoxylated phenolics may represent greater than 10% wt of the total phenolic compound content in the composition. Preferably, this may be greater than 20% wt. Preferably, this may be greater than 30% wt. In a further embodiment, honey produced from the method or plant contains at least 150 mg/kg of methoxylated phenolic compounds. Examples of principal phenolic compounds may be selected from the group consisting of:. phenyllactic acid, methoxylated phenyllactic acid, methoxylated benzoic acids, syringic acid methyl syringate, isomeric forms of methyl syringate, and combinations thereof. In one embodiment the free phenolic content may be measured indirectly by determining the sum of phenyllactic and 4-rnethoxyphenyllactic acids and derivatives thereof (particularly hydroxylated analogues). These may be increased in the plant nectar by 5-10,000 mg/kg. 10 WO 2010/082844 PCT/NZ2009/000300 Examples ofthese-compounds'are illustrated below:n COOH GOON ORO Phenyllactic acid 4-methoxyphenyllactic -acid. in ayoung honey these 6mpouids are understoodiby the inventors to typically account fdr more than three-quarters of the principal phenolic components. The inventors have found that, with no other influences other than age, honey tend to show an increase in predominance of benzoic acid compounds and their derivatives. Preferably, the methoxylated derivatives of benzoic acid noted above are benzoic acid, 2 rnethoxybenzoic acid, 4-methoxybenzoic acid and isomers of trimethoxybenzoic acid as shown below: COOH COOH COOH COOH oue OMe Benzoic acid 2-methoxybenzoic acid 4-methoxybenzoic acid Trimethoxybenzoic acid Hydroxylated benzoic acid derivatives (salicylic acid and 4-hydroxybenzoic acid) are also of interest although are present in less significant concentrations. Preferably, the third group of the principal phenolic components noted above include syringic acid and methyl syringate: COOH coOMe Me . OMe Meo oMe OH OH Syringic acid Methyl syringate These components are present as two isomers that are diagnostic and differentiate nanuka and kanuka honeys. In afurther embodiment, the free phenolicsr may also include a suite of other compounds auit with the tannin matrix in honeys. These range from relatively simple molecules such as garlic acid and methoxylated derivatives, abscisic acid, 'cinnamic acid, phenylacetic acid and 11 WO 2010/082844 PCT/NZ2009/000300 methoxylated and hydroxylated derivatives,.and methoxyacetophenone; to complexed polyphenolic molecules such as ellagicacid. Range .of:these molecules are illustrated below COOH 0

-O

1 ' - Ho OH Ho OH Meo. OH 00 .oH Gallic acid Cinnamic acid Ellagic acid Preferably, the nectar contains free, complexed or a mix of phenolic compounds sufficient to results in honey with 5mg/kg to 1 0,000mg/kg or higher depending on the preferred application. The inventors have found that honey bees perform about a ten-fold concentration of the nectar during the conversion into honey it is apparent three of these principal phenolic components are relatively more concentrated in the nectar than in the honey. This may be evidence of in vivo phenolic self-condensation reactions occurring as the honey bees perform nectar: dehydration. Such in vivo self-condensation reactions have been well described in the study of aging in wine (Monagas et al. 20041). In contrast syringic acid concentration is similar in nectar and fresh honey, indicating this molecule is mostly present as hydrolysable tannin in the nectar and the increased concentration in aged honey is due to tannin body degradation. The analysis of nectar components in various glasshouse conditions provides measurement of the plants production of the different components, and secondly production efficiency in different environments. This allows breeding selection to be tailored to fit the intended locations for plantation establishment. It should be appreciated from the above description that there are provided methods, plants and related honey with elevated, tailored and/or maximised concentrations of phenolic compounds and optionally also MGO in the plant nectar. Advantages of this manipulation should be apparent including increased potency in medical and nutritional applications. BRIEF DESCRIPTION OF THE DRAWINGS Further aspects of the present invention will become apparent from the following description that is given by Way of example only and with reference to the accompanying drawings in which: Figure 1 shows a graph illustrating the phenolic profile of rnonofloral manuka, kanuka and 27 Monagas, M.; Gomez-Cordoves C.; Bartolorne, B. 2004. Evolution of the phenolic content of red wines from Vlfis vinifera L during ageing in bottle. Food Chem. 95(3) 405-412. 12 WO 2010/082844 PCT/NZ2009/000300 "other honeys harvested in New:Zealandiand aged naturallyifor up to ten years; Figure 2 shows a graph illustrating the correlation between the sum of the principal phenolic components and rmethylglyoxal in monofloral manuka honey harvested in New Zealand and naturally aged; and, Figure 3 shows a graph illustrating the ratios of five principal phenolic acids in. honeys derived from Leptospermum species and varieties in New Zealand and Australia. BEST MODES FOR CARRYING OUT THE INVENTION The invention is now described with reference to various examples illustrating the medical and nutritional properties of the present invention. EXAMPLE 1 In this example, honey harvested from the indigenous New Zealand shrubs Leptospermum scoparium (manuka) and Kunzea ericoides (kanuka) are used to demonstrate the presence of free phenolic compounds and the way the concentration of these compounds change over time. Manuka and kanuka honeys were chosen to illustrate this effect as they contain relatively high levels of free phenolics and derivative compounds compared to other honey types. Figure 1 illustrates the concentration of the free phenolics present in five honey types of different ages. Relatively fresh (<3 months) manuka and kanuka honeys contain approximately 1000 mg. kg- of these compounds, whereas in comparison the other honey types of the same age contain considerably less than'100 mg. kg. Furthermore as the manuka and kanuka honeys are aged naturally, that is stored at room temperature following extraction from the honey comb, the concentration of the phenolic components increases approximately three-fold' over ten years to in the region of 3000 mg. kg 1 . However, the increase in free phenolic components' concentration illustrates a logarithmic curve; consequently much of the development of the phenolic profile occurs in the first five years of honey storage and aging. Table 1 below describes the concentrations of these components during the aging process. Whilst these compounds are common to manuka and kanuka honeys, the concentration of some coinponents differ significantly in these honeys. Table 1 - The phenolic profile and concentration of principal components mg/kg in monoloral rnanuka and kanuka honeys harvested in New Zealand and aged naturally for ten years. Values shown, mean i standard deviation 13 WO 2010/082844 PCT/NZ2009/000300 .. 0, - - o CL 75 4 2 8 7 2 5 18 0 3 0 4. .5 .*. *58. *3.- 1 4. 58. 4. 64 203 20 62 *31. Kanka .5 70.7 93.3 23 6.* 10.7 632424 S2 0. 0 57259 37. L.1 336 .0 59. 788 73* 183t4..2 31.1 *12.7 14.8 10. 26 .2 10 1 0 5 7.2 338 54 391 175.0 Manuka 0 1± 4.± 31.3v fr.m co 2±a 93± 14± 58.0 4.2 ±6.4 ±40.3 2.0 62 ±31.8 Kanuka 0.5 2 700.7±t 93.3± 2.3± 63.3± 103.7 963±t2 42.4± 26.1 15.5 0.8 8.5 ±11.9 0 23.4 5 2 1549± 307.0 3.4t 336.0 592.5 2788± 35.5±t 83.4 ±21.2 1.1 ±12.7 ±1 4.8 10.6 26.2 10o 1 .1680 .512 7.2 338 554 3091 17.0 The concentration of methylglyoxal in the manuka and kanuka honeys is also listed in Table 1. Mariuka honey, derived from Leptospermum scoparium, contains methyiglyoxal. As a manuka honey is aged, the concentration of free methylglyoxal also increases in the honey. This increase is understood to be due to a different mechanism to the increase in phenolics owing at least to the way the compounds develop when heated. It is understood by the inventors that the MGO increase may be due to conversion of DHA to MGO. Figure 2 illustrates the correlation between the concentration of methylglyoxal and the principal phenolic compounds in a naturally aged manuka honey. Methylglyoxal and total phenolic compounds do not correlate in kanuka honey because the methylglyoxal component is derived from Leptospermum scoparium, and the small amounts of methylglyoxal in the kanuka honeys represent insignificant manuka honey contamination. EXAMPLE 2 A further illustration of the presence of unique phenolic compounds in plant nectar used for honey manufacture is illustrated in Figure 3 which shows a comparison between manuka honey 14 WO 2010/082844 PCT/NZ2009/000300 .produced from Northland, Waikato arid.East Coast in New Zealand and a sampleafrom Queensland Australia. As can be seen in Figure , the ratio of plienolic compoundsallows separation-by region, and botanic source. The'doncentration of 2-methoxy-benzoic and tri-methoxy-benzoic acids is significantly elevated in honey derived from Leptosparmum polygalifoliurri in Queensland, -Australia. Pheryllactic acid is elevated in honey from Northland, New Zealand where variety is Leptospermun scopanrum var inanum. Elevated tri-methoxy-benzoic acid separates honey,, sourced from the Waikato wetlands and the East Coast of the North Island, New Zealand. EXAMPLE 3 In this example a range of honey samples were analyses to determine the antioxidant levels in the nectar derived honeys compared to control standards. Antioxidant activity was determined by the ABTS assay using a spectrophotometric method for antioxidant activity using the ABTS radical assay (expressed as Trolox Equivalent Antioxidant Capacity) based on the method of Miller & Rice-Evans (1997). All samples were diluted with warm water as required to bring into the appropriate range for the assay. The antioxidant activities of the various samples are given in Table 2. Table 2 - Antioxidant Levels for Honey Samples Tested Sample Description Antioxidant Activity by ABTS Assay (pmole TEAC/1 00g) Average Standard Deviation Far North, North Island NZ Honey (Fresh) 131.4 3.7 Far North, North Island NZ Honey (Aged) 256.2 4.0 Bush Blend Honey from Hokianga NZ (Fresh) 176.4 9.2 Bush Blend Honey from Hokianga NZ (Aged) 189.9 0.8 Waikato, NZ Wetlands Honey (Fresh) 143.8 1.5 19 Miller, N.J.; Rice-Evans, C.A. 1997: Factors influencing the antioxidant activity determined by the ABTS + radical cation assay. Free Radical Research 26(3): 195-199. 15 WO 2010/082844 PCT/NZ2009/000300 Waikato, NZ Wetlands Honey (Aged) 237.7 7.5 East Coast, North Island NZ Honey (Fresh) 153.9 4.3 East Coast, North Island NZ Honey (Aged) 243.7 3.4 Kanuka Honey (Fresh) 178.3 1.4 Kanuka Honey (1 Year Old) 148.5 7.4 Kanuka Honey (2 Years Old) 193.3 8.3 Kanuka Honey (3 Years Old) 239.6 11.4 Heated Manuka Honey 305.1 2.5 Clover Honey 49.7 3.2 Rewarewa Honey 215.9 3.4 Standard - 2-methoxybenzoic, 80 mg/kg 51.8 1.3 Standard - phenyllactic acid, 210 mg/kg 54.6 1.3 Standard - methylsyringate, 290 mg/kg 85.1 .2.7 Standard - gallic acid, 700 mg/kg 1695.4 58.3 Standard - syringic acid, 760 mg/kg 499.6 25.3 As can be seen in Table 2, the antioxidant levels increase in honey with age supporting earlier Examples. This effect occurs irrespective of region from which the honey has been collected. Also noted was that honeys known to have medical activity e.g. manuka honey, had moderate TEAC levels. Conversely, honeys known to have little medical activity e.g. rewarewa honey had higher TEAC counts. This variation in medical activity is understood by the inventors to be attributable to the phenolic levels (total TEAC count), but also the amount of methoxylated phenolic compounds. Manuka honey has been found by the inventors to have a high number of methoxylated phenolic compounds e.g. methoxybenzoic acid and methyl syringate. In contrast, honeys such as rewarewa.have been found to contain fewer methoxylated phenolic compounds and more non-methoxylated phenolics such as gallic acid. As noted in the above description, methdxylated compounds appear to have a greater degree of potency. 16 WO 2010/082844 PCT/NZ2009/000300 EXAMPLE 4 As noted above in Example 3, methoxylated phenolic compounds appear to have a greater presence in honeys (and hence nedtars from honeys) that are associated with greatermedica activity ng manuka honey. Afurther example is provided below demonstrating the quantity of methoxylated phenolic compounds in a variety of honeys and their comparative levels to further exemplify the presence of these methoxylated compounds in more 'active' honeys as opposed to less 'active' honeys. In this example a wide range of honeys were tested using the same criteria to measure the presence and concentration of 2-methoxybenzoic acid as a representative methoxylated phenolic acid. The results found are shown below in Table 3. Table 3 - Honey and Methoxylated Phenolic Compound Concentrations Sample 2-Methoxy Principal floral origin (and possible floral Geographic - Honey age BenoicAcid contaminate* origin (bear)[m/g Manukaa L scoparium var. incanum (Trifolium spp.) 0.1 Northland 32.7 Manuka L scopanum var. Incanum (Tifolium spp.) 0.5 Northland 28.9 Manuka" L scoparum var. incanum (Trifolium spp.) 0.9 Northland 29.0 Manukab L scoparum var. incanum (hive site not assessed) 2.5 Northiand 52.1 Manukab L scoparium var. incanurn (hive site not assessed) 3.5 Northland 50.7 Manukab L scoparium var. Incanum (hive site not assessed) 4.75 Northland 22.2 Manukab L scoperum var. incanum (hive site not assessed) 5 Northiand 14.8 Manukab L scoparium var. incanum (hive site not assessed) 5 Northland 36.3 Manuka" L scoparium var. incanum (Trifolum spp., Knighda 0.4 Northland 5.7 Manuka" L scopanrum var. Incanum (TDfolium spp. K. oxceisa, 0.75 Northland 4.3 Manuka. L scoparum var. linifolum (Trifolium spp., Weinmannia 0.25 Waikato 22.2 Manuka! L scopwrum var. nifolum (TrifOllum spp., Weinmannia 0.5 Waikato 23.3 Manuka' L scoparum var. unifaium (hive site not assessed) 4 Walkato 4.5 Manuka* L scoparium var. myrtifollum (Trifum spp., Knightda 0.5 Whanganul 1.2 Manuka' L scoparium var. triketoned (rfolum spp.) 0.1 East Coast 5.9 Manuka" L scoperlum var. triketoned (Tifoium spp.) 0.3 East Coast 6.4 Manuka" L scoparum var. triketone (Trifolium Spp.) 0.5 East Coast 6.4 Manuka L scoparium var. triketoned (hive site not assessed) 5.5 East Coast 1 A Manuka L scoperiurn (varety unknown, hive site not assessed) 1.5 Unknown 9.9 Kanuka Kunzea ericoldes (Tiffoliurn spp.) 0.1 . Northland Trace Kanukab. Kunrea edcoides (hive site not assessed) 1.5 Northland 0.7 Kanukab Kunzea ericoldes (hive site not assessed) 2.5 Walkato 0.3. Kanuka. Kurse ercoides (hive ste not assessed) 3.5 East Coast 11 17 WO 2010/082844 PCT/NZ2009/000300 Clover 0 TrfolIum spp. (hive site not assessed) 1 South Island Trae Rewarewa KnIgha exces (hive aste not assessed) 5 Bay of Plenty 0.4 Nectar"~ L scopsfurn var. incanum cultivar,4 samples - Bay of Plenty 17.7(8.6) ; Laptspermum Nichollsli deived gultWar 2 sample Bay of Plenty 7.6 (2.1) Neata,' Kunzea ericoldes, 1 sample - Bay of Plenty 0.5 "Samples collected from hive sites; b Aded samples from drums supplied by apiarisis and purchased as designated type; c Commercially labelled product; _ Unclassified L scoparium variety that carries an enhanced-triketone essential oil profile; Nectar samples collected from flowering specimen; Qualitative measurement. 5 As shown in Table 3, the concentration of 2-methoxybenzoic acid is higher in manuka origin honeys than either kanuka, clover or rewarewa derived honeys suggesting methoxylated phenolic compounds may be important to medical efficacy. EXAMPLE 5 10 In this example, tests were completed to confirm the presence of phenolic compounds in plant nectar from which honey is derived. The phenolic components can be isolated from the nectar of plant varieties and species. Table 4 below illustrates some of the components isolated mg/kg from two distinct cultivars of Leptospermum scoparium, and Kunzea ericoides. All of the phenolic compounds that are 15 present in the honeys are derived from these species and are present in the species' nectar. Table 4 - Phenolic components measured in cultivars of Leptospermum scoparium and Kunzea encoides (mg/kg) CL) CD E (C ~E- E C E 0 )% 0)0 . s0 8

.

-1 L. scopaiu 40 330 8.6911.8 - 3 o 'Bvr. L. scoparium 940. 330 8.9 91.3 321 cultivar 2 Kunzea 380 850 14.3- Trace 72 Nil ericoides Detected 18 WO 2010/082844 PCT/NZ2009/000300 Give e that the honey bees perform about aten-fold concertration of the nectar during the conversion into honey. it is apparent three of these principal components are relatively more co centrated in the nectat than in the honey: This is evidence of in vivo phenolic self condensatio rreations occurring as the hney bees erform nectar dehydration. S invivo self-condensation reactions have been well described in the study of aging in wine (Monagas et al. 20041). Incontrast syringic acid concentration is similar in nectar and fresh honey indicating that this molecule is mostly present as hydrolysable tannin in the nectar and-the increased concentration in aged honey may be due to tannin body degradation. The analysis of nectar components in various glasshouse conditions provides measurement of the plants production of the different components, and secondly production efficiency in: different environments. This allows breeding selection to be tailored to fit the intended locations for plantation establishment. EXAMPLE 5 A further qualitative example is now provided illustrating the impact of stress on plant nectar. In experiments completed by the inventors, manuka plants of a similar age and condition were split into two groups, one group being a control grown outdoors under standard growing conditions while the second group was grown in a greenhouse under elevated temperatures. The resulting 'stress' of the elevated temperatures increased the volume of nectar flow. qualitatively by at least a 1 0-fold level over the volume of nectar produced by non-stressed plants. This trial further illustrates the role of stress in plant nectar generation. EXAMPLE 6 In this example the link between honey tannin levels and nectar tannin levels for several plant species is described. Plant stress and the influence this has is also described. The phenolic content of nectar collected from 'Leptospermum scoparium (manuka) and fresh field honey sourced from areas that yield monofloral manuka honey Illustrate the relationship between nectar and honey constituents. Taking into account the concentration of nectar into honey by the honeybees (Apis mellifera), the principal phenolic components are present- in the nectar in similar proportions of that recorded in the fresh honey. Methylglyoxal is present. in 31 Monagas, M.; Gomez-Cordoves C.; Bartolome, B. 2004. Evolution of the phenolic content of red wines from Vilds vinifera L during ageing In bottle. Food Chem. 95(3) 405-412. 19 WO 2010/082844 PCT/NZ2009/000300 Lept6spermum'sdoparium nectar: .. Subsequent degradation of the tannin matrix would appear to be responsible for the increasing.; con centration of phenolic molecules in the. agin honey. Leptospermui scoparium (manuka) plants subjected td heat or water-deficit induced oxidative damage and aninbreased concentration of phenolic compounds in the nectar. This response is due to an increased level of both abscisic and salicylic acid in'the plant; and such nectar enhancements can be effected when the plants are subject to foliar applications of these, hormones. Ukewise the phenolic content of nectar harvested from Kunzea ericoides (kanuka) also contains the same principal phenolic components as the honey. The above findings illustrate that, at least for manuka and kanuka there is a link between phenolic levels in the nectar and that measured in the corresponding honey. The findings also illustrate that heat or drought stresses on plants influence concentration of phenolic compounds in the plant nectar and that this is not specific to one species of plant. EXAMPLE 7 In this example, a process of breeding plants to manipulate phenolic compound concentrations in the plant.nectar is described. One or more practical examples where the nectar of various plant sources (e.g. manuka, kanuka, clover, buckwheat, rewarewa etc.) are analysed for phenolics and/or MGO and plants subsequently selected and bred or even cross bred. Examples either in conjunction with the above or separate showing how other selection factors (growth rate, flower density etc) are measured and also taken into account. Determination as to what an ideal concentration or volume of phenolics and MGO in nectar is also considered. Selection of plants producing greater levels of methoxylated phenolic compounds can also be completed. The nectar produced-by Leptospermum scoparium (manuka) can be analysed and comparisons. made between different wild varieties and domestic cultivars. For example a population of the, wild variety Leptospermum scoparium var. incanum exhibits statistically significant variation in phenolic component production, and the cultivars bred from this variety are also significantly different from the parent population and each other. The nectar from a cultivar bred from Leptospermum scoparium var. incanum consistently yields 20 WO 2010/082844 PCT/NZ2009/000300 more thandouble the quantity of phenolic molecules and-methylglyoxal than genetically similar-: plants housedin common conditions., When the concentration of nectar components during the conversion into honey is taken into account it is apparent the phenolic components and methyiglyoxal are present in the nectar in concentrations that are equivalent to fresh manuka honey derived from the same plants; prior to the storage degradation of the central tannin matrix that binds a large proportion of these molecules in fresh honev. Coupled with standard morphological selection for improved growth rates and flowering density in common garden conditions, selection by nectar analysis allows improved cultivars to be isolated from breeding populations. For example apical meristem growth can vary from,250 mm to 600 mm per year, and flowering density ranges between 7 and 18 flowers per 10 mm of stem. The ideal concentration of the antioxidant phenolic components in nectar would compensate for the oxidative stress produced by methylglyoxal and hydrogen peroxide in the honey, particularly where the honey is used as a dietary supplement or as a wound healing agent. Specific antioxidant activity of the methoxylated phenolic components appears to commence at a concentration of between 150-200 mg/kg' in the application media. Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope of the claims herein. 21

Claims (16)

1. A method of producing honey with levels of tannin derived phenolic compounds elevated above an average concentration for a given honey type by the steps of: a. increasing the concentration of tannin derived phenolic compounds in the nectar of a plant or plants by subjecting the plant or plants to stress; and, b. producing honey from the nectar of the stressed plant or plants; wherein plant stress in step (a) is induced by artificially subjecting the plant or plants to conditions selected from the group consisting of: drought, pests, selective watering, pruning, lack of nutrient(s), UV exposure, heat, externally derived abscisic acid, externally derived salicylic acid, and combinations thereof.

2. The method as claimed in claim 1 wherein the plant or plants are also exposed to fungal material.

3. The method as claimed in claim 1 or claim 2 wherein the fungal material includes complex carbohydrate compounds associated with the cell wall of fungal cells.

4. The method as claimed in any one of the above claims wherein the phenolic compounds in the plant nectar are elevated by 5-10,000 mg/kg above an average level for a selection of plant species.

5. The method as claimed in any one of the above claims wherein step (a) of the method also increases the methylglyoxal (MGO) level of the honey derived from the plant or plants.

6. The method as claimed in any one of the above claims wherein the method also increases the level of phenyllactic acid and/or related methoxylated and/or hydroxylated derivatives of phenyllactic acid in the honey derived from the plant or plants.

7. The method as claimed in any one of claims wherein the sum of the phenolic compounds phenyllactic acid and 4-methoxyphenyllactic acids and derivatives thereof increases in the plant nectar by 5-10,000 mg/kg above an average concentration for a given selection of plants.

8. The method as claimed in any one of the above claims wherein the tannin derived phenolic compounds in the honey derived from the plant nectar are in a form selected from the group consisting of: a free form, a complexed form, and mixtures thereof.

9. The method as claimed in any one of the above claims wherein the tannin derived phenolic compounds are selected from the group consisting of: phenolic acids, phenolic salts, phenolic esters, related polyphenolic compounds, and combinations thereof.

10. The method as claimed in claim 9 wherein the tannins are hydrolysable tannin compounds.

11. The method as claimed in any one of the above claims wherein the phenolic compounds are methoxylated. 22

12. The method as claimed in claim 11 wherein methoxylated phenolic compounds are present in the honey product at a level greater than 150 mg/kg.

13. The method as claimed in any one of the above claims wherein the phenolic compounds are selected from the group consisting of: methoxylated benzoic acids, syringic acid, methyl syringate, isomeric forms of methyl syringate, and combinations thereof.

14. The method as claimed in claim 13 wherein the methoxylated derivatives of benzoic acid are selected from the group consisting of: 2-methoxybenzoic acid, 4-methoxybenzoic acid, trimethoxy benzoic acid, and combinations thereof.

15. The method as claimed in any one of the above claims wherein the tannin derived phenolic compounds increased in the plant nectar also include phenolic compounds selected from the group consisting of: gallic acid and methoxylated derivatives, cinnamic acid, phenylacetic acid, methoxyacetophenone, ellagic acid, and combinations thereof.

16. Honey produced by the method as claimed in any one of claims 1 to 15. 23