WO2010082844A1

WO2010082844A1 - Plant manipulation

Info

Publication number: WO2010082844A1
Application number: PCT/NZ2009/000300
Authority: WO
Inventors: Jonathan Mcdonald Counsell Stephens; Ralf-Christian Schlothauer
Original assignee: Comvita New Zealand Limited
Priority date: 2008-12-24
Filing date: 2009-12-23
Publication date: 2010-07-22
Also published as: AU2009337191B2; AU2009337191A1

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

PLANT MANIPULATION TECHNICAL FIELD

The invention relates to plant manipulation. More specifically, the invention relates to methods of 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 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 activity (Silver et al 199O²).

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 studies began to document the anti-bacterial activity of honey against a number of pathogens (Allen 1991⁵, Wϊllix 1992⁸). 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 (Leptospeπnum scoparium) have been shown to possess the highest levels of anti-bacterial activity (Molan 1992⁷) and to be active against a range of pathogens including Staphylococcus aureus, coagulase-negative Staphylococci, Entβrococci and Psβudomonas aeruginosa (Cooper 25

¹RnCh, 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

⁵ 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

• 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. 1999\ Cooper 2002², Cooper 2002^s, 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 1999, Molan 2001^s, AIi 1991 % 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 H. pylori, the causative agent of gastritis and the major predisposing factor for peptic ulcer disease, gastric cancer and B-cell MALT lymphoma (Somal 1994⁷, Osato 1999⁸, Mitchell 1999⁹). 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. 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. pylori has increased dramatically leading to an increasing number of treatment failures (Fishbach 2007¹⁰). 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 some countries and is commonly associated with treatment failure (Raymond 2007").

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 2008¹²). Given this environment, alternative treatment approaches are of interest.

20

' Cooper RA. Molan PC. Harding KG. (1999).Antibactβrial activity of honey against strains of Staphylococcus aureus from infected wounds. Journal of the Royal Society of Medicine. 92(6)283-5

^■ Cooper RA₁ Halas E, Molan PC. (2002).Thβ efficacy of honey in inhibiting strains of Pseudomonas aeruginosa from infected bums. J Burn Care Rβhabil 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 coagulasβ-negative staphylococci Journal of Antimicrobial Chemotherapy 56, 228-231

^■ Molan PC. Potential of honey AM J Clin Dermatol 2001 £;13-19

^■ AT AIi, MN Chowdhury, MS at Humayyd. (1991 Inhibitory effect of natural honey on Helicobacter pylori. Trap Gastroenterol,

' N Al Somal KE Coley, PC Molan and B Hancock. (1994).Suscβptibility 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 OY. 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.

^■ Josθttβ Raymond , Christophe Burucoa Olivier Pietrini Michel Bergeret Anne Dβcostβr Abdul Wann, Christophe Dupont and Nicolas Kalach (2007) Clarithromycin 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 hβlicobacter Volume 12 Issue 2 Page 157-163.

" Elvira Marvic, Silvia Wittmann, Gβrold Barth and Thomas Hβntel (2008) Identification and quantification of methylglyoxal as the dominant antibacterial constituent of Manuka (Leptospermum scoparium) honeys from New Zealand MoI. Nutr. Food Res. 2008, 52, 000 - 000 While the antimicrobial activity of honey has been reported to include osmolality, acidity, hydrogen peroxide and plant-derived components, more recent studies have shown that - osmolarity, acidity and hydrogen peroxide activity cannot account for 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 2002³ 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

¹ White, J.W., Schepartz, A.I. 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 Biophysica Acta 73, 57-.

² Ferreres et at Natural occurrence of abscisic acid in heather honey and floral nectar. J. Agric. Food Chem. 199644, 2053-2056.

³ NeIe Gheldof, Xiao-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. origin.

Gheldof et ai 2002a¹ 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 2003³ 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 2001" 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

NeIe Gheldof and Nicki J Engeseth (2002) Antioxidant Capacity of Honeys from Various Floral Sources Based on the Determination of Oxygen Radical Absorbancβ Capacity and Inhibition of in Vitro Lipoprotein Oxidation in Human Serum Samples J. Agric. Food Chem 200250, 3050-3055.

² Francisco A. Tomas-Barbβran, Frederico Ferreres, Cristina Garcia-vlguera, and Francisco Tomas- Lorente (1993) Flavonoids in honey of different geographical origin. Z Lβbensm Untβrs Forsch 196:38-44.

³ LJhu Yao, Nivβdita Datta, Francisco A. Tomas-Barberan, Federico Ferreres, Isabel Martos, Riantoπg Singanusong (2003) Flavonoids, phenolic acids and abscisic acid in Australian and New Zealand Leptospermum honeys. Food Chemistry 81 (2003) 159-168.

⁴ Francisco A Tomas-Barberan, Isabel Martos, Fedeπco Ferreres, Branka S Radovic and Elke Anklam (2001) HPLC flavonoid profiles as markers for the botanical origin of European unlfloral honeys. J Sd Food Agric 81:485-496. 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 and the types of compound present may 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.

Mβthoxytation

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 WaIIe 2006a¹) and also have much improved intestinal transcellular absorption (Wen and WaIIe 2006b²). 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

¹ Wen, X., WaIIe, T. (2006a) Methylatioπ protects dietary flavonoids from rapid hepatic metabolism. Xenobiotica 36: 387-397.

² Wen, X., WaIIe, T. (2006b) Methylated flavonoids have greatly improved intestinal absorption and metabolic stability. DrugMetab. Dlspos. 34: 1786-1792. 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 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.

DISCLOSURE OF THE INVENTION

The invention broadly relates 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.

Finding the above synergies was surprising as this goes against recent publications that suggest that mβthylglyoxal (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 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 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 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 sites. The best researched comparison for such aging is the development of flavour and aroma in red 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 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 ion is sensitive to age like phenolics as well as other influences e.g. heat and acidification.

The improved healing effects or potency 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 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.

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 earned in a tannin 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 increasing the concentration of phenolic compounds in the nectar of a plant or plants by subjecting the plant or plants to stress in order to produce a honey derived from the plant or plants with an elevated concentration of phenolic compounds.

In preferred embodiments, plants may be stressed by artificially subjecting the plant or plants to conditions selected from the group consisting erf: 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, plants may be selected based on their degree of stress in the natural environment

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 (MΘO) in the honey produced from the plant. As may be appreciated, not all plant varieties produce methylglyoxal but selected varieties such as Leptospeπvum 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-iu,uuυ mg/kg above a baseline level without stress being subjected to the plant.

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 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 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 (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 i greenhouse.

In the fifth embodiment, the plant or plants may also have an increased concentration of methylglyoxal (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 cultivars of 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 polyphenols 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. Preferably, the phenolic compounds are methoxylated. As noted above, the prior art teaches some useful properties attributable to methoxylated compounds. The inventors have found that honey which includes methoxylated compounds exhibit useful medical and nutritional effects. By way of example, the inventors have analysed the phenolics prominent in manuka (Leptospermum spp.) and kanuka (Kunsβa spp.) and a large number of these phenolics are methoxylated at one or more 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, methoxygallic 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 art mean that effects envisaged for medical and nutritional applications include:

* Greater bioavailability due to the methoxylated compounds be able to enter the cell fasten

* Longer bioavailability due to the methoxylated compounds having a much longer half life within cells to scavenge free radicals;

* Phase Il 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-methoxyphenyllactic acids and derivatives thereof (particularly hydroxylated analogues). These may be increased in the plant nectar by 5-10,000 mg/kg. Examples of these compounds are illustrated below:

Phenyllactic acid 4-methoxyphenyllactic acid

In a young honey these compounds are understood by the inventors to typically account for 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- methoxybenzoic acid, 4-methoxybenzoic acid and isomers of trimethoxybenzoic acid as shown below:

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:

Syringic acid Methyl synπgate

These components are present as two isomers that are diagnostic and differentiate manuka and kanuka honeys.

In a further embodiment, the free phenolics may also include a suite of other compouπαs am< with the tannin matrix in honeys. These range from relatively simple molecules such as gallic acid and methoxylated derivatives, abscisic acid, cinnamic acid, phenylacetic acid and mβthoxylated and hydroxylated derivatives, and methoxyacetophenone; to complβxed polyphenols molecules such as ellagic acid. A range of these molecules are illustrated below

Gallic aαd 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 10,000mg/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 monofloral manuka, kanuka, and 27

¹ Monagas, M.; Gomez-Cordoves C; Bartolome, B. 2004. Evolution of the phenolic content of red wines from VRis vlnlfβra L during ageing in bottle. Food Chem. 95(3) 405-412. "other honeys harvested' in New Zealand and aged naturally for up to ten years;

Fioure 2 shows a graph illustrating the correlation between the sum of the principal phenolic components and methylglyoxal 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 Leptospeπvum species and vaπeties 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'¹ of these compounds, whereas in comparison the other honey types of the same age contain considerably less than 100 mg. kg^"1. 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 components differ significantly in these honeys.

Table 1 - The phenolic profile and concentration of principal components mg/kg in monofloral manuka and kanuka honeys harvested in New Zealand and aged naturally for ten years. Values shown, mean ± standard deviation

The concentration of methylglyoxal in the manuka and kanuka honeys is also listed in Table 1. Manuka honey, derived from Leptospermum scopariυm, contains methylglyoxal. 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 produced from Northland, Waikato and East Coast in New Zealand and a sample from Queensland, Australia.

As can be seen in Figure 3, the ratio of phenolic compounds allows separation by region, and botanic source. The concentration of 2-methoxy-benzoic and tri-methoxy-benzoic acids is significantly elevated in honey derived from Leptospermum polygalifolium in Queensland, Australia. Phenyllactic acid is elevated in honey from Northland, New Zealand where variety is Leptospermum scoparium var. incanum. 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 analysed to determine the antioxidant levels in the nectar derived honeys compared to control standards.

Antioxidant activity was determined by the ABTS assay using a spectrophotometry method for antioxidant activity using the ABTS radical assay (expressed as Trolox Equivalent Antioxidant Capacity) based on the method of Miller & Rice-Evans (1997)¹.

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

19

¹ Miller, N.J.; Rice-Evans, CA. 1997: Factors influencing the antioxidant activity determined by the ABTS + radical cation assay. Free Radical Research 26(3): 195-199.

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 TEEAC 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, methoxylated compounds appear to have a greater degree of potency. EXAMPLE 4

As noted above in Example 3, methoxylated phenolic compounds appear to have a greater presence in honeys (and hence nectars from honeys) that are associated with greater medical activity e.g. manuka honey.

A further 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

^■ Samples collected from hive sites; Aged samples from drums supplied by apiarists and purchased as designated type; ^c Commercially labelled product; " Unclassified L scoparium variety that carries an enhanced triketone essential oil profile; ^θ Nectar samples collected from flowering specimen; ' Qualitative measurement.

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 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 curtivars of Leptospermum scoparium, and Kunzea ericoides. All of the phenolic compounds that are present in the honeys are derived from these species and are present in the species' nectar.

Table 4 - Phenolic components measured in curtivars of Leptospermum scoparium and

Kunzea encoides (mg/kg)

Given that the honey bees perform about a ten-fold concentration of the nectar during the - conversion into honey it is apparent three of these principal components are relatively more concentrated in the nectar than in the honey. This is evidence of in vivo phenolic self- condensation reactions occurring as the honey bees perform nectar dehydration. Sucn 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 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/

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 10-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 mellifβra), the principal phenolic components are present in the nectar in similar proportions of that recorded in the fresh honey. Methylglyoxal is present in 31

¹ Monagas, M.; Gomez-Cordovβs C; Bartolome, B. 2004. Evolution of the phenolic content of red wines from vWs vinifβra L during ageing in bottle. Food Chem. 95(3) 405-412. Leptospermum scoparium nectar.

Subsequent degradation of the tannin matrix would appear to be responsible for the increasing concentration of phenolic molecules in the aging honey.

Leptospermum scoparium (manuka) plants subjected to heat or water-deficit induced oxidative damage and an increased 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.

Likewise 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. incaπum 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 more than double the quantity of phenolic molecules and methylglyoxal than genetically similar, plants housed in common conditions.

When the concentration of nectar components during the conversion into honey is taken into account it is apparent the phenolic components and methylglyoxal 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^"1 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.

Claims

WHAT IS CLAIMED IS:

1. A method of increasing the concentration of phenolic compounds in the nectar of a plant or plants by subjecting the plant or plants to stress in order to produce a honey derived from the plant or plants with an elevated concentration of phenolic compounds.

2. The method as claimed in claim 1 wherein plant stress 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.

3. The method as claimed in claim 1 wherein the plant or plants are selected based on their degree of stress in the nautral environment.

4. The method as claimed in any one of claims 1 to 3 wherein the method also increases the methylglyoxal (MGO) level of the honey derived from the plant or plants.

5. 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.

6. The method as claimed in any one of the above claims wherein the 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.

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

8. The method as claimed in any one of the above claims wherein the phenolic compounds are derived from tannin compounds.

9. The method as claimed in claim 8 wherein the tannins are hydrolysable tannin compounds.

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

11. The method as claimed in claim 10 wherein methoxylated phenolic compounds are present in honey produced from the nectar at a level greater than 150 mg/kg.

12. The method as claimed in any one of claims 1 to 11 wherein the phenolic compounds are 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.

13. The method as claimed in claim 10 wherein the methoxyiated derivatives of benzoic acid are selected from the group consisting of: benzoic acid, 2-methoxybenzoic acid, 4- mβthoxybenzoic acid, trimethoxy-benzoic acid, and combinations thereof.

14. The method as claimed in any one of claims wherein the sum of the phenolic compounds phenyllactic acid and 4-methoxyphenyllactic acids and denvatives thereof increases in the plant nectar by 5-10,000 mg/kg.

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

16. A plant for use in honey production that has been subjected to the method as claimed in any one of claims 1 to 15.

17. Honey with elevated levels of phenolic compounds produced by the method as claimed in any one of claims 1 to 15.

18. A plant that has been stressed with an elevated concentration of phenolic compounds in the plant nectar of between 5 to 10,000 mg/kg in order to produce honey from the plant that has elevated levels of phenolic compounds.

19. The plant as claimed in claim 18 wherein plant stress is induced by artificially subjecting the plant or plants to conditions selected from the group consisting of: drought, pests, selective watering, pruning, lack of πιrtrient(s), UV exposure, heat, externally derived abscisic acid, externally derived salicylic acid, and combinations thereof.

20. The plant as claimed in claim 18 wherein stress occurs due to natural environmental conditions.

21. The plant as claimed in any one of claims 18 or 20 wherein the plant nectar also includes an elevated level of methylglyoxal (MGO).

22. The plant as claimed in any one of claims 18 to 21 wherein the phenolic compounds are in a form selected from the group consisting of: a free form, a complexed form, and mixtures thereof.

23. The plant as claimed in any one of claims 18 to 22 wherein the phenolic compounds are selected from the group consisting of: phenolic acids, phenolic salts, phenolic esters, related polyphenols compounds, and combinations thereof.

24. The plant as claimed in any one of claims 18 to 23 wherein the phenolic compounds are derived from tannin comDOunds.

25. The plant as claimed in claim 24 wherein the tannins are hydrolysable tannin compounds

26. The plant as claimed in any one of claims 18 to 25 wherein the phenolic compounds are methoxylated.

27. The plant as claimed in claim 26 wherein honey produced from the plant contains at least 150 mg/kg of methoxylated phenolic compounds.

28. The plant as claimed in any one of claims 18 to 27 wherein the phenolic compounds are 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.

29. The plant as claimed in claim 28 wherein the methoxylated derivatives of benzoic acid are selected from the group consisting of: benzoic acid, 2-methoxybenzoic acid, 4- methoxybenzσic acid, trimethoxy-benzoic acid, and combinations thereof.

30. The plant as claimed in any one of claims 18 to 29 wherein the sum of the phenolic compounds phenyllactic acid and 4-methoxyphenyllactic acids and derivatives thereof in the plant nectar is elevated by at least 5-10,000 mg/kg.

31. The plant as claimed in any one of claims 18 to 20 wherein the phenolic compounds elevated in the plant nectar also include phenolic compounds selected from the group consisting of: gallic acid and methoxylated derivatives, abscisic acid, cinnamic acid, phenylacetic acid, methoxylated and hydroxylated derivatives of phenylacetic acid, methoxyacetophenone, ellagic acid, and combinations thereof.

32. 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 cumVars of the plant that produce the highest amount of phenolic compounds in the nectar.

33. The method as claimed in claim 32 wherein the plant or plants are also selected based on the extent to which they produce elevated levels of phenolic compounds in the plant nectar when stressed.

34. The method as claimed in claim 32 or claim 33 wherein the plant or 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.

35. Honey produced from the nectar of a plant or plants that have been stressed to cause an increased concentration of phenolic compounds in the honey.

36. The honey as claimed in claim 35 wherein the concentration of phenolic compounds in the honey is increased by at least 5-10,000 mg/kg.

37. The honey as claimed in claim 35 or claim 36 wherein the concentration of methoxylated phenolic compounds in the honey is greater than 150 mg/kg.