WO2009007964A2 - Pectin methyl esterase-inhibiting polyphenolic flavonoids and use thereof - Google Patents

Abstract

The present invention relates to compositions comprising polyphenolic flavonoids in general and to polyphenolic flavonoids extracted from green tea in particular, useful to prevent or retard pectin degradation. Green tea polyphenols, especially catechins are now shown to inhibit the cell wall degrading enzyme pectin methyl esterase. The invention further provides methods of using these enzyme inhibitors, including agricultural uses to inhibit plant parasites and other plant pathogens, particularly to inhibit parasitic plants in or on a host plant, and as additives to processed foods and juices.

Description

PECTIN METHYL ESTERASE-INHIBITING POL YPHENOLIC FLAVONOH)S AND USE THEREOF

FIELD OF THE INVENTION The present invention relates to compositions comprising polyphenolic flavonoids in general and polyphenolic flavonoids extracted from green tea in particular, useful as inhibitors of pectin degradation. The invention further provides methods of using these compositions, including agricultural and horticultural uses to inhibit the development of parasites and other plant pathogens, particularly the development of parasitic plants, and as additives to processed foods and juices.

BACKGROUND OF THE INVENTION

Pectin methyl esterase (PME), which digests esterified homogalacturonan components of pectin found in plant cell walls, is a critical enzyme for tissue remodeling, growth, and fruit maturation, among other processes. Homogalacturonan, one of the two pectin polysaccharide backbones, is highly methyl-esterified when exported into cell walls and is subsequently de-esterified by the action of PME and other pectic enzymes. The control of methyl esterification levels by PMEs has been reported to have a direct effect on the regulation of a wide range of processes in plant physiology, including cell-to-cell adhesion and separation (e.g., abscission), cellular elongation, germination and seedling growth and fruit ripening. Some PMEs are ubiquitously expressed, whereas others are expressed in specific tissue regions or during specific life stages or events, such as pollination (Jiang LX et al., 2005 Plant Cell 17, 584-596), or parasitic plant haustorial formation (Nagar R et al., 1984 J Exp Bot 35, 1104-11 12). The data suggest that PMEs are encoded by a family of genes that are differentially regulated by cell type in response to specific developmental or environmental scenarios. International Patent Application Publication No. WO 2006/068603 discloses, for example, that modifying the expression of a pectin methyl esterase (PME) gene in transgenic plants results in improved biomass related property, such as an altered lignin content, lignin composition or extractability of lignin, and an altered fiber length.

In addition, enzymatic activity is regulated by direct interaction with endogenous inhibitors in plant tissue (Giovane A et al., 2004 BBA: Proteins and Proteomics 1696, 245-252; Raiola A et al., 2004, FEBS Lett 557, 199-203; WoIf S et al., 2003, FEBS Lett 555(3), 551-555). These protein inhibitors specifically interact with the enzyme active site region and hamper substrate access.

Parasitic plants comprise about 1% of flowering plant species and cause considerable agricultural losses, particularly in Africa and the Middle East by reducing growth and reproduction in their hosts (reviewed, for example, in Musselman LJ & Press MC 1995 In:. Press M C. Graves JD eds. Parasitic Plants. London, UK: Chapman & Hall, 1-13.; Riches CR & Parker C 1995 Wallingford, Oxon: CAB International). The similarity of parasitic plants to their hosts limits the use of many herbicides for parasite control, leaving methods such as weeding, fallow periods, sowing date modifications, and trap cropping, in which resistant crops are grown in infested soils to trigger germination and subsequent failure of parasites, as the most effective solutions for reducing parasite damage to susceptible crops. More recently, genetic engineering for resistant crops has become a possible option. International Application Publication No. WO 2006/098626 discloses the production of crop species that do not induce germination of parasitic plant seeds and therefore are resistant to these parasites. Inhibition of germination induction is achieved by silencing genes involved in the biosynthesis of the germination inducing compounds, strigolactones.

Previous work on parasitic plant invasion of host tissue has indicated that parasitic plants utilize cell wall degrading enzymes (CWDEs) such as PME; polygalacturonase

(e.g. Nagar et al., 1984, ibid; Bar Nun N & Mayer A m 1999 Phytochemistry 50, 719-

727; Veronesi C et al., 2005, 2005 Israel J. Plant Sciences 53, 19-27); xylanase and cellulase (Singh A & Singh M 1997 J. Plant Physiol. 150, 592-596). Parasites not only invade host tissue through the use of these enzymes, but may also utilize the degradation products of these enzymes to further enhance attachment to the host, as pectin degradation products can form mucilage that helps the parasite cling to the stem.

Antibody labeling has shown that the host tissue at the site of haustorial invasion of root parasites is depauperate in pectin (Losner-Goshen D et al., 1998 Annals of Botany 81,

319-326; Neumann U et al., 1999 Protoplasma 207(1-2), 84-97), suggesting active degradation by the parasite. Furthermore, it has been shown that extracts of stem peelings from hosts resistant to Cuscuta inhibit the activity of many cell wall degrading enzymes in vitro (Singh & Singh, 1997 ibid). Residual PME activity is problematic in a variety of food processing settings due to the degradation of texture and appearance of plant foods due to PME activity even after thermal deactivation techniques have been applied (e.g., Castaldo et al., 1996 Int J Food Sci and Technology 31, 313). Thus, PME inhibitors have been suggested as food additives for improving nutritional and taste quality (Giovane et al. 2004, ibid), although any additive inhibitors to foods must be safe for human consumption.

However, the use of proteinaceous inhibitors is complex and hence not trivial, Such that the use of small molecule inhibitors would be more tractable as applied enzyme inhibitors. To our knowledge, no small molecules have been implicated in inhibition of PME to date. Yet plants are adept at producing highly bioactive small molecules. Previous work using antisense technology to inhibit PME (e.g., Pilling J et al., 2004 Planta 219, 32-40) has shown systemic effects of PME inhibition, but antisense products are difficult to confine to particular plant parts or life stages, nor are they tractable for field experiments on non-model systems. Green tea extract is a potent inhibitor of activity or expression of a variety of enzymes in vivo and in vitro, including esterases (Okello EJ et al 2004 Phytotherapy Res 18, 624-627) although enzyme activation has also been found in some cases (Ayoub S &. Melzig MF 2006 J Pharmacy Pharmacol 58, 495-501). The enzyme inhibiting and inducing activities of green tea are mainly attributed to a group of polyphenolic flavonoids, particularly catechins. The catechins may inhibit enzymes by direct competitive interactions, by altering regulation of expression at the genetic level, or both. The gallate-containing catechins, epigallocatechin gallate (EGCG) in particular, have been implicated in inhibition of FabG and Fabl reductases (Zhang YM & Rock CO 2004 J Biol Chem 279, 30994-31001), carboxypeptidases such as angiotensin- converting enzyme (Persson IAL et al. 2006, Pharmacy and Pharmacology 58, 1139- 1144), as well as nitric oxide synthase (Singh R et al. 2002 Arthritis and Rheumatism 46, 2079-2086), oxidases (Morre DJ et al. 2003 Pharmacology & Toxicology 92, 234- 241) and matrix-degrading metalloproteinases in in vitro systems (Vankemmelbeke MN et al., 2003, Eur J Biochem 270, 2394-2403; Ahmed et al., 2004, J Pharma Experim Therap 308, 767-773).

Green tea extract has been shown to have various therapeutic effects. For example, U.S. Patent No. 5,135,957 discloses the use of tea polyphenols, particularly catechins, theaflavin and derivatives thereof for treating fungal infection of the skin (tinea). It has been also shown that green tea extracts, and particularly the tea-derived polyphenols, affect many cancer-related proteins and have anti-tumor properties.

U.S. Patent No. 6,410,061 discloses methods and compositions of treating cancer or solid tumors by administration of a therapeutically effective amount of particular combination of catechins selected from epigallocatechin gallate (EGCg), epicatechin (EC), epicatechin gallate (ECG) and epigallocatechin (EGC).

U.S. Patent No. 6,713,506 discloses how ester-bond containing tea polyphenols potently inhibit the proteasomal chymotrypsin-like activity in vivo and in vitro. That invention shows that treatment with tea-derived polyphenols is correlated with accumulation of cellular levels of both p27^KlP1 and IKB-α., two natural proteasome substrates, and Gi -phase arrest of tumor cells. The invention further discloses use of the polyphenol esters in prevention and treatment of conditions characterized by abnormal cellular proliferation. U.S. Patent Application Publication No. 20080075795 discloses the use of tea extract containing catechins, theaflavin and derivatives thereof for treating poultry infected with the avian influenza viruses.

Thus, catechins have previously been implicated as inhibitors of a variety of mammalian enzymes, but have not been shown to inhibit plant enzymes. Parasitic plants use esterases, among other enzymes, to degrade cell wall matrices in host tissue, a phenomenon that shares similarities with matrix degradation by metalloproteinases. However, nowhere in the background art has it been taught or suggested that the similar functions of these enzymes might be indicative of susceptibility to similar inhibitors.

Thus, it would be highly advantageous to have compositions and methods capable of inhibiting deleterious effects of pectin methyl esterase activity.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for inhibiting cell wall degrading enzymes (CWDEs). According to certain embodiments the compositions comprise potent inhibitors of the enzyme pectin methyl esterase (PME). Particularly, the compositions of the present invention comprise gallate-containing catechins selected from (-)-epigallocatechin gallate (EGCG), (-)-gallocatechin gallate (GCG). The compositions and methods of the present invention are useful in agriculture as well as in the food industry, for inhibiting deleterious activity of PME. Particularly, the catechin-containing compositions of the present invention are useful in inhibiting the development of plant-parasites or plant pathogens in or on the host plant, and in inhibiting or preventing pectin degradation in processed food products and beverages.

The present invention is based in part on the unexpected identification of novel inhibitors of pectin methyl esterase activity. It is now disclosed for the first time that green tea extract rich in flavonoids inhibits isolated PME activity in vitro, and inhibits parasitic plant PME extracts from both Cuscuta pentagona and Castilleja indivisa, representatives of two of the largest families of parasitic plants, the Cuscutaceae and Orobanchaceae. Furthermore, the present invention now discloses that the green tea extract inhibits C. indivisa germination and attachment to its native host Lupinus texensis.

It is further disclosed that the most active inhibitors of PME activity from among the catechin components found in green tea are EGCG and GCG. Thus, the catechins containing active galloyl esters were found to be the most active in PME inhibition.

According to some aspects, the present invention provides methods of controlling parasite or pathogen development in or on a plant comprising applying to the plant a composition comprising a pectin methyl esterase inhibitor. According to one aspect, the present invention provides a method of controlling parasite or pathogen development in or on a plant comprising applying to the plant a composition comprising a pectin methyl esterase inhibitor, wherein the pectin methyl esterase inhibitor comprises at least one polyphenol flavonoid.

According to certain embodiments, the composition comprises a plant extract. According to certain embodiments, the extract is a green tea extract. According to certain embodiments, the green tea extract comprises catechins. According to other embodiments, the green tea in enriched for catechins.

According to certain embodiments, the pectin methyl esterase inhibitor is a catechin. The catechin may be provided in the green tea extract, or it may be isolated or synthetic catechin.

According to typical embodiments, the PME-inhibiting catechin is a gallate containing catechin selected from the group consisting of (-)-epicatechin (EC), (-)- epicatechin gallate (ECG), (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCG) and (-)-gallocatechin gallate (GCG). According to particular embodiments, the gallate containing catechin component is a galloyl ester and the catechin is selected from EGCG, GCG and derivatives and salts thereof.

According to certain embodiments, the composition applied to the plant is an agricultural composition further comprising an agriculturally acceptable diluent or carrier. According to one embodiment, the composition further comprises at least one additional active ingredient selected from the group consisting of an herbicide and a pesticide. The pesticide can be any one of a bactericide, fungicide, nematicide, and insecticide.

According to certain embodiments, the plant parasite is selected from the group consisting of parasitic angiosperms, nematodes, plant-parasitic fungi and plant-parasitic insects. According to other embodiments, the plant pathogens are selected from bacteria and fungi.

According to certain embodiments, the parasitic angiosperm is a member of a family selected from the group consisting of Cuscutaceae, Orobanchaceae,

Loranthaceae and Viscaceae (mistletoes). According to typical embodiments, the plant parasite is selected from Cuscuta pentagona (dodder) and Castilleja indivisa (Indian paintbrush).

According to additional embodiments, the composition is formulated in a form selected from the group consisting of a solution, an aerosol, a suspension, an emulsion, a particulate form, a microbead, a nanoparticle and a powder.

According to further embodiments, the composition is applied to the plant in a form selected from the group consisting of spraying, dripping, and dispersing.

According to certain embodiments the compositions may be applied in agricultural settings to prevent attachment of a plant parasite to potential hosts, or to prevent development of a plant parasite or pathogen on the plant host, thus improving agricultural yields. According to certain embodiments the compositions are exogenously applied to the host crops. According to certain embodiments, the compositions are applied to the crop seeds before planting. According to specific embodiments the compositions are applied to the roots of host crops. According to other embodiments the compositions are applied to the aerial part of the host crop, including to stem, leaves, and fruit.

According to additional aspects the compositions of the present invention are useful in the food industry for prevention of cloud loss and degradation in processed food products and juices.

According to one aspect, the present invention provides a method for retarding or preventing pectin degradation in a processed food product or beverage comprising adding to the processed food or beverage a composition having a pectin methyl esterase inhibiting activity. Advantageously, the present invention now discloses that plant extracts comprising polyphenol flavonoids, being suitable for use in foods and beverages are highly efficient in inhibiting the activity of methyl pectin esterase.

According to certain embodiments, the plant extract is a green tea extract. According to other embodiments, the green tea extract comprises catechins, particularly gallate-containing catechins described hereinabove. According to further aspects the present invention provides compositions for the application of EGCG, GCG, derivatives or salts thereof or green tea polyphenolics in the field and/or in the food industry. According to certain embodiments, the compositions are useful in methods of controlling plant parasites and/or pathogens in agricultural or silvicultural settings. According to other embodiments, the compositions are useful in inhibiting or preventing pectin degradation in processed food or beverages.

According to one aspect, the present invention provides an agricultural composition comprising at least one gallate-containing catechin selected from the group consisting of EGCG, GCG in an amount effective in inhibiting the activity of an isolated PME by at least 20%. According to certain embodiments, the present invention provides an agricultural composition comprising at least one gallate-containing catechin selected from the group consisting of EGCG and GCG in an amount effective in inhibiting the activity of an isolated PME by at least 20% for controlling plant-parasite development in or the plant.

According to another aspect, the present invention provide a food-grade composition comprising at least one gallate-containing catechin selected from the group consisting of EGCG and GCG in an amount effective in inhibiting the activity of an isolated PME by at least 20%.

According to certain embodiments, present invention provide a food-grade composition comprising at least one gallate-containing catechin selected from the group consisting of EGCG and GCG in an amount effective in inhibiting the activity of an isolated PME by at least 20% for inhibiting or preventing pectin degradation in processed food or beverages.

According to certain embodiments, the agricultural or food grade composition comprises plant extract, particularly green tea extract.

According to certain embodiments, the compositions comprise a combination of EGCG and GCG. According to other embodiments, the composition comprising a combination of EGCG and GCG further comprises at least one additional catechin, particularly at least one gallate-containing catechin. According to one embodiment, the concentration of the combined total amount of EGCG and GCG is at least equal to the amount of the at least one additional catechin. According to additional embodiments, the at least one catechin is present in the compositions of the present invention in an amount effective in inhibiting the activity of an isolated PME by at least 30%, at least 40%, at least 50% or more.

According to other embodiments, the concentration of the at least one catechin is in the range of from about 0.01% to about 10% by weight. According to other embodiments, the catechin concentration is in the range of from about 0.03% to about

7% by weight. According to yet further embodiments, the catechin concentration is in the range of from about 0.06% to about 6% by weight.

The compositions of the invention comprising the inhibitors of CWDEs may comprise any suitable excipients, diluents and surfactants as are well known in the art of food and agricultural sciences. According to various embodiments the compositions may be used in formulations selected from a solution, an aerosol, a suspension, an emulsion, a dispersible solid, a particulate form, a microbead, a nanoparticle, and a powder.

According to specific embodiments the formulation will be in sustained release, controlled release or in slow-release form.

According to yet another aspect, the compositions and methods of the present invention are useful to inhibit esterase activity of cell wall degrading enzymes, including PME and cutinases, in tissue invasive systems across taxa. According to specific embodiments the compositions and methods of the invention are useful for inhibiting the invasive capacity of parasitic plants, nematodes, fungal and bacterial parasite as well as non-parasitic pathogens in plants.

These and additional features and uses of the present invention will be better understood in conjunction with the figures, examples and claims that follow.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows sample image of agarose plate showing inhibition of various dilutions of PME by PP60.

FIG. 2 demonstrates the effect of varying PME concentration (activity) and varying PP60 concentration on stain diameter in agarose plates. Figure 2A: Relationship of diameter of staining and PME concentration; Figure 2B: relationship of diameter of staining of a consistent PME solution with the addition of varied PP60 concentration, with 95% confidence limits.

FIG. 3 shows inhibition of parasitic plant PME extracts by PP60. Mean diameter of staining showing one-way ANOVA results (effect of treatment F₇₂₈ =1470.9, pO.OOOl) and Tukey's HSD test results for PME solution (1 mg/ml), C. pentagona and C. indivisa PME extract with PP60 (50 mg/ml) added. Diamonds are 95% confidence intervals. Non-overlapping circles in the Tukey's HSD test show significant differences.

FIG. 4 demonstrates the effect of individual catechins on PME activity. Mean diameter of staining showing one-way ANOVA results (treatment main effect, F_{5 18} =162.4, pO.OOOl) and Tukey's HSD test results for PME solution (0.75 mg/ml) with PP60 components added. Diamonds are 95% confidence intervals. Non-overlapping circles in the Tukey's HSD test show significant differences.

FIG. 5 illustrates PME inhibition by EGCG measured by fiuorometry. The figure shows comparison of baseline PME esterase activity (top line, in black) with the inhibition of PME activity by EGCG (0-4.0 mM). The series of lines show decreasing production of the cyano-acetate fluorescent product (λ_em = 465 nm) in PME solutions with increasing concentration of EGCG. The inset shows the relationship between inhibitor concentration and the ratio of fluorescence of the solution versus the baseline. K; = 420 μM ± 20 (curve fit r²=0.995).

FIG. 6 presents docking model results showing the interaction of EGCG with the catalytic site of PME. The spheres represent distribution of the ligand's geometric centers around the binding site area. The number of spheres (100) corresponds to the number of the random docking runs. Catalytic residues Dl 36, Dl 57, W252 and W227 are highlighted.

FIG. 7 presents lineweaver-Burk Plot for interaction of PME with EGCG, indicating competitive interaction. This analysis was limited to a small range of substrate concentrations due to quenching problems at higher concentrations. FIG. 8 presents fluorometric data showing fluorescence resonance energy transfer (FRET) between PME and EGCG. Figure 8A: Effects of sequential EGCG addition to PME solution on inherent fluorescence intensity, showing decreased fluorescence by the PME (peak at approximately 340 nm), and concomitant FRET signal showing an increase in EGCG fluorescence emission at approximately 390 nm due to very close proximity to the PME tryptophans. Figure 8B: Active site cleft of PME (PDB code:lgq8) defined by residues D136, D157, are marked in thick lines; W227 and W252 marked in stripped lines are located at the rim of the active site. Figure 8C: Structure of EGCG.

FIG. 9 shows average number of seedlings germinated per Petri dish at a given time for L. texensis (host) (Figure 9A) and C. indivisa (parasite) (Figure 9B) in cold, wet stratified seeds treated with varying levels of catechins, green tea infusion, or water.

Error bars represent standard error for number of seeds per Petri dish within a treatment.

FIG. 10 demonstrates host-parasite attachment. Figure 1OA: Proportion of C. indivisa attached to host in the varying catechin levels experiment at 21 days after parasite addition, and proportion of attachment of two different C. indivisa plants within each pot 35 days after parasite addition. Figure 1OB: Proportion of C. indivisa attached in the fertilization effects versus catechin experiment at 20, 28, 36 and 44 days after parasite addition to host pots.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS The present invention discloses for the first time that compounds in green tea can be used to inhibit pectin methylesterase (PME) activity. The most active inhibitors of PME activity were gallate-containing catechins, particularly EGCG and GCG.

Due to their PME inhibition activity, the compositions of the present invention are useful for controlling the development of plant parasites and pathogens in or on the plant, as well as for controlling PME activity in processed food and beverages. Definitions

As used herein, the term "pectin methyl esterase" (PME) refers to a plant enzyme that hydrolyzes the methyl ester of pectin. The enzyme cleaves the ester bonds that form the crosslinks between pectic polysaccharides, exposing the pectin to further degradation by other pectic enzymes. A skilled artisan would recognize that by "tea" it is meant an aqueous extract, usually with hot or cold water, of certain plant leaves. Many types of plants are suitable for tea preparation. Preferably, commercially available tea, i.e. dried and in certain instances, fermented leaves of the plant Camellia sinesis (green tea or black tea) is used. Many commercial tea products are decaffeinated, but are nevertheless suitable for the preparation of tea-derived polyphenol compounds of the instant invention.

As used herein, the term "green tea" refers to an aqueous extract made solely from leaves of the plant Camellia sinesis.

As used herein, the term "plant extract enriched for catechins", particularly "green tea enriched for catechin" refers to an extract containing at least about 40% by weight, about 50%, about 60%, or about 70% by weight or more at least one type of catechin.

According to typical embodiments, the green tea extract enriched for catechins comprises about 60% catechins by weight.

As used herein, the terms "catechin" or "catechins" refer to polyphenols antioxidant plant metabolites, specifically fiavonoids. Catechin and epicatechin are epimers, with (-)-epicatechin and (+)-catechin being the most common optical isomers found in nature. Epigallocatechin and gallocatechin contain an additional phenolic hydroxyl group when compared to epicatechin and catechin, respectively. Catechin gallates are gallic acid esters of the catechins.

As used herein the term "about" refers to ±10%. It is required that any numeric value in the application will be referred to as if it is preceded by the term "about".

Because of the role played by cell wall degrading enzymes in the invasion of parasites to plant, it is now disclosed that inhibition using exogenous compounds such as green tea extracts provide a novel approach to control plant parasites as well as nonparasitic plant pathogens. It is also disclosed that the inhibitors of cell wall degrading enzymes are useful in preventing pectin degradation in food products and beverages. The data disclosed herein suggest that while EGCG or GCG can be identified and utilized as an isolated compound, even simple green tea extract is effective.

Among the many uses of the compositions and methods disclosed herein the following are considered some non limiting examples:

1. Use of plant extracts containing polyphenol flavonoids, particularly catechins, in the food industry, for the prevention of cloud loss and degradation of processed food products and juices. Conveniently, the catechin-containing plant extract used in a green tea extract, which is suitable for animal consumption, including human consumption. According to certain embodiments the green tea extract is enriched for catechins, particularly EGCG, GCG or combination thereof. 2. Field application of EGCG and/or GCG or green tea polyphenolic flavonoids in solution or in slow-release form, e.g., in agricultural settings to prevent attachment of parasitic plants to potential hosts, or development of the parasite on the host, thus improving agricultural yields. Green tea extracts can be used to inhibit PME activity at the surface of potential hosts' roots or aerial parts, providing the opportunity to produce a low-cost, non-toxic method of pest control in under-developed countries where access to agricultural chemicals is limited by financial considerations.

3. Application of EGCG and/or GCG or green tea polyphenolic flavonoids to inhibit esterase activity of cell wall degrading enzymes, including PME and cutinases, in tissue invasive systems across taxa, potentially including fungal and bacterial pathogens in plants.

Thus, according to one aspect, the present invention provides a method of controlling parasite or pathogen development in or on a plant comprising applying to the plant a composition comprising a pectin methyl esterase inhibitor, wherein the pectin methyl esterase inhibitor comprises at least one polyphenol flavonoids. According to certain embodiments, the composition comprising a pectin methyl esterase inhibitor comprises a plant extract. According to yet another aspect, the present invention provides a method of retarding or preventing pectin degradation in a processed food product or beverage comprising adding to the processed food or beverage a composition having a pectin methyl esterase inhibiting activity. According to certain embodiments, the composition having pectin methyl esterase inhibiting activity comprises a plant extract comprising polyphenol flavonoids. According to typical embodiments, the plant extract is enriched for catechins.

According to certain embodiments the catechin (either isolated or present in the plant extract, particularly green tea extract) is gallate containing component, particularly gallate containing component selected from (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCG) and (-)- gallocatechin gallate (GCG). According to other embodiments, the gallate containing component is galloyl ester. According to certain embodiments, the galloyl ester is selected from EGCG and GCG. According to other embodiments, the galloyl ester is GCG.

In some cases synergistic effects of catechins may exist. In particular, combinations of catechins, e.g., EGCG and GCG, optionally with certain other compounds will be useful compositions for inhibiting the cell wall degrading enzymes.

The inhibition activity of EGCG and/or GCG may be useful in protection against various types of tissue invaders that use PME, cutinases, and non-specific esterases to invade their plant hosts. These include parasitic angiosperms, nematodes, parasitic as well as non parasitic fungi (e.g. Wang F et al., 2006 Int. J. MoI. Sci. 7, 346-357; Sugui

JA et al., 1998 Physiological and Molecular Plant Pathology 52, 213-221) and bacterial pathogens (e.g. Davies KA et al., 2000 Physiological and Molecular Plant Pathology 57, 63-75; Fett F et al., 1992 Applied and Environ Microbiol 58, 2123-2130; Purdy RE et al. Biochemistry 14, 2832-2840; U.S. Patent Application Publication No.

20060134286). Overexpression of PME inhibitors in Arabidopsis thaliana has already been shown to inhibit fungal infection (Lionetti V et al., 2007 Plant Physiol. 143, 1871

-1880). According to the present invention, exogenous application of an inhibitor such as EGCG and/or GCG may enhance resistance to these plant parasites and pathogens.

Among the potential uses for PME inhibitors is their use as additives for juices and other processed foods. In citrus juice cloud particles impart the characteristic color and flavor, and although the chemical composition of these particles is known, the details of their stabilization are not well understood. Pectin, a major component of orange juice cloud, is thought to play an important role in juice destabilization: in the presence of the active enzyme pectin methylesterase (PME), pectin forms calcium pectate complexes and causes the precipitation of cloud particles.

Recent research has focused on the changes occurring to orange juice cloud particles after addition of pectinase and PME (Croak & Corredig 2006 Food Hydrocolloids, 20(7), 961-965). The particle size of juice cloud did not change with addition of pectinase. On the other hand, at the natural pH (3.8) of the juice, the addition of PME caused aggregation of the cloud particles within a few minutes, and the amount of enzyme added affected the kinetics of the aggregation. A higher amount of enzyme was necessary to cause the same effects at pH 6.0, while at low pH (2.5) cloud particles showed faster aggregation and a higher particle size, possibly because of a decreased surface charge at this pH. The results obtained by dynamic light scattering provided evidence of a direct effect of the PME on the juice cloud and suggest that PME inhibitors would provide a useful tool to prevent losses due to the mechanism of cloud aggregation and precipitation.

In particular, it is important to note that the present invention provides PME inhibitors that are themselves derived from plant extracts, such as green tea extract flavonoids. These would provide additives acceptable to the food industry. For certain applications it will be important to optimize the composition so as not to impart any undesirable flavors to the food or beverages being treated.

Green tea extract has been shown as a potent inhibitor of activity or expression of a variety of enzymes in vivo and in vitro (Ahmed et al., 2004, ibid; Aucamp J et al., 1997 Anticancer Res. 17, 4381-4385; Morre et al., 2003, ibid; Okello et al., 2004, ibid; Persson et al., 2006, ibid; Vankemmelbeke et al., 2003, ibid; Zhang & Rock, 2004, ibid), including esterases (Okello et al., 2004, ibid). Particularly, gallate-containing catechins are highly effective. The present invention discloses for the first time that surprisingly, gallate-containing catechins are also active inhibitors of the plant enzyme pectin methyl esterase. Previous work has shown that at low pH, catechins can chelate Zn²⁺ and form insoluble compounds with enzymes. However, since PME does not contain Zn²⁺ at the active site, the binding mechanism to PME may be novel. Without wishing to be bound by any theory or mechanism of action it is postulated that the binding of EGCG in the catalytic cleft of PME results in inhibition of the enzyme activity. Specifically, extended docking of the EGCG catechin molecule to large surface area of the methyl esterase reveals that this flexible ligand molecule prefers clustering at the enzyme active site (Figure 6). The binding energy values (ΔG) calculated for 100 different docking positions range from -6.37 to -3.38 kcal/mol. Interestingly, the average ΔG value of -4.47 kcal/mol corresponds to the binding affinity value -800 μM, which is of the same order of magnitude as the measured inhibition constant. The binding mode of EGCG is characterized by ligand-protein interaction between the catalytic residues D136, W252 and W227 and Ql 13 and E253, and more specifically, a stacking interaction with W252 the binding interaction of EGCG to PME may be due to competitive interactions.

Proteinaceous PME inhibitor (PMEI) from kiwi fruit was shown to cause inhibition by direct contact with the relatively wide active binding site cleft of PME, thus covering the binding site access point (D'Avino et al., 2003 Proteins: Struct Funct Genet 53, 830-839; Di Matteo A et al., 2005 Plant Cell 17, 849-858). The fluorescence (also called Forester) resonance energy transfer (FRET) behavior exhibited in samples of commercial citrus extract and EGCG in increased concentrations (Figure 8), in which emissions of fluorescence from the binding site tryptophan of PME are absorbed by EGCG and re-emitted at a peak of around 390 nm, suggests that the aromatic system in EGCG is interacting closely with tryptophan groups located on the PME molecule. Previous work by D'Avino et al. ( 2003 ibid) has suggested that most of the fluorescence observed in PME is due to the two tryptophans at the catalytic site, as the other tryptophan residues in PME are minimally exposed to solvent (<14%). Therefore, the FRET data disclosed herein suggest increased shielding of the exposed tryptophans at the catalytic site as a result of interaction with EGCG, providing circumstantial evidence that EGCG is interacting directly at the catalytic site of PME.

The teaching of the present invention is exemplified by green tea extract inhibition of the plant parasite Casteilleja indivisa. Castilleja indivisa is a facultative parasite, and as such is an excellent model system for studying parasitism inhibition as its ability to grow without attachment to a host enables investigation into effects of enzyme inhibitors over time. Furthermore, seeds of this facultative parasite do not require a host-derived signal for germination and establishment. Thus, effects of exogenous application of enzyme inhibitor are not complicated by the need for germination stimulants. The present invention now shows that a single application of high catechin treatment (about 5 g/L PP60, green tea extract that is at least 60% catechins by weight) in cold stratifying seeds resulted in significantly delayed germination in seeds of the host plant {Lupinus texensis). The delay in onset of germination for L. texensis was approximately 30 days. No significant delay in C. indivisa germination was identified.

This difference may be attributed to the longer cold stratification requirement for germination in C. indivisa. The longer stratification period required for imbibition and germination most likely results in greater dissipation of catechin efficacy over time and therefore reduced effect on germination overall. Second application of catechin- containing green tea extract to C. indivisa closer to germination may further reveal the effect of stratification on the catechin inhibition. The reduced coefficient of variation for average number of days to germination in L. texensis suggests that once germination began in the high catechin treatment, more seeds germinated in a shorter period of time, thereby "catching up" with the low catechin (lg/L PP60) and water treatments, since there was no significant difference among treatments with regard to average number of germinate seeds per plate at the end of the experiment. This suggests that catechin addition should occur after host germination but before parasite germination is likely to begin. The advantage to this single initial treatment method is that one strong treatment at the beginning of the growing season should slow parasite germination and establishment and therefore give crops/hosts a head start, similarly to the recently suggested strategy of transplanting seedlings rather than planting seeds into fields to reduce parasite damage (Oswald A et al., 2001 Weed Science 49(3), 346-353). Early infection has a stronger effect on host allocation to stem tissue and grain production than later infection (Gurney A L et al., 1999 New Phytologist 143(3), 573-580.; Van Ast A & Bastiaans L 2006 Weed Research 46(3), 264-274). Thus, any delay in seed germination of parasites would have a positive impact on host performance by reducing the duration of parasitism. Moreover, the high catechin treatment inhibited parasitism between C. indivisa and L. texensis, delaying attachment by at least four days. This provides a second opportunity to reduce the duration of parasitism in crop plants. The final parasite testing in both plant growth experiments showed that most parasites eventually successfully attached, suggesting that parasitism in this system or these growing conditions is delayed, not eliminated. Delay in attachment by even a few days may actually allow hosts to tolerate parasitism in some systems (Gurney et al., 1999, ibid). The observed delay in attachment and significantly lower parasitism through five weeks after parasite addition suggests that in the case of holoparasites, which require host attachment to survive past the first week after germination, the inhibitory effect of the high catechin treatment may be sufficient to prevent parasitism altogether in a high proportion of hosts.

Given the 2% nitrogen content of Polyphenon 60, there was an obvious fertilization effect in the highest catechin treatment during the varying catechin levels experiment. It was therefore examined whether nutrient availability alone was responsible for the observed delay in parasitism. However, the catechin versus nitrogen experiment showed conclusively that adding nitrogen alone, while providing a greater fertilization effect than PP60 (suggesting that the form of nitrogen in PP60 is not as available as that in the nitrogen treatment), did not influence timing of parasitism in this system, thus showing that the nitrogen content of the catechin treatment presented herein was not the reason for the observed delay in parasitism. Previous work has shown that catechins exuded from the roots of Centaurea maculosa can inhibit seed germination in a variety of species (Bais H P et al., 2003 Science 301, 1377-1380), and that germination in Centaurea itself is delayed by catechin, although survival is not affected (Perry L G et al., 2005 J Ecology 93(6), 1126-1 135.); Bais et al. 2003, ibid) hypothesize that the catechin acts as a phytotoxicant, and that the mode of allelopathy by Centaurea maculosa is related to this phytotoxicity. However, the present invention now discloses that catechins can be successfully used to inhibit pectin methyl esterase. Taken with the findings of Ren and Kermode showing that pectin methyl esterase (PME) activity is a critical step in breaking seed dormancy in yellow cedar, and given the conservation of cell wall degrading enzymes across taxa and the fact that PME is important in tissue growth and remodeling in general, the compositions and methods of the present invention may advantageously be used to inhibit germination of deleterious parasitic plants.

Furthermore, parasitic plants are known to utilize cell wall degrading enzymes such as PME during host invasion, often resulting in nearly complete destruction of pectins in the area surrounding the haustorial invasion (Losner-Goshen et al., 1998 ibid; Neumann et al., 1999 ibid). Without wishing to be bound by any specific mechanism or theory, the mode of attachment inhibition observed in the experiments presented herein is a result of the ability of catechins, particularly EGCG, to inhibit PME activity at the interface between host and parasite. According to additional aspects, the present invention provides agricultural or food grade compositions comprising at least one gallate-containing catechin selected from the group consisting of EGCG and GCG in an amount effective in inhibiting the activity of an isolated PME by at least 20%.

According to certain embodiments, the present invention provides an agricultural composition comprising at least one gallate-containing catechin selected from the group consisting of EGCG and GCG in an amount effective in inhibiting the activity of an isolated PME by at least 20% for controlling plant-parasite development in or the plant.

According to other embodiments, present invention provide a food-grade composition comprising at least one gallate-containing catechin selected from the group consisting of EGCG and GCG in an amount effective in inhibiting the activity of an isolated PME by at least 20% for inhibiting or preventing pectin degradation in processed food or beverages.

According to certain embodiments, the at least one catechin is present in the compositions of the present invention in an amount effective in inhibiting the activity of an isolated PME by at least 30%, at least 40%, at least 50% or more.

Inhibition of PME activity can be measured by any suitable method as is known to a person skilled in the art. According to certain embodiments, PME activity and its inhibition according to the teachings of the present invention is measured in pectin/agarose medium plate assays based on Downie et al. (Downie B et al., 1998 Anal. Biochem 264, 149-157) as described in the Example section hereinbelow. According to other embodiments, the enzymatic activity of PME and its inhibition is measured by monitoring the fluorescence emission during the hydrolysis of a cyano- acetate as a substrate as described in details in the Example section hereinbelow.

Catechin preparation The extracts, polyphenolic compounds or combination of compounds derived therefrom are generally prepared by methods known in the art. Tea extracts containing high concentrations of EGCG, EGC, and other naturally occurring tea-derived polyphenols are commercially available, e.g. from Sigma-Aldrich Inc. Alternatively, green tea extracts can be produced by methods well known in the art. For example U.S. Patent Nos. 6,268,009, 6,063,428 and 5,879,733 to Ekanayake et al., describes the preparation of a green tea extract having improved clarity and color. The green tea extract is obtained by treating the extract at a temperature in the range of 25⁰C to 6O⁰C with an amount of a food grade cation exchange resin effective to remove metal cations present in the extract. The treated extract is then contacted with a nanofiltration membrane. However, the process described in Ekanayake Patent No. 5,879,733 is not suitable to separate EGCG from a mixture of tea catechins.

U.S. Patent No. 4,613,672 to Hara describes a process for the preparation of pure EGCG which process includes the following steps: Tea leaves are extracted with hot water or with aqueous solutions of 40-75% methanol, 40-75% ethanol or 30-80% acetone. The obtained extract is washed with chloroform, and the washed extract is dissolved in an organic solvent. The organic solvent is distilled off, and the concentrated extract component is subjected to high speed liquid chromatography using a reverse- phase partition column with a developer of acetone/tetrahydrofuran/water (0-25:0- 35:65-85, vol %), whereby each of (-) epicatechin, (-) epigallocatechin, (-) epicatechin gallate and (-) epigallocatechin gallate is isolated from one another. US Patent No. 7,012,149 to Burdick et al. discloses a process for making (-)- epigallocatechin gallate (EGCG) by subjecting a green tea extract to chromatography on a macroporous polar resin, eluting EGCG from the resin with a polar elution solvent, optionally concentrating the eluate, optionally regenerating the resin by desorbing the remaining catechins, and optionally concentrating the desorbed catechins. With regard to chemical synthesis of the catechins of the present invention, reference is made to Thompson et al., 1972, J. Chem. Soc. Perkin Trans. I., 1287, for discussions on the synthesis on gallocatechin and catechin oligomers. The synthesis of EGCG is discussed extensively in Li et al., 2001, Enantioselective Synthesis of Epigallocatechin-3 -gallate (EGCG), the Active Polyphenol Component from Green Tea, Organic Letters, 3, 739-741.

Furthermore, while the invention is described with respect to tea-derived polyphenol compounds or analogs, from this disclosure the skilled organic chemist will appreciate and envision synthetic routes to obtain and/or prepare the active compounds of the present invention. Accordingly, the invention comprehends synthetic tea polyphenols or their salts and/or their derivatives and/or their synthetic precursors which include, but are not limited to glycosides, gallates, esters, and the like. That is, the active ingredients of the compositions of the present invention can be isolated from tea or from other plant species as well as prepared by synthetic routes.

With regard to the synthesis of polyphenols of the invention, the skilled artisan will be able to envision additional routes of synthesis, based on this disclosure and the knowledge in the art, without undue experimentation, for example, based upon a careful retrosynthetic analysis of the polymeric compounds, as well as the monomers. For instance, given the phenolic character of the compounds, the skilled artisan can utilize various methods of selective protection/deprotection, coupled with organometallic additions, phenolic couplings and photochemical reactions, e.g., in a convergent, linear or biomimetic approach, or combinations thereof, together with standard reactions known to those well-versed in the art of synthetic organic chemistry, as additional synthetic methods for preparing the inventive compounds, without undue experimentation. In this regard, reference is made to Carruthers W Some Modern Methods of Organic Synthesis, 3rd ed., Cambridge University Press, 1986; March J Advanced Organic Chemistry, 3rd ed., John Wiley & Sons, 1985; and van Rensburg H et al., 1996 Chem Comm 24, 2747-2748.

Formulations of the polyphenolic compounds, combinations thereof and compositions comprising the same can be prepared with standard techniques well known to those skilled in the agricultural, food, and pharmaceutical arts, in the form of a liquid, spray and solid forms for immediate or slow-release of the active compounds.

Formulations and application

The tea extract or the purified catechins can be used by themselves or may be formulated according to the intended use. When used to control plant parasites it can be formulated into various formulations such as an emulsifiable concentrate, a suspension, a dust, a granule, a wettable powder, a water-soluble powder, a liquid formulation, a flowable concentrate, a water dispersible granule, an aerosol, a paste, an oil-based formulation and a concentrated emulsion in water in combination with carriers, surfactants and other adjuvants which are commonly used for formulation as agricultural adjuvants. They are blended usually in such proportions that the active component is between 0.1 and 95% by weight of active compound, preferably between 0.5 and 90%.

The carrier used for the above formulation may be classified into a solid carrier and a liquid carrier. The solid carrier may include, for example, animal and plant powders such as starch, activated charcoal, soybean powder, wheat flour, wood flour, fish flour and powdered milk; and mineral powders such as talc, kaolin, bentonite, calcium carbonate, zeolite, diatomaceous earth, white carbon, clay and alumina. The liquid carrier may include, for example, water; alcohols such as isopropyl alcohol and ethylene glycol; ketones such as cyclohexane and methyl ethyl ketone; ethers such as dioxane and tetrahydrofuran; aliphatic hydrocarbons such as kerosene and light oil; aromatic hydrocarbons such as xylene, trimethylbenzene, tetramethylbenzene, methylnaphthalene and solvent naphtha; halogenated hydrocarbons such as chlorobenzene; acid amides such as dimethylacetamide; esters such as glycerin esters of fatty acids; nitriles such as acetonitrile; and sulfur-containing compounds such as dimethyl sulfoxide. The carrier used is preferably a solid carrier or a liquid carrier.

The surfactants used may include, for example, metal salts of alkylbenzenesulfonic acids, metal salts of dinaphthylmethane disulfonic acids, salts of alcohol sulfates, alkylarylsulfonates, lignin sulfonates, polyoxyethylene glycol ethers, polyoxyethylene alkyl aryl ethers or polyoxyethylene sorbitan monoalkylates, alkyl polyglycoside; anionic surfactant selected from polyarylphenol polyalkoxyether sulfates and polyarylphenol polyalkoxyether phosphates.

The other adjuvants may include, for example, sticking agents and thickeners such as carboxydimethylcellulose, gum arabic, sodium arginate, xanthan gum, guar gum, tragacanth gum and polyvinyl alcohol; antifoaming agents such as metal soap; or physical property improvers or coloring agents such as fatty acids, alkyl phosphates, silicone and paraffin, and are preferably guar gum or xanthan gum.

When these formulations are practically used, they may be used directly or after diluted with a diluent such as water to a predetermined concentration. Various formulations containing the compounds of the present invention, whether diluted or not, may be applied by conventional methods, i.e., application methods (such as spraying, misting, atomizing, dusting, granule application, submerged application and seeding box application), soil treatment (such as mixing or drenching), surface application (such as painting, dressing and covering) or dipping.

When used for controlling PME activity in foods, edible composition may be produced by optionally blending green tea extract or isolated catechins with commonly employed food materials such as glucose, fructose, sucrose, maltose, sorbitol, stevioside, corn syrup, lactose, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, dl-a-tocopherol, sodium erythorbate, glycerine, propylene glycol, glycerine fatty acid esters, polyglycerine fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, gum arabic, carrageenan, casein, gelatin, pectin, agar, vitamin B family, nicotinic acid amide, calcium pantothenate, amino acids, calcium salts, colorants, flavoring agents and preservatives.

Foods as used herein is intended to mean any food, food composition, food ingredient, or food product, whether comprised of a single ingredient or a mixture of two or more ingredients, whether liquid, liquid containing, or solid, whether primarily carbohydrate, fat, protein, or any mixture thereof, whether edible per se or requiring preliminary conventional steps like cooking, mixing, cooling, mechanical treatment, and the like.

The concentration of the active component can be suitably adjusted and determined in accordance with the type of formulation, and the method, the purpose, the season or the site of application

Needless to say, the compounds of the present invention are sufficiently effective when used alone. However, they may be used, if necessary, for agricultural uses, in combination or in admixture with fertilizers or other agrochemicals such as insecticides, miticides, nematicides, fungicides, antivirus agents, attractants, herbicides and plant growth regulators, and such combined use can sometimes produce improved effects.

The following examples are provided in order to illustrate certain embodiments of the present invention. It is to be understood that they are to be construed in a non- limitative fashion. EXAMPLES Materials and Methods

Pectin infused agarose plate method

Pectin/agarose medium plates were used to test for enzymatic activity in solutions of PME and PME combined with the hypothesized inhibitor based on the description of Downie et al. (Downie B et al.,1998 ibid). 0.1% w/v -94% esterified citrus pectin (Sigma Aldrich) was added slowly to 0.1 M citrate/0.2M potassium phosphate buffer (pH 7) while stirring. Agarose (Metaphor intermediate melting point, FMC Bioproducts Catalog # 50181) at 2% (w/v) was added slowly, and the mixture was heated until the agarose dissolved. The mixture was cooled to 60°C, at which point sodium azide (0.05% w/v) was added to prevent bacterial growth. This solution was then pipetted into 100 mm square Petri plates (25 ml/plate) and allowed to cool in an isolated chamber. The plates were then sealed and placed upside-down in refrigeration for a minimum of 20 minutes. In preparation for each experiment, two mm diameter wells were created in the agarose medium using a cork-borer. Each well was filled with 15 μL of solution in the patterns described below. For a given experiment, stock solutions of PME (citrus PME lyophilized powder, -35% protein, 198 units/mg protein, protein molecular weight 33954 Da; Sigma Aldrich, unless otherwise noted) and/or green teas extract containing at least 60% catechins (Polyphenon 60, or PP60 Sigma Aldrich, St. Louis MO) or individual catechin components were used, each of which was filtered at 0.2 μm unless otherwise noted. For each agarose plate well, these solutions were mixed individually in 96-well plates for transfer into the agarose plates. Once the solution had been added to each well, the plates were sealed with parafilm, placed in ziplocks, and incubated for approximately 16 hours at 30⁰C (after Downie et al., 1998, ibid). All plates for a given experiment received identical incubation periods. The plates were removed from incubation, rinsed with HPLC-grade water, and filled with 0.05% ruthenium red solution and incubated for 45 minutes. The plates were then drained, rinsed several times with HPLC grade water, and the darker ruthenium red staining around each well indicating the presence of de-esterified pectin (Figure 1) was measured on the back of the plate using electronic calipers to 0.01 mm (Mitutoyo, Japan). Concentration-dependent inhibition of PME by green tea catechins

The experiment examined the effects of PME concentration on the diameter of observed staining in the plates and the effect of PP60 solution on that activity. A set of PME solutions created by serial dilution was utilized: 1 mg/ml of lyophilized PME powder, 0.75 mg/ml, 0.5 mg/ml, 0.25 mg/ml, and 0.1 mg/ml. These were diluted 1 :1 with HPLC grade water, or 1 : 1 with 100 mg/ml solution of PP60 in individual cells in a 96-well plate, and then transferred from each cell in to the agarose plate wells. One well of each solution was placed on each of 5 plates, plus controls (water and PP60 alone).

A further experiment investigated the relationship between PP60 concentration and inhibition of PME activity using 1 mg/mL PME solution combined 1 : 1 with five treatment solutions: 100 mg/ml PP60, 50 mg/ml PP60, 20 mg/ml PP60, 1 mg/ml PP60, and water (control/calibration). These were mixed from stock solutions in individual cells in a 96-well plate, and then transferred from each cell into an agarose plate well. Four plates were used with two replicates of each solution per plate.

Preparation of parasitic plant PME extracts.

An experiment was designed to examine the effect of PP60 on extracts of PME from Castilleja indivisa, a root hemiparasite and from Cuscuta pentagona, a stem holoparasite. Cuscuta pentagona cuttings grown on Vicia faba were placed in water tubes and exposed to Coleus spp. stem for 4 days, then harvested and extracted. Castilleja indivisa were harvested from soil and roots were rinsed in HPLC-grade water. For the two species, 95.8 mg Cuscuta pentagona cuttings and approximately 165 mg of freshly harvested and rinsed Castilleja indivisa root material were placed into 400 μl and 600 μl respectively of 0.1 M citrate/0.2 M phosphate buffer at pH 7 with 3 g of 2.3 mm chrome steel beads. The tissue was ground for 10 seconds on high in a Beadbeater (Biospec products), then allowed to incubate for approximately three hours at room temperature (after Downie et al., 1998, ibid). A portion of the resulting slurry was diluted approximately 1 :1 with buffer and used without filtration. The following solutions were mixed individually in cells in 96-well plates and then transferred to individual wells in the three agarose plates: C indivisa PME extract + buffer (2/plate), C. indivisa PME extract + PP60 (50 mg/ml) (2/plate), C. pentagona PME extract + buffer (2/plate), C pentagona PME extract + PP60 (50 mg/ml) (2/plate), PME 1.0 mg/ml + buffer (1/plate), PME 1.0 mg/ml + PP60 (1/plate), buffer control (1/plate), and PP60 + buffer (1/plate).

Test for inhibitory effects of individual catechins.

This experiment tested individual polyphenolic flavonoid components of PP60 (individual catechins purchased from Sigma Aldrich, St. Louis, MO) for differential inhibition of commercially available PME. PME solution was made up to 1 mg/ml in 0.1 M citrate/0.2 M phosphate buffer at pH 7. This solution was filtered (0.4 μm PTFE filter, Acrodisc), then diluted to 0.75 mg/ml. All PP60 components were diluted in buffer to 20 mg/ml, then filtered (0.4 μm PTFE filter, Acrodisc) to remove particulate while maximizing activity in solution. Maximum concentration of each solution was EC: 34.45 mM, EGC: 32.65 mM, ECG: 22.61 mM, GCG: 21.82 mM, EGCG: 21.82 mM. The following solutions (four plates, 2/plate) were mixed individually in a 96-well tray prior to placement in agarose plate wells: PME + buffer, PME + EGC, PME + ECG, PME + EGCG, PME + GCG, PME + EC.

Fluorometry of PME Interaction with EGCG The enzymatic activity of PME and inhibition by EGCG were measured at 37°C by monitoring the hydrolysis of a cyano-acetate as a substrate (provided by Guomin Shan and Bruce D. Hammock, University of California, Davis) at λex = 340 nm and λem = 465 nm as described by (Shan and Hammock, 2001). The standard assay mixture contained 20 mM Tris, pH 7.0 and 100 mM NaCl. The steady-state kinetic data were processed and analyzed by linear and nonlinear regression using Kaleidagraph 3.5. The initial velocities were calculated by using a linear fit followed by non-linear fit to the Michaelis-Menten equation as shown in Equation 1 :

Vi = V_max S/(K_m + S) where Vj is the initial velocity, V_max is the maximum velocity, S is the substrate concentration, and K_n, is the Michaelis-Menten constant.

The inhibition constant measurement was determined by pre-incubating PME

(2μM) with increasing concentrations of EGCG ranging from 0 to 4.0 mM. Incubation was allowed at 37°C for 30 min before steady state measurement. The cyano-acetate substrate was added to a final concentration of lOμM to initiate the assays.

Measurements of PME activities were performed at 37°C. The Kj values were calculated using Equation 2:

Vi = V_max S / (K_m (l+ I/Ki) + S)

The kinetic data measured in different substrate concentrations were subjected to Lineweaver-Burk analysis. When used for determining the type of enzyme inhibition, the Lineweaver-Burk plot can distinguish competitive, noncompetitive and uncompetitive inhibitors. Competitive inhibitors may be characterized by possessing same y-intercept as uninhibited enzyme but exhibiting different slopes and x-intercepts between the two data sets.

To test whether the interaction of EGCG with PME occurs at the substrate binding site, the inherent fluorescence of PME, and PME fluorescence with incremental additions of EGCG were measured. In the unbound state, the binding site of the PME molecule has two exposed tryptophan residues that absorbs light at 295 nm and emits light at approximately 340 nm (D'Avino et al., 2003, ibid), whereas the other tryptophans in plant PMEs appear to be deep within the folded structure of the protein and not accessible to the solvent; these therefore may not contribute strongly the tryptophan fluorescence of the molecule nor to the observed quenching resulting from interaction with inhibitors (D'Avino et al., 2003, ibid). The EGCG molecule absorbs light at 340 nm and emits radiation at 390 nm. Thus, if the EGCG is bound very close to the tryptophan residue of PME, fluorescence resonance energy transfer (FRET) can occur, in which EGCG absorbs the tryptophan fluorescence. If this occurs, a shift should be observed in the peak emission spectrum of the complex from 340 nm to 390 nm during the addition of EGCG, and the emission at 390 should increase as the emission at 340 decreases.

For the FRET experiment, a solution of 0.5 mg/ml of lyophilized citrus PME powder (Sigma- Aldrich, St. Louis, MO) was dissolved in 0.1 M citrate/0.2 M phosphate buffer at pH 7 (approximately 10.3 μM). A serial dilution of EGCG was prepared ranging from 1.0 to 10.0 mM. Fluorescence was measured in a quartz cuvette

(Spectrosil quartz, Starna Cells, Inc., Atascadero, CA) on a Jasco J-810

Spectropolarimeter (Jasco, Easton, MD) at λex=295 nm, and λem=320 to 450 nm. After measuring a baseline scan of the PME solution to confirm peak PME λem=340 nm

(D'Avino et al., 2003, ibid), sequential 2 μL aliquots of concentrated EGCG solution

(increasing concentration by 20.0 nM per aliquot) were added to the cuvette and mixed gently, then incubated in the dark for 2 minutes prior to scanning. Additions were performed until changes in PME fluorescence leveled off. Background fluorescence of the buffer was subtracted from all spectra.

Molecular docking analysis To explore possible interactions of EGCG molecule in the PME active site, the

AutoDock protocol was employed, which has been shown to reproduce ligand-protein binding modes efficiently (Morris D J et al., 1998 J. Comput. Chem. 19, 1639-1662). The docking algorithm used in this study treats ligands as flexible molecules, while keeping protein conformation restrained. All 12 non-cyclic σ bond torsions of the EGCG molecule were free to rotate. Partial charges on the ligand atoms were calculated by semi-empirical AMI method (MOPAC7 program). Gasteiger partial charges were applied to enzyme molecule. A cubic grid 50 A * 50 A x 50 A around the active site was built by the autogrid program. Grid point step 0.375 A was taken. A Lamarckian genetic algorithm coupled with local search was used for docking with the default values as implemented in AutoDock4.

The number of docking runs was set to 100. To generate input files, AutoDockTools (ADT) program (http://www.scripps.edu/pub/olson- web/doc/autodock/tools.html) was used.

Comparison of tomato and citrus PME inhibition by EGCG The pectin degradation activity of two different PME enzyme types was tested in combination with varying concentrations of EGCG. Commercially available citrus PME (Sigma Aldrich, St. Louis, MO) was made up to 0.5 mg/ml in 0.1 M citrate/0.2 M phosphate buffer at pH 7, then filtered (0.2 μm PTFE filter, Acrodisc) to remove any remaining particulate. Tomato PME was extracted and purified as previously described (Vovk and Simonovska, 2007). EGCG powder (Sigma Aldrich, St. Louis, MO) was dissolved in the buffer to a concentration of 40 mM by warming the tube very slightly under running tap water, then serially diluted to 30, 20, 10, 5, 2.5, and 1 mM. Five plates were used for each PME source, each plate containing one well with buffer, one with EGCG alone, one with the PME solution and buffer, and one each of the PME solution plus each of the EGCG solutions. All solutions for each well were mixed individually in 96-well trays prior to placement in the agarose plate wells. Germination trials

To investigate the effects of green tea catechins on germination rates of host and parasite, seeds of C. indivisa and L. texensis (Native American Seed Farm, Junction, Texas) were used for a germination trial. There were five treatments: 5 g/L, 2 g/L or 1 g/L of green tea extract that is at least 60% catechins by weight (Polyphenon 60 or PP60, Sigma Aldrich, St. Louis, MO), 8 g/L green tea infusion steeped for 10 minutes (organic and non-GMO; TC Bauer Co., Schaumberg, IL), or a water control (all solutions made in HPLC grade water). Ten Petri plates per treatment for each species were lined with filter paper which was then soaked with 70% ethanol in HPLC grade water and allowed to dry to surface sterilize the paper. After drying, 20 seeds were placed in each Petri dish, and 5 and 0.35 mL of the treatment solutions were added to wet the filter paper under the host and parasite seeds, respectively. The Petri dishes were sealed with parafilm and placed in the dark at 4°C. Germination was monitored daily for 98 days (14 weeks). A follow up experiment on C. indivisa utilized an identical setup to that described above except that it included only four treatments (5 g/L and 1 g/L PP60, 8 g/L green tea, and water), and the 0.35 mL treatments were applied at the start of stratification and at 18 days after the start of stratification (two applications of the treatment).

Parasite attachment inhibition studies Two parasitism inhibition studies were performed, one with varied catechin treatments and one in which the highest catechin treatment from the first experiment was compared with a nitrogen treatment. For both parasitism inhibition studies, C. indivisa seeds were placed in 10x150 mm Petri plates on moist filter paper, which were then parafilmed and stored in opaque envelopes at 4°C for 30 days prior to planting. At 21 days of C. indivisa stratification, L. texensis seeds were counted and allowed to imbibe for 5 min in 20 mL water with 85.5 mg (+/- 0.05mg) rhizobial inoculum (Prairie Moon Nursery, Winona, MN) for Lupinus. The seeds, water and inoculum were transferred to moist filter paper in a Petri dish, parafilmed and stored at 4°C for 7 days prior to planting. At planting, L. texensis seeds were sown in 40-cell flats (2 seeds/cell) in 2:1 Promix HP:sand (Promix from Premier Horticulture Inc., Quakertown, PA) and grown in growth chambers (Conviron, Winnipeg, Canada) with the following settings: 16 hour days, 22/17°C, and 70% RH. One week later, C. indivisa seeds were sown in undivided flats in 2:1 promix:sand under the same conditions as L. texensis. All flats were watered three times daily for 4 minutes using automated sprinklers (DIG Corp., Vista, CA). Flats were rotated weekly among chambers (3 chambers for L. texensis) and/or within chamber (1 chamber for C. indivisa). Twenty-eight days after seed planting, L. texensis seedlings were transplanted to 4-inch pots, centered on one half of the pot. The pots were distributed among three growth chambers (for the varying catechin level experiment) or two chambers (catechins and nitrogen experiment) and randomized among chambers weekly. Thirty-five days after initial L. texensis planting (one week after transplanting into pots), C. indivisa were transplanted into the 4-inch pots with L. texensis. Four C. indivisa were transplanted into the pots with the L. texensis, evenly spaced on the opposite half of the pot from the L. texensis. Host and parasite length by width (and height if bolting occurred) were measured weekly during randomization.

Parasite attachment inhibition - varying catechin levels The first parasitism inhibition study investigated the efficacy of varying catechin treatments (as PP60) or green tea infusion in inhibiting C. indivisa parasitism on L. texensis. Soil treatments were begun two days after C. indivisa transplant into the pots. Pots containing one L. texensis and four C. indivisa plants received one of five treatments in tap water: high, medium or low catechin extract treatment (5 g/L, 2 g/L or 1 g/L PP60), 8 g/L green tea infusion, or a water control. The initial assignment was 56 pots per treatment, 28 with and 28 without parasites (to separate effects of treatment and parasite addition on host size). Due to parasite mortality the actual sample size for each treatment/parasite combination ranged from 23 to 28 replicates and the actual number of parasites tested at each harvest ranged from 19 to 25 per treatment. Each pot received 60 mL of its designated treatment twice per week, poured evenly on the soil surface, avoiding the aboveground portions of the plants to the extent possible.

Parasite attachment inhibition - fertilization effect versus catechins

The second parasitism inhibition experiment was designed to clarify whether the

2% nitrogen found in the high catechin treatment could cause the parasite inhibition observed in the varying catechin level experiment. Soil treatments were begun two days after C. indivisa transplant into the pots. Pots containing one L. texensis and four C. indivisa plants received one of three treatments in tap water: 1) catechin treatment (5 g/L PP60), 2) 100 ppm N (NH₄NO₃ in water; the equivalent of the 2% nitrogen in the PP60 solution assuming all of the nitrogen in the PP60 solution was available to the plants), and 3) a water control. There were 53 hosts per treatment, 27 with and 26 without parasites. Due to parasite mortality, the actual number of parasites tested at each harvest ranged from 16 to 25 replicates per treatment. Each pot received 60 mL of its designated treatment twice per week, poured evenly on the soil surface, avoiding the aboveground portions of the plants to the extent possible. Attachment was tested at 21, 29, 37 and 45 days after parasite addition.

Measuring attachment success Castilleja indivisa forms underground attachments to the roots of L. texensis.

Thus, attachment is not readily visible from above ground. However, L. texensis produces alkaloids that are not found in the parasite by itself but are taken up and can be detected in parasites that are attached to L. texensis via the Dragendorff reagent colorimetric test (Harborne J 1984 Phytochemical Methods: A Guide to Modern Techniques of Plant Analysis. London: Chapman and Hall). Leaf tissue from C. indivisa was macerated and extracted in hydrochloric acid (1 N) for at least 2 days, followed by addition of NaOH (10 N) to bring the pH to at least 12, then by washing with dichloromethane to extract the alkaloids from the basic aqueous layer (Harborne, 1984, ibid; Wink, 1993 M 1993 In: P. Waterman ed. Methods in Plant Biochemistry. London: Academic Press, pp. 197-239). This dichloromethane layer was spotted on filter paper and sprayed with Dragendorff reagent. A red/orange stain indicates the presence of alkaloids in the parasite and therefore attachment (e.g., Adler L S & Wink, N 2001 Biochemical Systematics and Ecology 29, 551-561). Because of the small size and delicacy of C. indivisa at early stages of growth, one entire C. indivisa from each pot was sampled for the first two attachment tests in both of the inhibition experiments. In the varying catechin levels experiment, the last two parasites were tested using individual leaves to allow for concurrent sampling and later re-sampling. For the fertilization versus catechins experiment, the entire randomly selected C. indivisa was harvested from each pot at each sampling.

Parasite attachment inhibition - varying catechin levels

For the experiment involving varying catechin levels, the sampling to test for attachment occurred at 14, 21 and 35 days after parasite addition. At 35 days, both remaining C. indivisa were tested by sampling individual leaves to test for dependence of parasite attachment at one position on parasite attachment at another position The attachment of one parasite in a given pot did not significantly influence the attachment of the other parasite in the same pot (N=121, χ² = 36.48, p<0.001 ; model also included treatment as a main effect, χ² = 0.01, p=0.92) , suggesting that measurements of different parasites from within an individual pot over time should give reliable estimates of average attachment for each treatment without being confounded by lack of independence. Parasites unconnected at 35 days were re-tested at 49 days after parasite addition.

Statistical analyses

Inhibition of PME activity

Data were analyzed using JMP Statistical Software (V. 5.0.1.2, SAS Institute). A Standard Least Squares fit model was used to test two models; one in which treatment was nested within plate (where there was replication within plates) and, if plate did not contribute significantly to variance, one in which only treatment was tested for effects on staining diameter. In the one case in which a plate effect was found (in the individual catechin experiment) the effect of treatment was tested both by excluding the outlying plate and by averaging the diameter of the treatments within plate and performing the one-way ANOVA for effects of treatment on stain diameter. No qualitative differences in results were detected. Plate level averages are presented. Tukey's HSD post-hoc tests were used to compare among treatments.

Inhibition of germination and attachment

Statistical analyses were performed using JMP 7.0 (SAS Institute). For the germination experiment, "acceleration of failure time" parametric survival models were performed to test for the effects of catechin treatment on average number of days to first germination (replicated at the Petri dish level within treatment). One way ANOVAs were performed to test differences among final germination ratio in each treatment differed at the end of the experiment. For average days to attachment in the attachment experiments, the last day of testing was used as the minimum estimate of days to attachment for unattached plants, and planned contrasts were used to compare effects of each treatment with water. For both attachment experiments, one way ANOVAs and non-parametric Kruskal-Wallis tests were performed on average days to first attachment due to violations of normality. The two tests did not differ with regard to significance of tested effects; therefore the more common ANOVAs are reported. For the attachment experiment using varied catechin levels and for the catechins versus nitrogen experiment, ANOVAs were used to analyze Lupinus texensis size as a response to catechin treatment and presence of parasitism and to analyze C. indivisa size as a response to treatment and attachment to the host. For post-hoc analyses, planned contrasts were used for average days to attachment to compare treatments with the control (water), and Tukey's HSD tests were used to analyze the relationship among categories in the ANOVAs for size analyses. Within a given harvest date, C. indivisa attachment rate differences among treatments were analyzed using Chi Square tests. Both Likelihood Ratio and Pearson's Chi Square tests were performed and did not differ in significance; therefore, the more common Pearson's results are shown. In the varying catechin levels experiment, the ANOVA for effects of treatment and neighbor attachment on parasite attachment was performed using Nominal Logistic Fit because the response variable (attached or not) is categorical.

Example 1: Effect of Green Tea Extract on Citrus PME Activity

Effects of PME concentration

Enzymatic activity of solutions of commercially available citrus PME and PME plus PP60 was tested using ruthenium red staining of pectin-infused plates of agarose medium (Downie et al.,1998, ibid). A serial dilution of PME solution was incubated in agarose plates with and without PP60.

The one-way ANOVA showed a significant effect of catechin addition on staining diameter (Fj i_>48=l 024.2, p<0.0001). Ruthenium red stain of de-esterified pectin was observed around each well that contained control PME samples, with diameter dependent upon PME concentration (Figure 2a). No de-esterification was observed around any of the wells containing PP60, indicating loss of enzymatic function with addition of PP60.

A Tukey's post-hoc test comparing diameters among PME solutions showed no difference between 1 mg/mL PME and 0.75 mg/mL PME, but significant differences among all other PME treatments. All solutions of PME with PP60 added showed a significant decrease in staining diameter, with no staining evident around the wells (no difference in staining from buffer or buffer plus PP60), indicating a loss of PME activity with PP60 addition regardless of PME concentration. Effects of green tea extract concentration

The inhibition of citrus PME by PP60 was further demonstrated by the change of the staining diameter upon addition of increasing PP60 concentrations. Variable PP60 concentration with a standard PME solution significantly affected staining diameter (F_5>42=230.1, p<0.0001; Figure 2b). A Tukey's HSD post-hoc test showed significant differences among all PME+PP60 combinations and the PME control (1 mg/mL), indicating no overlap in PME activity level among these treatments. The PP60 dilution series showed a linear reduction in PME activity with PP60 concentration (r² = 0.9281, p O.0001; Figure 2b). Samples with PP60 added at 100 mg/mL showed no PME activity (no ruthenium red staining). This confirms that PME activity is inhibited by PP60 and that this inhibition is concentration dependent and can be observed on agarose plates.

Example 2: Effect of Green Tea Extract on the Activity of PME Extracted from Parasite Plant

Extracts of parasitic plant PME were combined with PP60 solution in an agarose plate experiment to determine if PP60 inhibition of PME is consistent across taxa, and specifically if catechins could inhibit PME activity in parasitic plants. PME activity in Castilleja indivisa root extracts and Cuscuta pentagona haustorial region extracts as well as the 1 mg/ ml PME standard were significantly reduced by the addition of PP60 (F_7>28= 1420.9, pO.OOOl ; Figure 3). Activity was completely inhibited by PP60 addition at 50 mg/ml (no difference observed in stain diameter in wells filled with buffer only or with buffer plus PP60). A Tukey's HSD post-hoc test showed significantly lower PME activity in samples with PP60 added compared with PME controls (without PP60). This experiment shows both that green tea catechin inhibition of PME is consistent and effective on various PME types, and that green tea catechins may be used to inhibit PME activity in parasitic plants.

Example 3: Catechin Type and PME Inhibition

Given the consistent inhibition of PME by the combined catechins in PP60, the individual catechins of green tea were utilized to determine the most active constituents of the PP60 extract in the inhibition of citrus PME. Addition of individual catechins significantly affected PME activity overall (F_5>42 = 222.1, pO.OOOl), and the individual components of PP60, comprising EGCG, GCG, EC, ECG and EGC, affected PME activity differently (Figure 4), with the gallate ester-containing EGCG and GCG having the most significant inhibitory effect on PME (stain diameter decrease from 1.82 cm to 1.51 and 1.39 cm, respectively). The Tukey's HSD test among treatments showed that under the condition of this particular experiment, no significant difference in stain diameter was observed upon addition of EC, ECG, EGC compared to control (Figure 4). This suggests that catechins containing active galloyl esters are the most active PME inhibitors in green tea extracts. The dominance of EGCG and GCG in inhibition of PME is consistent with studies in other systems showing the gallate-containing catechins, EGCG in particular, as inhibitors of FabG and Fabl reductases and carboxypeptidases as well as nitric oxide synthase, oxidases and matrix-degrading metalloproteinases (MMPs) in in vitro systems. Example 4: Fluorometric Analyses of Enzymatic Activity

Fluorometric analyses of en2ymatic activity were used to test the interaction of PME with EGCG. The activity of PME with and without EGCG on an artificial synthetic substrate with a fluorescent product was used to estimate the Kj for PME interaction with EGCG. Fluorometric measurements of enzymatic degradation of cyano- acetate substrate (Shan and Hammock, 2001) showed significant reduction in purified tomato {Solanum lycopersicum L.) PME activity with the addition of EGCG (Figure 5). No background fluorescence of the cyano-acetate substrate was found in the absence of PME (data not shown), indicating the stability of this substrate in the absence of enzymatic degradation. Using this technique, the estimated K; is 420 μM ± 20 (r²=0.995); this is higher than the Kj of the endogenously produced PME inhibitor protein from kiwi, which has a Kj of 0.053 μM at pH 7 (D'Avino et al., 2003, ibid). However, it should be noted that the Kj value for the EGCG and PME measured by this cyano-acetate substrate will likely differ from that of PME interactions with pectin, as the cyano-acetate is a much smaller substrate than the esterified pectin network normally degraded by PME.

The Lineweaver-Burk plots of EGCG interactions with PME shows no change in y-intercept with increasing inhibitor concentration, but does show a change in slope and x-intercept with increasing EGCG concentration, suggesting a competitive inhibition mechanism (Figure 7). The experiments presented herein were limited to a small range of substrate concentrations due to quenching problems at higher concentrations; thus, our ability to definitively analyze the type of inhibition is limited. However, given the results, and without wishing to be bound to a specific mechanism or theory, competitive interaction is the most appropriate interpretation.

The inherent fluorescence of PME with and without EGCG was used to investigate the site of EGCG binding to PME as well. Inherent PME fluorescence in commercial citrus extract was reduced with increasing EGCG concentration, with a concomitant rise in emission at the peak EGCG wavelength of 390 nM (Figure 8). This suggests that fluorescence (also called Forester) resonance energy transfer (FRET), between the PME tryptophans (^_n, = 340 nm) and the EGCG (λ_eX = 340 nM and λ_em = 390 nm) is taking place.

Example 5; In vitro Inhibition of PME from Different Plant Sources Using agarose plate assays, it was confirmed that EGCG inhibits PME from two different plant families, Solanaceae (Solarium lycopersicum, tomato) and Rutaceae (citrus). Purified tomato PME (14 μM) and commercially available citrus PME (approximately 5 μM) were combined in agarose plates with varying concentrations of EGCG solution or buffer. This experiment showed a concentration-dependent relationship between EGCG addition and PME activity, as reflected by staining diameter. EGCG treatments had a significant effect on PME activity for both citrus PME (F_9,4o = 874.36, p<0.0001) and tomato PME (F₉,₄₀ = 2245.29, p<0.0001). There was a significant negative linear relationship between concentration and stain diameter for both citrus (Diameter = 1.2 - 0.00077*Conc, r² = 0.87) and tomato PME solutions (Diameter = 1.96 - 0.001 1 *Conc, r²=0.94).

Taken together, these results combined with the other experiments presented here show that green tea extract has consistent inhibitory activity against PME from widely differing plant sources (i.e., citrus, tomato, and the two parasitic plants), suggesting that its inhibitory role is related to a highly conserved region of PME structure. The inhibition of citrus and tomato PME extracts by EGCG in vitro, combined with the successful inhibition of parasitic plant PME extracts with PP60, suggest that EGCG act as a non-specific pan-inhibitor for PME. The docking analyses, in conjunction with the Lineweaver-Burk analysis and the fluorometry data showing FRET between EGCG and PME indicate direct interaction of EGCG at the catalytic binding site of PME and competitive inhibition. Such interactions may be mediated via hydrophobic interactions at the Sl pocket residing at the catalytic site as suggested by Smith et al. (2004). Example 6: Green Tea Extract Effect on Seed Germination

Germination of the host plant Lupinus texensis began as early as 10 days after treatment and germination continued through day 98, the last day of the experiment (Figure 9a). Catechin treatment had a significant effect on average days to first seedling germination of L. texensis (Table 1), most likely due to the difference between the onset of germination in the high catechin treatment (approximately 40 days) compared to the other treatments (approximately 10 days; Figure Ia). Germination of seeds in the high catechin treatment (PP60 5 g/L) began significantly later than in the other treatments. However, once germination began in the high catechin treatment, it rapidly increased, allowing seeds in the high catechin treatment to "catch up" to that in the other treatments. Thus, at the end of the experiment, there was no significant difference in the average number of germinated seeds per plate in each treatment (F_{4 44}=O.87, p=0.49; Figure 9a ). The low catechin treatment may enhance germination between days 50 and 70 (Figure 9a).

Germination of the facultative parasite C. indivisa, began 27 days after treatment and continued through day 98, the final day of the experiment (Figure 9b). Unlike the L. texensis seeds, catechin treatment had no significant effect on average days to onset of germination in C. indivisa (Table 1), although there appears to be a slight inhibitory effect on later germination (Figure 9b). At the end of the experiment, there was no significant difference in the average number of germinated seeds per plate in each treatment (F_{4 44}=1.49, p=0.22). The green tea infusion may enhance germination in this species between days 60 and 75 (Figure 9b).

Table 1 : Parametric survival models for average days to first seedling germination in L. texensis (Weibull distribution) and C. indivisa (log-normal distribution)

Example 7: Parasite Attachment Inhibition - Varying Catechin Levels

At the first attachment sampling date, 14 days after parasite addition, there were no C. indivisa attached to L. texensis. At the second sampling, 21 days after parasite addition, catechin treatment had a significant effect on attachment (Table 2). Plants receiving the water treatment showed the greatest attachment rate (34.6%) followed by low catechin treatment (23.1%), green tea infusion (15.4%), and medium catechin treatment (8.0%; Figure 10a). No parasites receiving the high catechin treatment were attached at 21 days. At 35 days after parasite addition, the two remaining C. indivisa in each pot were both tested by sampling individual leaves and allowing the plants to continue growing. L. texensis receiving high catechin treatment showed a significantly lower parasite attachment rate, at approximately 20% for the high catechin treatment compared to approximately 90% for all other treatments (ranging from 84.6-96.3%; Figure 10a). Parasites at both positions showed similar attachment rates for each treatment, indicating that replicate parasites within a pot are representative of the treatment effect at each time point. Plants unconnected at 35 days were re-tested at 49 days after C. indivisa were transplanted into L. texensis pots. Nearly all of the plants were attached at 49 days. To get a minimum estimate of average time to attachment, we utilized the test date on which attachment occurred in a given pot or the last date of the experiment if no attachment was found. Based on this analysis, attachment was delayed by a minimum of approximately 7.3 days in the high catechin treatment compared to water (46.7 days versus 39.4 days on average to attachment).

Table 2: Pearson's Chi Square test for effects of treatment on C. indivisa attachment to L. texensis at each harvest in the two attachment experiments

Table 2a: Varying catechin levels

Table 2b: Fertilization effects versus catechins

Host plant size (length by width as described the material and methods hereinabove) was noticeably different between treatments and between plants parasitized with C. indivisa and unparasitized plants. By 21 days, catechin treatment had a significant effect on host plant size (Table 3), probably due to an unintentional fertilization effect caused by 2% Nitrogen content in PP60. At 21 days after parasite addition, hosts receiving high catechin treatment were significantly larger than plants receiving water (31% larger) or low catechin treatment (25% larger) according to the Tukey's HSD test. At 35 days, hosts receiving the high catechin treatment were significantly larger than plants in all other treatments (112 cm² vs. 50-75.8 cm²), and hosts receiving medium catechin treatment were significantly larger (75.8 cm²) than the low catechin treatment (59.8 cm²), green tea infusion (56.6 cm²), and water treatments (50 cm²). At 49 days, host size was significantly different among all the PP60 treatments and water, with the high, medium and low catechin treatments corresponding to the greatest, intermediate and lower increase in size (138, 81.7 and 64.3 cm², respectively), and plants receiving green tea infusion treatment being in between and not significantly different from the low catechin treatment and water (56 cm²and 50.7 cm², respectively). By 35 days, the effect of parasite addition on host plant size was also significant

(Table 3), with host plants grown with parasites showing 15% less leaf area than hosts grown alone. In all cases where parasite addition had a significant effect, hosts with parasites were significantly smaller than those without (regardless of attachment).

No initial size differences were observed among C. indivisa at a given position or by treatment when parasites were transplanted into the host pots (one way ANOVAs, not shown). Attached parasites were significantly larger (by a factor of three) than unattached parasites by day 35 (Table 4), presumably as a result of the availability of resources from their host. At each parasite harvest, the overall size of the parasites in the water and PP60 treatments did not differ significantly according to the Tukey's HSD test, although the parasites in medium and low catechin treatment were significantly larger than those in water, suggesting that while host size was significantly increased by catechin addition, the additional resources were not readily available to the parasite because of the reduced attachment rate in the highest catechin treatment. Parasites sampled at day 49 were not analyzed for size by treatment because only a few parasites that had been unattached previously and were of substantial size were left; therefore, the sampling would not have been representative of all parasites receiving a given treatment.

Table 3: Standard Least Squares Means ANOVA testing effects of treatment and parasite treatment (present or not) on host plant size- Table 3a: Varying catechin levels

Table 3b: Fertilization effects versus catechins

Days 7-49 refer to days after parasite addition to the host pots. Example 8; Parasite Attachment Inhibition - Fertilization Effect versus Catechins

At the first parasite sampling date, 20 days after parasite addition, treatment had a significant effect on attachment (Table 2). Plants receiving water treatment showed the greatest attachment rate (7/21, or 33.3%), followed by the nitrogen treatment (5/25, or 20%; Figure 10b). No parasites receiving the high catechin treatment were attached at 20 days (0/18). At 28 days after parasite addition, plants receiving the water and nitrogen treatments had essentially the same attachment rate (14/31 or 66.7 and 13/19, or 68.4%, respectively), while plants receiving the catechin treatment showed a significantly lower parasite attachment rate (7/21, or 33.3%; Figure 10b). At 36 days after parasite addition, treatment continued to have a significant effect on attachment rate (Table 2), with the highest attachment rates in plants receiving water (95%) or nitrogen (100%) and significantly lower attachment in the catechin treatment (62.5%). Nearly all of the plants were attached at 44 days, with no significant differences observed among treatments. Using means calculated as in the previous experiment, we found a minimum delay in attachment of approximately 4 days in plants receiving the high catechin treatment compared to the water control treatment. Plants in the nitrogen treatment showed no delay in attachment. The reduced delay caused by the catechin treatment compared to the previous experiment is most likely a function of the different sampling timing in the two experiments, since harvests were performed with a two weeks time interval in the varying catechin levels experiment and only a one week time interval in the nitrogen versus catechin experiment, but the combination of the Chi Square test for attachment rate at each harvest and the lag in average days to attachment show that attachment is consistently delayed by high catechin soil amendments. Host plant size was again noticeably different between treatments and between parasitized and unparasitized plants. By 20 days, treatment had a significant effect on host plant size (Table 3), and this significant effect was maintained through day 44. At each date, plants receiving the nitrogen treatment were significantly larger than plants receiving the catechin treatment, and both of these were significantly larger than plants receiving only water according to the Tukey's HSD.

By 36 days, host plants grown with parasites showed 16% less leaf area than hosts grown alone (Table 3). In all cases where parasite addition had a significant effect, hosts with parasites were significantly smaller than those without (regardless of attachment) according to a Student's t-test. However, the significant treatment x parasite interaction term at 36 and 44 days indicates that the size reduction in the presence of the parasite was found only in the nitrogen treatment; in both the catechin and water treatments, size did not differ significantly between host plants grown with or without parasites (Tukey's HSD).

No initial size differences were observed among C. indivisa at a given position or by treatment when parasites were transplanted into the host pots (one way ANOVAs, not shown). Attached parasites were significantly larger than unattached parasites by day 20 (Table 4). However, by day 36, the effect of attachment on parasite height was no longer significant, perhaps due to the overriding effect of the treatments, particularly nitrogen, on size. At all harvests, parasites in the nitrogen treatment were significantly larger than those in the water treatment according to the Tukey's HSD test. The nitrogen treated plants were significantly larger than plants in the catechin treatment in all cases except the third harvest, at which the catechin treated plants were intermediate to the nitrogen and water treated plants.

Table 4: Standard Least Squares Means ANOVA testing effects of treatment and parasite attachment on parasite's own size at each harvest

Table 4a: Varying catechin levels

Table 4b: Fertilization effects versus catechins

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

1. A method of controlling parasite or pathogen development in or on a plant comprising applying to the plant a composition comprising a pectin methyl esterase inhibitor, wherein the pectin methyl esterase inhibitor comprises at least one polyphenol flavonoid.

2. The method of claim 1 wherein the at least one polyphenol flavonoid is a catechin.

3. The method of claim 1 wherein the composition comprising a pectin methyl esterase inhibitor comprises a plant extract.

4. The method of claim 3 wherein the plant extract is a green tea extract.

5. The method of claim 4 wherein the green tea extract comprises at least one catechin.

6. The method of claim 1 or 5 wherein the catechin is gallate containing catechin.

7. The method of claim 6 wherein the gallate containing catechin is selected from the group consisting of EC, ECG, EGC, EGCG, GCG and derivatives and salts thereof.

8. The method of claim 6 wherein the gallate containing component is a galloyl ester.

9. The method of claim 8 wherein the catechin containing galloyl ester is selected from the group consisting of EGCG, GCG and derivatives and salts thereof.

10. The method of claim 9 wherein the catechin is GCG or a derivative or a salt thereof.

11. The method of claim 1 wherein the parasite is selected from the group consisting of a parasitic plant, an insect, a nematode and a fungus.

12. The method of claim 1 , wherein the parasite is a parasitic plant.

13. The method of claim 12, wherein the parasitic plant is member of a family selected from the group consisting of Cuscutaceae, Orobanchaceae, Loranthaceae and Viscaceae.

14. The method of claim 13 wherein the parasitic plant is selected from Cuscuta pentagona and Castilleja indivisa.

15. The method of claim 1 wherein the pathogen is selected from the group consisting of fungal and bacterial plant pathogens.

16. The method of claim 1 wherein the composition is formulated in a form selected from a solution, an aerosol, a suspension, an emulsion, a particulate form, a microbead, a nanoparticle, and a powder.

17. The method of claim 1 wherein the composition is applied to the plant in a form selected from spraying, dripping, and dispersing the composition.

18. The method of claim 1 wherein applying the compositions includes applying to plant parts selected from roots, stems, leaves, fruits, and seeds.

19. The method of claim 18, wherein the compositions are applied to seeds before planting.

20. The method of claim 18 wherein the compositions are applied to the roots.

21. The method of claim 1 , wherein the plant is a crop plant.

22. The method of claim 21 , resulting in improvement of the crop yield

23. A method of retarding or preventing pectin degradation in a processed food product or beverage comprising adding to the processed food or beverage a composition having a pectin methyl esterase inhibiting activity.

24. The method of claim 23 wherein the composition having pectin methyl esterase inhibiting activity comprises a plant extract comprising polyphenol flavonoid.

25. The method of claim 24 wherein the plant extract is a green tea extract.

26. The method of claim 25 wherein the green tea extract comprises catechins.

27. The method of claim 23, wherein the composition having a pectin methyl esterase inhibiting activity comprises catechins.

28. The method of claim 24 or 27 wherein the catechins are gallate containing catechins.

29. The method of claim 31 wherein the gallate containing catechins are selected from the group consisting of EGCG, GCG and derivatives and salts thereof.

30. The method of claim 31 wherein gallate containing catechin is GCG or a salt or derivative thereof.

31. The method of claim 23 wherein the beverage is a fruit juice.

32. The method of claim 31 wherein adding a pectin methyl esterase inhibitor reduces cloud loss.

33. The method of claim 25 wherein the processed food product is selected from the group consisting of a foodstuff to which pectin is added and a foodstuff that naturally contains pectin.

34. An agricultural composition comprising at least one gallate-containing catechin selected from the group consisting of EGCG, GCG a derivative and a salt thereof in an amount effective in inhibiting the activity of an isolated PME by at least

20%.

35. The agricultural composition of claim 34, wherein said composition comprises a combination of EGCG and GCG.

36. The agricultural composition of claim 35, further comprising at least one additional catechin.

37. The agricultural composition of claim 36, wherein the concentration of the combined total amount of EGCG and GCG is at least equal to the amount of the at least one additional catechin.

38. The agricultural composition of claim 34, wherein the concentration of the at least one catechin is in the range of from about 0.01% to about 10% by weight.

39. The agricultural composition of claim 34, said composition comprises a green tea extract.

40. The agricultural composition of any one of claims 34-39, for controlling plant- parasite development in or the plant.

41. A food grade composition comprising at least one gallate-containing catechin selected from the group consisting of EGCG, GCG, a derivative and a salt thereof in an amount effective in inhibiting the activity of an isolated PME by at least 20%.

42. The food grade composition of claim 41 , wherein said composition comprises a combination of EGCG and GCG.

43. The food grade composition of claim 42, further comprising additional catechin.

44. The food grade composition of claim 43, wherein the concentration of the combined total amount of EGCG and GCG is at least equal to the amount of the at least one additional catechin.

45. The food grade composition of claim 41, wherein the concentration of the at least one catechin is in the range of from about 0.01% to about 10% by weight.

46. The food grade composition of claim 41, said composition comprises green tea extract.

47. The food grade composition of any one of claims 41-47, for inhibiting or preventing pectin degradation in processed food product or beverages.