THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from US provisional patent application number 61/525,299 filed August 19, 201 1 the content of which is hereby incorporated by reference is its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to the fields of organic stevia rebaudiana extract, zero or low calorie natural sweeteners. Specifically, the present invention relates to the composition of an improved stevia rebaudiana extracts to reduce after-taste. Alternatively, the invention relates to a process for the generation of steviol recovered from unwanted wastes of steviol glycosides extraction from stevia leaves. Still, the present invention relates to formulations consisting of stevia extract mixed with natural sweeteners having antioxidant activity, such as maple syrup, honey, and agave, to increase its functional properties. Specifically, a formulation containing active metabolites steviol combined with abscisic acid, a bioactive molecule found in maple syrup, is proposed to act as TRPV1 regulator to play a role in pain management, anti-inflammation, oxidative stress and energy modulation.

BACKGROUND OF THE INVENTION

[0003] Sweeteners are widely used both by consumers and by the food and beverage industry. Consumers use them as an ingredient in various food items and also as a means for customizing the sweetness of beverages, fruit, yogurt, and the like. The food and beverage industry uses such sweeteners in prepared beverages and other food items. Such sweeteners include both caloric and low-caloric sweeteners. Caloric sweeteners include sucrose, fructose, and glucose. Recently, low-calorie (or non-calorie) sweeteners have gained increased popularity (see Figure 1). In many instances, they can be used as substitutes for caloric sweeteners and are often referred to as "sugar substitutes". Common sugar substitutes include saccharin, aspartame, and sucralose. In parts of Asia, compounds extracted from stevia rebaudiana (hereinafter stevia) plants have also been used as sugar substitutes for some time. For example, Japan has marketed stevia as an alternative to artificial sweeteners since 1970. In the 1980s, China began producing stevia commercially, becoming the main supplier to Japan. [0004] Although stevia leaves have been predominantly studied for their sweetening properties, they are composed of, by dry weight: about 6.2% protein; about 5.6% lipids; about 52.8% total carbohydrates; about 15% steviol glycosides and about 42% of water- soluble substances. Additionally, stevia leaves are a significant source of potentially health benefiting phytochemicals. Over 100 sfev/ ^' a-derived phytochemicals have been discovered thus far. The constituents of stevia leaves can be divided into two categories: sweetening constituents and non-sweetening constituents.

[0005] The sweetening constituents represent about 14% by weight of dried leaves and are mostly diterpene glycosides based on the kaurene skeleton. In particular, steviol glycosides are responsible for the sweetening properties of stevia. The most abundant form of steviol glycosides in stevia leaves are steviosides followed next by rebaudiosides. The rest of the sweetening compounds are present in much smaller quantities. Stevioside was isolated in 1931 and has also been produced from the hydrolysis of stevioside as well as isosteviol. Subsequent studies have led to the isolation of seven other sweet glycosides of steviol. Among these, rebaudioside A is considered the sweetest and the most stable, and it is less bitter than stevioside. Rebaudioside A represents a much smaller percentage of the total weight of stevia leaves and thus chemical processes aimed at producing higher yield of rebaudioside A (Reb A) have been developed. Rebaudioside E (Reb E) is as sweet as stevioside, and rebaudioside D (Reb D) is as sweet as rebaudioside A, while the other glycosides are less sweet than stevioside. The sweetness of high potency sweeteners compared to sucrose is summarized in Table 1.

[0006] Stevioside is the most prevalent glycoside in stevia, comprising 6-18% of stevia leaves. Typical proportions, on a dry weight basis, for the four major glycosides found in the leaves of wild stevia plants are: 0.4-0.7 % dulcoside A; 1-2% rebaudioside C; 2-4 % rebaudioside A; and 5-10 % stevioside. Table 2 summarizes the sweetness of the principal steviol glycosides found in stevia leaves compared to sucrose.

Table 1 : Sweetness of high potency sweeteners in Sucrose Equivalence (%SE) in water

Table 2: Comparison of the sweetness potential of the principal steviol glycosides found in stevia leaves (compared to sucrose).

[0007] The non-sweetening constituents of stevia leaves that have been identified include labdanediterpene, triterpenes, sterols, flavonoids, volatile oil constituents, pigments, gums and inorganic matter. Table 3 summarizes the main non-sweetening constituents found in stevia leaves. Table 3: Stevia-derived molecules classified by chemical group and affinity

[0008] Commercially available stevia extracts contain a high percentage of the glycoside diterpenes stevioside (CAS no. 57817-89-7) and rebaudioside A (CAS no. 58543-16-1), the principal sweetening compounds, and smaller amounts of other steviol glycosides (see Figures 2A and 2B). The exact composition of the extracts depends on the composition of the stevia leaves from which they originate, which in turn are influenced by factors such as soil, climate, cultivation methods, harvest time, as well as on manufacturing process including the extraction and purification methods used. The impurities present in stevia extracts are primarily due to other compounds that are co-extracted from stevia leaves, such as pigments and saccharides. Regulatory submissions from countries in different parts of the world suggest that the main components of commercially available extracts of stevia contain, as the main components, stevioside and rebaudioside A. The amounts of these compounds range from about 10-70% for stevioside and about 20-70% for rebaudioside A. Furthermore, most commercially available stevia extracts have a total steviol glycoside content of more than 90%, with the two main steviol glycosides (stevioside and rebaudioside A) making up about 80% of the extracts.

[0009] Taste issues of stevia of delayed onset of sweetness, low maximal response, bitterness and lingering sweetness have all been challenging. Because of these taste issues, most stevia formulations are not composed of only steviol glycosides. Indeed, blending caloric sweeteners with stevia offers a solution, which provides the upfront sweetness and mouthfeel that stevia extracts lack. Blending stevia with sugars offsets the low maximal response to sweetness and products can then be developed with high degree of sweetness. Sugars used in stevia formulations include sucrose, sugar cane high-fructose-glucose corn syrup, and sugar alcohols such as erythritol, mannitol, xylitol and sorbitol. When sugars are replaced with stevia extracts, they are typically used at only 0.02 to 0.06 percent of the sugar's original mass.

[0010] According to Phillips et a/. , (1987) the bitter aftertaste in stevia extracts is due to the presence of essential oils, tannins, and flavonoids. However, Soejarto et a/. , (1983) concluded that the sesquiterpene lactones are responsible for the bitter aftertaste, while Tsanava et a/. , (1991) suggested that caryophyllene and spathulenol contribute decisively to the aftertaste. Nevertheless, along with providing sweetness, it appears that at least a portion of the aftertaste is attributable to stevioside and rebaudioside A, although the contribution of rebaudioside A is significantly less than that of stevioside (Jakinovich et a/. , 1990).

[0011] Different methods exist for improving the taste of stevia extracts (e.g., diminishing its bitter aftertaste). The methods involve enzymatic modification of stevioside by pullanase, isomaltase (Lobov et a/. , 1991), β-galactosidase (Kitahata et a/. , 1989), or dextrin saccharase (Ghanta et a/. , 2007). Another method involves adding thaumatin, a natural protein that is a low calorie flavor modifier, which is extracted from the fruits of the katemfe (thaumatococcus daniellii) tree from the West African rain forest. Furthermore, exposure to low pH (acidic conditions, including citric acetic and malic tartaric acid) have been reported to enhance the sweetness of stevia extracts. Finally, the addition of plant (Ogawa) extracts which are natural stevia optimizers, have the ability to mask the unpleasant aftertaste of stevia.

[0012] The diterpene known as steviol is the aglycone of stevia's sweet glycosides. Steviol glycosides (stevioside, Rebaudioside A-F, dulcoside) extracted from stevia leaves are metabolized into steviol by the gastrointestinal tract. Although the metabolite steviol is not responsible for the sweetening property of steviol glycosides, it is however considered the active or functional ingredient of stevia. Following metabolism, steviol is found in peripheral blood. Steviol is likely to be responsible for beneficial effects associated with steviol glycosides that have been reported on human health such as anti-inflammatory, antimicrobial, anti-diarrheal, diuretic, anti-tumor, antihypertensive and antihyperglycemic properties. Steviol is more potent than stevioside. For example, steviol is at least 10 times more potent than stevioside to modulate specific immunomodulatory and secretory activities. It is proposed that steviol can be used as pharmaceutical drug or nutraceutical ingredient. Indeed, a patent application from DSM Nutraceuticals describes steviol as an active ingredient to improve cognitive functions, such as learning, memory and alertness, as well as psychosocial pressure (see WO 2009/071277).

[0013] As most of the interest in stevia has been directed to its potential as a low calorie sweetener, little is known about the antioxidant potential of stevia extracts. Stevia leaves are known to contain vitamins A and C, as well as flavonoids such as kaempferol and quercetin. Tadhani et a/. , (2007) have reported that a water-based extract from stevia leaves have an antioxidant activity of 9.66 to 38.24 mg equivalent of gallic acid, ascorbic acid, beta-hydroxy acid (BHA) and Trolox® (6-hydroxy-2,5,7,8-tetramethylchroman-2-carJboxy/; ^' c acid) per gram on a dry weight basis. Furthermore, Xi et a/. , (1998) have proposed that stevioside could have an anti-oxidant activity.

[0014] There is an increasing interest for natural sweeteners. This interest stems partially from increasing consumer demand for such products, but also from the rise of a variety of businesses selling natural products and requiring suppliers of such products to certify that natural ingredients are used in any products being supplied. Such natural sweeteners also often provide functional properties that are useful for maintaining health and other physiological activities important for the well-being of individuals.

[0015] Stevia formulation with other natural sweeteners that have functional properties such as anti-oxidant properties, (for example: maple syrup, agave and honey) are not commercially available. In this invention, we propose novel formulations of stevia containing natural sweeteners that have antioxidant properties (maple syrup, agave, honey, corn syrup).

[0016] For example, maple products are typically viewed as sweeteners with energetic and sugar-containing nutritional potentials. However, maple syrup, native to North America, is much more than a concentrated sugar solution. It contains organic acids, amino acids, minerals, and a wide variety of unidentified compounds, chemicals formed during the evaporation process that contribute to color and taste. Although maple syrup has been predominantly known for its sweetening properties, there is now evidence that it also has anti-cancer properties, possibly due to its antioxidant properties. Published scientific studies have shown the antioxidant and antimutagenic activity of phenolic compounds found in maple syrup. Further investigation into maple syrup yields 3 new lignans, a new phenylpropanoid, and 26 other phytochemicals. Two studies related to maple sap and maple syrup from Quebec showed that phenolic compounds interfere with three essential phenomena involved in the development of tumors: oxidation, inflammation and angiogenesis, the process of developing new blood vessels. Early 2010, a research group identified 20 compounds, 13 of which were newly discovered. Several of these antioxidant compounds newly identified have also been reported to have anti-cancer, anti-bacterial, and anti-diabetic properties. Maple syrup contains important quantity of abscisic acid, a phytohormone that stimulates insulin release through pancreatic cells and that increases sensitivity of fat cells to insulin, which makes it a potent weapon against metabolic syndrome and diabetes according to researchers from Rhode Island University There is evidence that dietary abscisic acid can help ameliorate glucose tolerance and obesity-related inflammation in mice fed with high fat diets, and that it may also play a role in reducing the risk of atherosclerosis and IBD by suppressing the inflammatory conditions related with these diseases. Anti-hyperglycemic activity of maples products has also been proposed, and it has been shown that dietary abscisic acid can enhance glucose tolerance in mice fed with high- fat diets.

[0017] Compounds such as abscisic acid that can be obtained from sources other than maple but which can also be found in maple-derived extracts, are known to have health benefiting effects such as anti-inflammatory properties. WO2007/092556 relate to compositions containing abscisic acid for treating and/or preventing diseases associated with expression of peroxisome proliferator activated receptor γ (PPARy). Numerous diseases are listed including diabetes, glucose intolerance, insulin resistance, obesity-related inflammation, inflammatory bowel disease, multiple sclerosis, allergies, and cardiovascular disease. WO 2010/088375 appears to disclose compositions for enhancing the efficiency of a bioactive compound (e.g., anti-diabetic drugs such as thiazolidinediones) by combining it with abscisic acid. However, the above documents do not appear to disclose "maple" or maple-derived extracts as an ingredient or as a possible source of abscisic acid.

[0018] Thus, there remains a need for an stevia extract that has no or low aftertaste. Particularly, this new stevia extract is organic certifiable.

[0019] There also remains a need for a stevia extract that, when formulated with one or more other natural sweeteners with functional properties results in an improved functional ingredient.

[0020] There also remains a need for a methodology for the low cost production of steviol, that can be used as a nutraceutical or pharmaceutical compound.

[0021] The present invention seeks to address these and other needs.

[0022] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

[0023] In a first embodiment, the invention relates to a composition of stevia extract comprising at least 95% of total steviol glycosides having low bitterness and/or low or no aftertaste.

[0024] In an alternative embodiment, the composition comprises Stevioside: Rebaudioside A and Rebaudioside C in a ratio of about 1 1 :2:1. Alternatively, the composition comprises from about 65-70% Stevioside, from about 20-25% Rebaudioside A and from about 5-6% Rebaudioside C.

[0025] In yet another embodiment, the steviol glycosides as defined hereinabove comprise a maximum of 69% Stevioside. In yet another embodiment, the steviol glycosides as defined hereinabove, comprise at least 22% of Rebaudioside A.

[0026] In one embodiment, the invention relates to a composition of stevia extract with low bitterness and low or no aftertaste comprising at least 95% of total steviol glycosides containing a maximum of 6% Rebaudioside C.

[0027] In one aspect, the present invention provides a composition of steviol glycosides having low or no aftertaste essentially consisting of: a maximum of 69% Stevioside, at least 22% of rebaudioside A and a maximum of 6% Rebaudioside C to achieve at least 95% of total steviol glycosides.

[0028] In one embodiment, there is provided a composition of steviol glycosides having low or no aftertaste essentially consisting of: about 69% Stevioside, about 23% of rebaudioside A and about 6% Rebaudioside C.

[0029] In one embodiment, there is provided a composition of steviol glycosides having low or no aftertaste essentially consisting of: 68.7% Stevioside, 23% of rebaudioside A and 5.96% Rebaudioside C.

[0030] In one embodiment, the stevia extract of the present invention is organic certifiable. In another aspect, the present invention provides a composition comprising the stevia extract of the present invention and a nutritionally, nutraceutically or pharmaceutically acceptable carrier. In another aspect, the present invention provides a food product, food supplement, food additive or nutraceutical comprising the stevia extract of the present invention.

[0031] In a second aspect, the invention relates to such a stevia extract in admixture with a natural sweetener having enhanced antioxidant properties. In another aspect, the present provides a composition according as defined hereinabove, in admixture with a natural sweetener selected from the group consisting of: maple syrup; honey; agave syrup; and corn syrup; or byproducts thereof. More particularly, the stevia extract is an admixture with maple syrup or byproducts thereof (for example, maple butter, maple toffee or maple sugar).

[0032] In another aspect, the present invention provides a method of preventing or treating a disease associated with oxidative stress by administering to a patient a composition (extract, formulation or food products as defined herein) comprising a combination of stevia and another natural sweetener as defined above.

[0033] In a third aspect, the present invention provides a process for the production of steviol from a stevia extraction waste product. In one embodiment, the process comprises the steps of obtaining waste product from a stevia extraction process, and carrying enzymatic hydrolysis on steviosides present in said waste product to generate purified or enriched steviol.

[0034] In a further aspect, the invention, provides a composition comprising a mixture of steviol and abscisic acid having synergistic properties. In another aspect, the present invention provides a composition comprising the mixture of steviol and abscisic acid in admixture with a nutraceutically or pharmaceutically acceptable carrier. In another aspect, the present invention provides a nutraceutical or pharmaceutical comprising the mixture of steviol and abscisic acid of the present invention.

[0035] In another aspect, the present invention provides a method of treating a disease associated with pain management, inflammatory conditions and oxidative stress by administering to a patient having the disease a composition (extract, formulation or food products as defined herein) comprising a combination of steviol and abscisic acid of the present invention.

[0036] In another aspect, the present invention provides a method for the neuroprotection or the immunoprotection of a mammal comprising administering a composition (an extract, formulation or food product) as defined herein.

[0037] Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

DETAILED DESCRIPTION OF THE INVENTION

Brief description of the figures

In the appended figures:

[0038] Figure 1. Classification of various sweeteners, their molecular composition in sugar molecules and other associated molecule. The proportion of each sugar molecule in each sweetener composition is expressed in percentage (%).

[0039] Figure 2. Representation of structures of stevioside (A) and related compounds (B). In rebaudioside D and E, R1 is composed of 2 p-Glc-p-Glc(2→1). Rebaudioside A, B, C, D, E and F comprise an additional R2 sugar moiety on carbon 3 of the first β-Glc. Rebaudioside F has one β-Glc substituted by -β-Xyl. Glc and Rha correspond, respectively, to glucose and rhamnose sugar moieties.

[0040] Figure 3. Scheme outlining an exemplary extraction method to obtain an organic stevia extract containing at least 95% steviol glycosides used for this invention as described in detail in WO2012/006742.

[0041] Figure 4. Descriptions of various stevia extracts containing at least 95% steviol glycosides and varying taste profiles.

[0042] Figure 5. HPLC profile of the best tasting stevia extract containing 95% steviol glycosides from sample EC20101 1 1 -05.

[0043] Figure 6. Maple syrup act in synergy with steviol glycosides (stevioside, RebA) to enhance the antioxidant potential of the formulation, as shown with higher bars than the sum of maple syrup+stevioside/RebA. The antioxidant potential were measured using the ORAC method.

[0044] Figure 7. A novel process to produce steviol from stevia extraction waste products.

[0045] Figure 8. Steviol and abscisic acid act in synergy to increase antioxidant activity. ORAC values of steviol and abscisic acid alone are lower than steviol and abscisic acid combined together.

[0046] Figure 9. Steviol from stevia combined with abscisic acid found in maple syrup act in synergy to potentiate neuroprotection in human dopaminergic SH-SY5Y neuronal cells.

[0047] Figure 10. Neuroprotection of steviol + abscisic acid is conserved over time. ROS levels in human dopaminergic SH-SY5Y cells were measured, and levels obtained using steviol + abscisic acid were comparable to control levels.

[0048] Figure 11. Steviol from stevia combined with abscisic acid found in maple syrup act in synergy to potentiate immunoprotection in human Jurkat T cells.

[0049] Figure 12. Steviol from stevia combined with abscisic acid found in maple syrup act in synergy to potentiate immunoprotection in human macrophage-like PMA-treated THP1 cells.

[0050] Figure 13. Steviol and abscisic acid prevented the generation of ROS in Jurkat T cells.

[0051] Figure 14. ROS production from THP1-Jurkat co-cultured increased in a dose- dependent manner when Jurkat T cells pretreated with various concentrations of steviol (SVO) or abscisic acid (ABS).

[0052] Figure 15. Increased viability of Jurkat cells treated with steviol+abscisic by MTT assay.

[0053] Figure 16. Proposition that the combination of steviol with abscisic acid causes the creation of a novel TRPV1 regulator.

[0054] Figure 17. A proposed model for the mode of action of steviol mixed with abscisic acid regulating TRPV1 activity. [0055] Figure 18. Another proposed model for the mode of action of steviol mixed with abscisic acid regulating TRPV1 activity.

[0056] Figure 19. Steviol mixed with abscisic acid reduces levels of pro-inflammatory cytokine TNF-alpha from macrophage-like cells.

[0057] Figure 20. Steviol (SVO) mixed with abscisic acid (ABS) increases ATP synthesis in human dopaminergic cells SH-5YSY as shown by enhanced levels of intracellular ATP levels. This suggests that SVO+ABS enhance the energetic potential of cells and modulate neurotransmission.

[0058] Figure 21. The effect of capsaisin, a TRPV1 agonist, on SVO and ABS effect was determined by measuring levels of ATP secreted from SH-5YSY human dopaminergic cells. Results show that the combination of steviol with abscisic acid prevents the increases in ATP secretion induced by capsaicin when cells are treated with SVO or ABS separately. This provide evidence that SVO+ABS act as TRPV1 inhibitor.

[0059] Figure 22. Proposed model for the mode of action of SVO+ABS on immune and neuronal cells

Description of illustrative embodiments

Definitions

[0060] In the present description, a number of terms are extensively utilized. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.

[0061] The use of the word "a" or "an" when used alone or in conjunction with the term "comprising" in the claims and/or the specification may mean "one" but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one".

[0062] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. In general, the terminology "about" is meant to designate a possible variation of up to 10%. Therefore, a variation of 1 , 2, 3, 4, 5, 6, 7, 8, 9 and 10 % of a value is included in the term "about".

[0063] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, un-recited elements or method steps.

[0064] The term "subject" or "patient" as used herein refers to an animal, preferably a mammal, and most preferably a human who is the object of treatment, observation or experiment.

[0065] "Mammal" includes humans and both domestic animals such as laboratory animals and household pets, (e.g. cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non- domestic animals such as wildlife and the like.

[0066] As used herein, the term "purified" refers to a compound (e.g., steviosides, rebaudioside A, quercetin and the like) having been separated from a component of the composition in which it was originally present. The term purified can sometimes be used interchangeably with the term "isolated". Thus, for example, "purified or isolated quercetin" has been purified to a level not found in nature. A "substantially pure" compound or molecule is a compound or molecule that is lacking in most other components (e.g., 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100% free of contaminants). By opposition, the term "crude" means compounds or molecules that have not been separated from the components of the original composition in which it was present. Therefore, the terms "separating", "purifying" or "isolating" refers to methods by which one or more components of the biological sample are removed from one or more other components of the sample. A separating or purifying step preferably removes at least about 70% (e.g., 70, 75, 80, 85, 90,

95, 96, 97, 98, 99, 100%), more preferably at least about 90% (e.g., 90, 91 , 92, 93, 94, 95,

96, 97, 98, 99, 100%) and, even more preferably, at least about 95% (e.g., 95, 96, 97, 98, 99, 100%) of the other components present in the sample from the desired component. For the sake of brevity, the units (e.g., 66, 67...81 , 82, ...91 , 92%....) have not systematically been recited but are considered, nevertheless, within the scope of the present invention.

[0067] The compounds and extracts described herein can be formulated as pharmaceutical compositions by formulation with additives such as pharmaceutically acceptable excipients, pharmaceutically acceptable carriers, and pharmaceutically acceptable vehicles, or as nutraceutical or nutritional formulations with additives such as nutraceutically or nutritionally acceptable excipients, nutraceutically or nutritionally acceptable carriers, and nutraceutically or nutritionally acceptable vehicles. As used herein, the term "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar unwanted reaction, such as gastric upset, dizziness and the like, when administered to human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compounds of the present invention may be administered. Sterile water or aqueous saline solutions and aqueous dextrose and glycerol solutions may be employed as carrier, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

[0068] The compounds, compositions and extracts of the present invention can be prepared as nutritional formulations such as foods, including medical or functional foods and dietary supplements. A "medical or functional food" is defined as being consumed as part of a usual diet but which has been demonstrated to have physiological benefits and/or to reduce the risk of a disease or condition such as a chronic disease, beyond basic nutritional functions. A "dietary supplement" is defined as a product that is intended to supplement the human diet and is typically provided in the form of a pill, capsule, tablet, or like formulation. By way of example, but not limitation, a dietary supplement may include one or more of the following ingredients: vitamins, minerals, herbs, botanicals, amino acids, dietary substances intended to supplement the diet by increasing total dietary intake, and concentrates, metabolites, constituents, extracts or combinations of any of the foregoing. Dietary supplements may also be incorporated into food stuffs, such as functional foods designed to promote health or to prevent disease or disorders. If administered as a medicinal preparation, the composition can be administered, either as a prophylaxis or treatment, to a patient in any of a number of methods. The subject compositions may be administered alone or in combination with other pharmaceutical agents and can be combined with a physiologically acceptable carrier thereof. The effective amount and method of administration and aim of the particular formulation can vary based on the individual subject, the stage of the disease or condition, and other factors evident to one skilled in the art. In the case of a pharmaceutical formulation as well as a nutraceutical formulation, during the course of the treatment, the concentration of the subject compositions may be monitored (for example, blood plasma levels may be monitored) to insure that the desired level is maintained.

[0069] The term "nutraceutical" has been used to refer to any substance that is a food or a part of a food and provides medical or health benefits, including the prevention and treatment of disease or condition. Thus, a nutraceutical is a product isolated or purified from foods that is generally sold in medicinal forms not usually associated with foods. A nutraceutical is demonstrated to have a physiological benefit or provide protection against chronic disease. Hence, compositions falling under the label "nutraceutical" may range from isolated nutrients, dietary supplements and specific diets to genetically engineered designer foods, herbal products, and processed foods such as cereals, soups and beverages. In a more technical sense, the term has been used to refer to a product isolated or purified from foods, and generally sold in medicinal forms not usually associated with food and demonstrated to have a physiological benefit or provide protection against chronic disease. Suitable nutraceutically acceptable excipients may include liquid solutions such as a solution comprising a vegetable- and/or animal-and/or fish-derived oil.

[0070] As used herein, the terms "disease" and "disorder" may be used interchangeably or may be different in that the particular malady or condition may not have a known causative agent (so that etiology has not yet been worked out) and it is therefore not yet recognized as a disease but only as an undesirable condition or syndrome, wherein a more or less specific set of symptoms have been identified by clinicians.

[0071] The compounds, compositions and extracts of the present invention may be administered as either a food or a food supplement. For example, when provided as a food, the extracts of the present invention are combined with material primarily made up of protein, carbohydrate and/or fat that is used in the body, preferably a human body, to sustain growth, repair, and vital processes, and to furnish energy. When provided as a food supplement, the compositions comprise selected substances such that they can be eaten at or about the same time as a food. The food supplements are generally eaten within about one hour before or after the food is eaten, typically within about one-half hour before or after the food is eaten, preferably within about 15 minutes of when the food is eaten, and further preferably within one to five minutes of the time the food is eaten. The food supplement can also be eaten at the same time as the food, or even with the food.

[0072] As used herein, the term "stevia" refers to the plant stevia rebaudiana, commonly known as sweetleaf, sweet leaf, sugarleaf, or simply stevia unless otherwise indicated. The phrase "stevia extract" refers to a sweetener-rich extract derived from the leaves of the stevia rebaudiana plant.

[0073] "Sweetness" refers to the measure of how sweet a substance is when tasted. Sucrose (i.e., table sugar) is the prototypical example of a sweet substance and is generally the reference point (equivalent to "1 ") used to measure sweetness, for example, in terms of percentage sucrose equivalence or in terms of a fold increase in sweetness compared to sucrose. As used herein, the term "high sweetness" refers to a fold increase of at least 250 as compared to that of sucrose. In one embodiment, the present invention provides a stevia extract with a fold increase in sweetness of at least 250 as compared to that of sucrose. In another embodiment, the present invention provides a stevia extract with a fold increase in sweetness of at least 300 as compared to that of sucrose. In yet another embodiment, the present invention provides a stevia extract with a fold increase in sweetness of about 250 to about 400 as compared to that of sucrose.

[0074] As used herein, the phrase "hot water" in the context of an extraction solvent refers generally to water having a temperature of 50°C to 100°C.

[0075] A "natural product" refers to naturally-occurring compounds that are end products of secondary metabolism; often, they are unique compounds for particular organisms or classes of organisms. An "all-natural product" refers to a product made with and/or from only natural compounds or products.

[0076] "Organic certification", "organic certifiable" or the like refers to a certification process for producers of organic food and other organic agricultural products. In general, any business directly involved in food production can be certified, including seed suppliers, farmers, food processors, retailers and restaurants. Requirements vary from country to country, and generally involve a set of production standards for growing, storage, processing, packaging and shipping that include, for example: avoidance of most synthetic chemical inputs (e.g. fertilizer, pesticides, antibiotics, food additives), genetically modified organisms, irradiation, and the use of sewage sludge; use of farmland that has been free from synthetic chemicals for a number of years (often, three or more); keeping detailed written production and sales records (audit trail); maintaining strict physical separation of organic products from non-certified products; and undergoing periodic on-site inspections. In some countries, certification is overseen by the government, and commercial use of the term organic is legally restricted.

[0077] An "organic food" refers to a food made with ingredients derived from crops obtained from organic farming and made in a way that limits or excludes the use of synthetic materials during production. Organic agricultural methods are internationally regulated and legally enforced based in large part on the standards set by the International Federation of Organic Agriculture Movements (IFOAM). For greater clarity, unless otherwise specified the use herein of the term "organic" preceding any plant, animal or food product thereof refers to a product made with ingredients derived from crops obtained from organic farming and made in a way that limits or excludes the use of synthetic materials during production. For example, "organic stevia" refers to a stevia plant or extract thereof derived from organic farming or organic-certifiable production methods.

[0078] "Generally recognized as safe" or "GRAS" refers to an American Food and Drug Administration (FDA) designation that a chemical or substance added to food is considered safe by experts, and may be exempted from some other regulatory requirements. As used herein, a "biosolvent" is a solvent obtained from biological or organic sources. In one embodiment, the present invention utilizes a GRAS solvent. In another embodiment, the present invention utilizes a GRAS biosolvent. Examples of GRAS solvents or biosolvents included within the scope of the present invention are ethanol, sorbitol and mannitol. An example of a solvent or biosolvent not present on the FDA GRAS list is methanol.

[0079] As used herein, an "organic certifiable solvent" refers to a solvent that is permitted for use by organic certification agencies without, by itself, voiding organic certification. Currently, two solvents that satisfy this criteria are water and ethanol (preferably derived from organic plants). The phrase "organic certifiable solvent" as used herein should not be confused with the phrase "organic chemical solvent". The latter relates to the field of organic chemistry and refers herein to any solvent containing at least one carbon atom that does not include an "organic certifiable solvent" as defined above. For example, according to the present invention, ethanol (especially derived from organic plants) is considered an "organic certifiable solvent" but is not comprised within the definition of "organic chemical solvent". In contrast, according to the present invention, methanol is considered an "organic chemical solvent" but not an "organic certifiable solvent".

[0080] As used herein, an "organic certifiable stevia extract" refers to an extract from stevia rebaudiana obtained without the use of an organic chemical solvent as defined above, or of any other substance which would otherwise void organic certification. In one embodiment, the present invention relates to such an organic certifiable stevia extract. In another embodiment, the levels of purity of extracts of the present invention defined or referred to elsewhere, when more specifically relating to the methods of the instant application, also apply to the organic certifiable stevia extract as defined here.

[0081] As used herein, the phrase "sweetening compounds", "sweetener compounds", "sweetener" or the like generally refers to an additive (artificial or natural) which increases the basic taste of sweetness of a product to be ingested and can be considered as a sugar substitute (with or without additional calories). In one embodiment, these phrases refer to the sweetness-enhancing compounds from stevia, including glycosides, steviosides and rebaudiosides. Furthermore, stevia is the only known natural sweetener with zero calorie.

[0082] "Antioxidant compounds" refers to any molecules capable of slowing or preventing the oxidation of other molecules that may cause oxidative stress and may damage or kill cells. Oxidative stress is thought to be associated with many human diseases. In one embodiment, an antioxidant compound of the present invention is steviol or abscisic acid.

[0083] A "food additive" refers to any substance added to foods during processing thereof to improve characteristics such as color, texture, flavor, and/or conservation. A "food supplement", "dietary supplement" or "nutritional supplement", refers to a preparation intended to provide nutrients, such as vitamins, minerals, fiber, fatty acids or amino acids, that may be missing or may not consumed in sufficient quantities in an individual's diet.

[0084] "Medical food" refers to any food that is specially formulated and intended for the dietary management of a disease that has distinctive nutritional needs that cannot be met by normal diet alone.

[0085] A "functional food" is similar in appearance to, or may be, a conventional food that is consumed as part of a usual diet, and is demonstrated to have physiological benefits and/or reduce the risk of chronic disease beyond basic nutritional functions, i.e. they contain a bioactive compound.

Harvesting and processing of stevia leaves

[0086] The harvested raw material for extraction (i.e., stevia leaves) is dried and grinded. Drying of the stevia leaves can be done under the sun or via other drying methods. In one embodiment, drying of the stevia leaves involves the application of heat. In another embodiment, the drying process involved exposing the leaves to a temperature between 25°C and 50°C. In another embodiment, the stevia leaves are crushed or grinded (e.g., manually or with the aid of a machine) into a powder. Other techniques for drying and grinding stevia leaves would be within the grasp of the skilled person. Grinding of the raw material raises the extraction yield but may also increase the level of impurities while also potentially making filtration more difficult. Consequently, the skilled person will adapt this grinding step in accordance with the final method used and the desired levels of purity.

Extraction of grinded and dried stevia leaves with GRAS solvent [0087] Following harvesting and processing, the grinded and dried stevia leaves are extracted with an appropriate GRAS solvent of the present invention. According to the present invention, a number of different parameters can influence the overall yield, quality and/or purity of the desired final product. These parameters include, but are not limited to, the identity of the chosen GRAS solvent; the temperature and time at which the chosen natural solvent is used; the ratio of raw material to solvent (raw material:solvent) that is employed; the number of successive extractions performed; the chosen method of purification of the desired products and the conditions related thereto. The skilled person will understand that these parameters are not necessarily mutually exclusive, and that a particular choice relating to one parameter may or may not affect the choice of other parameters. For example, the identity of the chosen natural solvent, and the temperature thereof, can affect the optimal ratio of raw material to solvent that is required to obtain the desired results.

Purification of the compounds of interest

[0088] A number of purification means can be employed to purify the compounds of interest from a stevia extract, including sweetening compounds such as stevioside and rebaudioside A. According to the present invention, separate purification methods can be employed to, for example, maximize the yield and/or purity of either the sweetening compounds.

[0089] To purify sweetening compounds of interest, two purification means can be used in succession. For example, chromatographic purification using different gel columns in succession can be employed. The first column can be an anionic exchange column used in order to remove non-sweetening components such as chlorophyll and flavonoids. In this case, the sweetening compounds of interest (e.g., rebaudioside A and stevioside) are not retained in the column and flowthrough. In a subsequent step, a second means of purification is used (e.g., a reverse phase column) to absorb and retain the rebaudioside A and stevioside allowing further non-sweetening component to flow through and be removed. The skilled person will recognize that the first and second means of purification can each be repeated subsequently one or more times on the same sample in order to remove an even higher amount of pigments or other impurities to obtain a final product of even higher purity. In this regard, the skilled person will be able to adapt the number of times the first and/or second means of purification is repeated according to the desired level of purity and yield.

[0090] In one embodiment of the present invention, a strongly basic anion exchange resin- based chromatographic column can be employed as a first means of purification. In another embodiment, the first means of purification can be a strongly basic (type I) anion exchange resin, for example, for decolorizing intermediate to light solutions, and removing metal ions. In another embodiment, the first means of purification can be an Amberlite® IRA-900 or an equivalent column thereof. The IRA-900 column is a strongly basic, macroreticular resin of moderately high porosity with benzyltrialkylammonium functionality which is useful in decolorizing and removal of organic materials. The matrix is made of styrene-divinylbenzene (macroreticular) with a particle size of 16 to 50 mesh. In another embodiment, the first means of purification can be an Amberjet® 4200 or 4600 column. In general, this chromatographic step removes the pigments from the extract. These pigments can then be eluted if desired by the addition of, for example, HCI (e.g., 1 N HCI, 2 bed volumes or 2 column volumes) and ethanol (e.g., 94%) to the column.

Formulation of the organic stevia extracts of the present invention

[0091] An organic stevia extract of the present invention (e.g., containing the sweetening compounds of interest) can be formulated with or without enhanced antioxidant properties. If enhanced antioxidant properties are desired, organic stevia extract of the present invention can be supplemented with one or more natural sweetener having antioxidant properties, such as: maple syrup, honey, agave syrup and/or corn syrup.

Production of steviol from steviosides or other steviol qlvcosides-containinq wastes

[0092] Steviol can be obtained by enzymatic hydrolysis of steviol glycosides using pectinase (Melis et al., 2009). Microbial hydrolysis of steviol glycosides has also been described. Bacteria species can be used to perform the hydrolysis of steviol glycoside into steviol via their beta-glucosidase (Renwick et al., 2008).

[0093] The present invention provides a process for the production of steviol from a stevia extraction waste product. In one embodiment, the process comprises the steps of obtaining waste product from a Rebaudioside A purification process, and carrying enzymatic hydrolysis on steviosides present in said waste product to generate purified or enriched steviol. Particularly, the hydrolysis is carried out with pectinase, beta-glucosidase, or beta- galactosidase.

Formulation of steviol of the present invention

[0094] The steviol can be formulated with abscisic acid (a bioactive molecule found in maple syrup) to further en enhanced its functional properties. This synergistic mixture can be used for the prevention or treatment of pain, inflammatory conditions and oxidative stress such as for example, neuroprotection or immunoprotection. Applications of the present invention

In a particular aspect, the present invention provides a method of treating a disease associated with pain management, inflammatory conditions and oxidative stress by administering to a patient having the disease a composition (extract, formulation or food products as defined herein) comprising a combination of steviol and abscisic acid of the present invention. Particularly, the pain may be associated with acute migraine or headache- related conditions.

[0095] In another aspect, the present invention provides a method for the neuroprotection or the immunoprotection of a mammal comprising administering a composition (an extract, formulation or food product) as defined herein.

[0096] In another aspect, the present invention provides use of the composition according to the invention as a Ca ⁺² flow modulator (stimulator or inhibitor), ATP modulator (stimulator or inhibitor), or a TNF modulator (stimulator or inhibitor). Alternatively, the composition may be used for the prevention of aging-related symptoms in a mammal. Particularly, the aging- related symptoms are: Alzheimer's disease or Parkinson's disease.

Anti-oxidant

[0097] Antioxidant reduce oxidative damage caused by metabolic reactions that use oxygen for the respiratory chain, which generates the majority of cellular ATP via oxidative phosphorylation (OXPHOS). When the oxygen is reduced to water by the electron transport chain, reactive oxygen species (ROS) are generated and may cause oxidative damage to molecules, such as lipids, proteins, and OXPHOS enzymes. Direct damage by oxidation deforms many mitochondrial proteins. Deformation decreases proteins affinity for substrates or coenzymes in respiratory chain and, thereby, decreases their function. Furthermore, the lacks of the structural protection of histones and repair mechanisms makes the mtDNA quite susceptible to ROS. Oxidative damages are the cause of several disorders, such as neurodegenerative diseases (Parkinson, Alzheimer), diabetes, obesity, chronic fatigue syndrome, migraine, and muscle pain. Diets deficient in antioxidants can accelerate cell death, mitochondrial decay contributing to the development of chronic diseases, and other dysfunction. Antioxidant deficiency increases ROS levels and oxidative stress, and consequently leads to mitochondrial dysfunction and age-associated diseases, including metabolic syndrome. [0098] Because the organic stevia extract of the present invention has sweetening potential, optionally with antioxidant activity, it can be of special interest to the food industry, for example, as a food additive, a food supplement, and/or a functional food, or for the nutraceutical or pharmaceutical industry used as excipients. Data herein suggest that an antioxidant stevia-based formulation can also be added in medical food, for applications in the prevention of chronic diseases (such as, for example, diabetes, obesity or metabolic syndrome).

Anti-inflammation

[0099] Sugars are present in an endless number of food products, and their excessive intake can be damaging for health. To prevent sugar intake excess, natural sweeteners that do not contain any calories, like stevia, can provide an interesting alternative. The use of stevia sweetener as food additive promises a revolution in the food industry. Applicant developed novel formulations of stevia-based sweetener that will provide an alternative to the use of sugar for people concerned with sugar intake and the prevention of chronic inflammatory diseases. Data show that the combination of steviol, the metabolite of stevia, with abscisic acid, an active ingredient found in maple syrup, promotes an anti-inflammatory response by causing a reduction in the pro-inflammatory cytokine TNF-alpha.

[00100] TNF-alpha is a cytokine involved in systemic inflammation and it is best known for its role in the acute phase reaction. It is produced chiefly by activated macrophages and other antigen-presenting cells, although it can be produced by other cell types as well. TNF is an endogenous pyrogen able to induce fever. TNF can also induce apoptotic cell death, sepsis cachexia, inflammation, and inhibit tumorigenesis and viral replication. Dysregulation of TNF production has been implicated in a variety of human diseases, including Alzheimer's disease, cancer, major depression, and inflammatory bowel disease.

Pain management

[00101] TRPV1 is a nociceptor-specific ion channel that can evoke pain sensation upon activation or sensitization. TRPV1 is activated by capsaicin found in hot chili pepper, acidification (protons) or by noxious heat greater than 43°C. Upon tissue damage and the consequent inflammation, a number of inflammatory mediators, such as various prostaglandins and bradykinin, are released. These agents increase the sensitivity of nociceptors to noxious stimuli. This manifests as an increased sensitivity to painful stimuli (hyperalgesia) or pain sensation in response to non-painful stimuli allodynia. Most sensitizing pro-inflammatory agents activate the phospholipase C pathway. Phosphorylation of TRPV1 by protein kinase C have been shown to play a role in sensitization of TRPV1 , the cleavage of PIP2 by PLC-beta can result in disinhibition or TRPV1 and consequently contribute to the sensitivity of TRPV1 to noxious stimuli. TRPV1 activity becomes desensitized upon prolonged exposure to capsaicin. Extracellular calcium ions are required for this phenomenon, thus influx of calcium and the consequential increase of intracellular calcium mediate this effect. Various signaling pathways such as calmodulin and calcineurin, and the decrease of PIP ₂ , have been implicated in desensitization of TRPV1. The analgesic effect of capsaicin is associated with TRPV1 inhibition. Antagonists of TRPV1 are known to reduce pain and have shown efficacy in reducing nociception from inflammatory and neuropathic pain models. Numerous TRPV1 antagonists have been developed and can be used in the treatment of neuropathic pain associated with multiple sclerosis, chemotherapy or amputation, as well as pain associated with inflammatory response of damaged tissue such as in osteoarthritis. An application for TRPV1 antagonists in asthma treatment has been proposed. TRPV1 receptor is also a prime therapeutic target for the treatment of migraine since it is known that the development of headaches involves the excitement of the trigeminovascular system followed by the neurogenic inflammation in the dura matter of the CNS.

Neuroprotection and immunomodulation

[00102] This invention relates to the improvement of human neuroprotection because of the impact of steviol-abscisic acid on a model of microglia-T cell interaction. Ingested food products are absorbed and metabolized at the mucosal surfaces of the gastrointestinal tract. Immune cells, such as T lymphocytes, the majority of which reside in the sub-epithelium layer of the gastrointestinal mucosa, can respond to metabolized by-products derived from food. Upon stimulation, T lymphocytes can migrate back to the lymph nodes and enter the blood stream to circulate throughout the body via the peripheral blood system. Under pathological inflammatory conditions, the permeability of blood vessels and accessibility to the normally "immune-privileged tissue", such as the central nervous system (CNS), can be modulated, giving better access to circulating T lymphocytes to migrate into the tissue. T lymphocytes can contribute to the pathology of classical inflammatory diseases of the CNS such as multiple sclerosis (MS). Inflammation of the CNS is also thought to play roles in the pathology of classical neurodegenerative disorders such as Parkinson's and Alzheimer's disease. In all these conditions, activated T lymphocytes enter into the CNS in either large (in MS) or subtle (Parkinson's and Alzheimer's disease) quantities. Upon entering into the nervous system, the T lymphocytes can either damage neurons directly or they interact with microglia to promote the upregulation of several inflammatory molecules, which further promotes neuroinflammation and potential neurotoxicity. Indeed, we have documented that while activated T cells or microglia that are cultured independently produce relatively low amounts of inflammatory cytokines, their co-culture resulted in the substantial elevation of tumor necrosis factor-a (TNF-a), and interleukin (IL)- 1 β, IL-6, IL-10 and IL-12 (Chabot et a/. , 1997; Chabot et a/. , 1999). The clinical relevance of the T cell - microglia interaction is suggested by our observation that all established (interferon-β and glatiramer acetate) or experimental (minocycline and IVIG) MS therapies that we have applied to the co-culture inhibited their interactions (Chabot and Yong, 2000; Chabot et a/. , 2002). Therefore, reducing the capacity of T lymphocytes to engage microglia or to kill neurons represents interesting approaches to the prevention of neurological disorders. Because of the presence of anti-inflammatory phytochemicals in stevia-derived products, the impact on neuroimmunological processes involved in inflammatory conditions of the CNS was studied. The neuroprotective potential of a maple, stevia or maple-stevia product may provide a tool for the promotion of these products. This potential novel application may also help design better approaches to prevent CNS diseases.

Energy modulation

[00103] Understanding the mechanisms of energy regulation is crucial for the design of novel health strategies as people are looking for way to maintain an optimal energy level for the accomplishment of their everyday tasks. Within the human body, energy produced and used by the metabolism is in the form of ATP (adenosine triphosphate), an energy-rich biomolecule and universally used for energizing cellular activities. ATP is produced in mitochondria, the powerhouse of the cell and most complex organelle. ATP is the unique molecule responsible for the storage of energy and its use for the majority of basic metabolic processes. Because plants and microorganisms capture and use energy by the same reaction, and the amount of biomass is large, the formation and use of ATP is the principal net chemical reaction occurring in the whole world (Boyer, 1998). This is clearly a very important reaction.

[00104] The intracellular role of ATP has been recognized for many years; it is an energy source for many important reactions. ATP is produced by oxidative phosphorylation (OXPHOS) process in mitochondria using simple and complex sugars or lipids (fatty acids) as an energy source. The majority of cellular ATP is generated by OXPHOS. A small part of ATP is generated from the reaction of glycolysis in the cytosol of the cell. Under basal conditions, ATP is present intracellular^ in concentrations of 3-5 mM (Fitz, 2007). The cell stabilizes its energy charge by adjusting the rate of ATP synthesis to the state of energy demand. The ATP level is a crucial parameter of cell homeostasis.

[00105] ATP has also a major role in cells signalling in being released from certain presynaptic neural cells and functioned as a neurotransmitter. Apparently, evolution has assured that ATP is indispensable outside the cells (extracellular ATP), and as a signalling molecule between them. ATP, are considered as the phylogenetically most ancient epigenetic factors sustaining many biological effects. Extracellular ATP at low micromolar concentrations can influence biological processes including platelet aggregation, vascular tone, neurotransmission (peripheral and central), cardiac function and muscle contraction (Gordon, 1986). ATP can interact with specific receptors on the surface of many different cells. ATP are also release from cells in response to mechanical stress. Recent studies on immune cells demonstrate that extracellular release of ATP has dramatic cytotoxic properties and a wider role in neurodegenerative processes is possible. Furthermore, ATP is believed to excite gustatory primary afferent fibers and adjacent cells in the taste bud. Taste buds are specialized sensory organs in the mouth, on the surface of the tongue, that transduce chemical stimuli into signals that are transmitted to the central nervous system via primary gustatory afferent fibers.

[00106] The present invention is illustrated in further details by the following non- limiting examples.

EXAMPLES

Example 1 : Extraction of steviol glycosides from Stevia leaves

[00107] An exemplary process for producing steviol glycosides in accordance with the invention is presented in Figure 3. According to a variation of the process of Figure 3, the stevia leaves were collected and dried. Once dried, the leaves were grinded and subsequently heated for 4 hours at 1 10°C. The grinded and dried leaves were then subjected to two hot water extractions with a concentration of starting material of 7% (w/v): the first being at a temperature of 85°C for 30 min, and the second being at a temperature of 60°C for 30 min.

[00108] The solution was then treated with electrocoagulation. After 7 hours of electrolysis, the coagulate thus formed was separated from the liquid. The solution thus obtained was still greenish but is exempt of matter in suspension and many undesirables substances. [00109] Two successive chromatographic purifications using different gel columns were employed to purify the sweetening compounds of interest (i.e., stevioside and rebaudioside A) from the water-based extract obtained above. Following extraction, the first purification was carried out with an Amberlite® IRA-900 column (2 cm diameter x 35 cm length) to remove the pigments from the water-based extract, which remain fixed on the resin in the column. The flow rate of the water-based extract through the column was adjusted to 4 mL/min. A volume of 35 mL resulted in the maximum concentration of sweetening compounds in the eluate. A volume of 100 mL enabled the recovery of 90% of the sweetening compounds. The pigments were then removed from the IRA-900 column with 1 N HCI and ethanol (94%).

[00110] The second step of the purification was carried out using a XAD-2 column on which the sweetening compounds and the antioxidant compounds were retained. An 80% ethanol eluate solution was superior to solutions of 60% and 94% ethanol, since it allowed for a more a rapid desorption of the sweetening compounds.

[00111] Alternatively, an additional step of continuous centrifugation at 2 000 g and diafiltration (50% of initial volume was added to the retentate using water as a solvent and a 2 kD nitrocellulose membrane or a 10 kD polysulfone membrane for ultrafiltration) was employed before purification of the water-based extracts with the IRA-900 column, the extract was clarified but these additional steps did not greatly improve the purity.

Example 2: Different compositions of stevia extracts give slightly different tastes

[00112] Figure 4 shows different embodiments of the composition enriched in steviol glycosides when produced according to the different conditions according to the general process of Example 1. Particularly, it can be seen that the conditions of the specific steps may vary and be adjusted according to the skill of one in the art and obtain highly purified stevia extract.

[00113] Particularly, it can be seen that under specific conditions line 3, sample EC20101 1 1 1-05 gave the best tasting profile with a content of steviol glycosides of 97.66% purity. Specifically, this extract was composed of 68.7% stevioside; 23% Rebaudiosides A and 5.96% Rebaudiosides C.

Example 3: HPLC analysis of the extract having best taste profile

[00114] Sweetening compounds were separated and quantified using an inverse phase column (C18: 5 μηι x 250 x 4.6 mm). The mobile phase was 68% methanol and 32% water in an isocratic mode. The chromatography was carried out at room temperature at the flow rate equal to 1 mL/min under a pressure of between 2500 and 2700 psi. The chromatography lasted 20 minutes and sweetening compounds (steviosides and rebaudioside A) had a retention time of 15.4 minutes. The standard used was obtained from Chromadex®.

[00115] Figure 5 shows the HPLC profile of the Stevia extract having the best taste profile as described in Example 2.

Example 4: Formulation of stevia extract with other natural sweeteners having increased antioxidant properties.

[00116] Figure 6. Maple syrup act in synergy with steviol glycosides (stevioside, RebA) enhances the antioxidant potential of the formulation, as shown with higher bars than the Sum of maple syrup+stevioside/RebA. Maple syrup with sucralose did not enhance the antioxidant potential of the formulation. The sum of maple syrup The antioxidant potential were measured using the ORAC method.

Example 5: Process for the production of Steviol.

[00117] Figure 7 provides a novel process to generate steviol via the waste products generated during the process for the extraction of stevia or the purification of RebA. Liquid stevia extract containing at least 95% steviol glycosides undergoes refining via membrane filtration. The resulting unwanted steviol glycosides are hydrolyzed enzymatically to generate steviol.

Example 6: Steviol mixed with abscisic acid has neuroprotective properties.

[00118] Figure 8. Steviol from stevia combined with abscisic acid act in synergy to potentiate neuroprotection in human dopaminergic SH-SY5Y neuronal cells. Cellular ROS levels were obtained by measuring the oxidation of CM-H ₂ DCFDA (5-(and 6)-chlromethyl- 20,70-dichlorodihydrofluresceindiacetate; Invitrogen) a cell-permeant indicator. Cells were treated for 1 hour with 5μΜ CM-H ₂ DCFDA. After removing the CDFDA solution, cells were treated with various adaptogenic formulations prepared with ROS buffer (HBSS containing 2% FBS). After 30min of exposure with samples, a first reading was taken using the synergy HT (Biotek) plate reader. Various concentrations of AAPH (100mM, 40mM, 16mM,6,4mM and 2,5 mM) were then added, and readings (excitation: 485nm, emission: 530nm) were taken every 30 minutes for 2 hours. The fluorescence intensity is an indicator of H ₂ 0 ₂ intracellular level so values are expressed in Relative Fluorescence Unit (RFU). Levels of ROS production obtained from using steviol + abscisic acid were comparable to control levels. Steviol and abscisic acid were both used at a concentration of 25μΜ. Neuroprotection is preserved with increasing AAPH levels. Steviol alone and abscisic acid alone enhance ROS levels.

[00119] Figure 9. Neuroprotection of steviol + abscisic is conserved over time. ROS levels in human dopaminergic SH-SY5Y cells were measured. While individual treatment of steviol or abscisic acid enhanced cellular levels ROS levels, levels obtained using steviol + abscisic acid were comparable to control levels indicating that the mixture of steviol and abscisic acid prevents the increase in ROS levels normally induced by both compounds. Similarly, Figure 10 provides evidence that, when mixed together, steviol and abscisic acid act in synergy to decrease ROS production and that neuroprotection is conserved over time.

Example 7: A mixture of steviol and abscisic acid has immunoprotective properties.

[00120] Figure 11. Steviol from stevia combined with abscisic acid found in maple syrup act in synergy to potentiate immunoprotection in human Jurkat T cells. DCFH-DA at 10uM was incubated with cells at 37°C during 1 hour under constant agitation. Cell suspension was washed 3 times to removed non-incorporated DCFH-DA. Cells were plated in 96-well plate at a density of 200000 cells per well in HBSS supplemented with FBS 2%. Levels of ROS production obtained from using steviol + abscisic acid were comparable to control levels. Steviol and abscisic acid were both used at a concentration of 6.25μΜ. Neuroprotection is preserved with increasing AAPH levels. Steviol alone and abscisic acid alone enhance ROS levels.

[00121] Figure 12. Steviol from stevia combined with abscisic acid found in maple syrup act in synergy to potentiate immunoprotection in human macrophage-like PMA-treated THP1 cells . Cells treated with 20μΜ PMA were previously plated in 96-well plate at a density of 100000 cells per well. Cells were washed 3 times with HBSS to remove complete culture medium and replace by the assay buffer HBSS/FBS 2%. DCFH-DA at 5μΜ was incubated with cells at 37°C during 1 hour. Adherent cells were washed 3 times to removed non-incorporated DCFH-DA. Levels of ROS production obtained from using steviol + abscisic acid were comparable to control levels. Steviol and abscisic acid were both used at a concentration of 6.25μΜ. Neuroprotection is preserved with increasing AAPH levels. Steviol alone and abscisic acid alone enhance ROS levels. Example 8: A mixture of steviol and abscisic acid has synergistic antioxidant properties.

[00122] Figure 13. Jurkat T cells were pre-treated with steviol (SVO 25mM), abscisic acid (ABS 25mM) or steviol mixed with abscisic acid (SVO+ABS 25mM) for 3 hours prior to co-culture with THP-1 cells. THP-1 were treated for 48h with PMA to induce their differentiation into macrophage-like cells as previously described (Chabot et al, 2001). Jurkat T cells were rinsed before co-culture with PMA-treated THP-1 cells. Treatment with steviol or abscisic acid enhanced the production of ROS from THP1 -Jurkat co-cultures, whereas the combination of steviol and abscisic acid prevented the generation of ROS. Data are representative of 2 separate experiments.

[00123] Figure 14. ROS production from THP1 -Jurkat co-cultured increased in a dose-dependent manner when Jurkat T cells pretreated with various concentrations of steviol (SVO) or abscisic acid (ABS). The combination of steviol with abscisic acid prevented the increased production of ROS from THP1 -Jurkat co-cultures 30 minutes after the addition of ROS inducer (AAPH). Data are representative of 2 separate experiments.

[00124] Figure 15. MTT assay demonstrate that Jurkat cells treated for 3 hours with steviol+abscisic acid increased the viability of their subsequent THP1 -Jurkat co-culture in accordance with Chabot S et al. 2001 . Treatment of Jurkat T cells with steviol or abscisic acid had no effect on the viability of THP1 -Jurkat co-cultures. This data suggest that the combination of steviol and abscisic acid enhance the viability of co-cultures generating lower level of ROS.

Example 9: Mixture of steviol with abscisic acid generate a TRPV1 regulator.

[00125] Figure 16. Molecule of steviol and abscisic acid are shown separately. We propose that the combination of steviol with abscisic acid causes the creation of a novel TRPV1 regulator. Known agonist and antagonist of TRPV1 show resemblance to a potential molecule that may be generated by mixing steviol with abscisic acid together. Physiological pathways associated with TRPV1 regulation are shown in Figures 17 and 18. Functional applications of steviol combined with abscisic acid creates an inhibitor to oxidative stress in immune and neuronal cells models: such as pain, inflammation , ATP modulation, or modulating calcium flow (influx and/or efflux). Example 10: Mixture of steviol with abscisic acid has anti-inflammatory properties.

[00126] Figure 19 show the anti-inflammatory property of steviol-abscisic acid, suggested by the decrease in TNF-alpha production from macrophage-like PMA treated THP-1 cells. Levels of TNF-alpha in the conditioned media were measured using an ELISA kit purchased at R&D systems.

Example 11 : Mixture of steviol with abscisic acid has energy modulatory activity

[00127] Figure 20. Data show that the mixture of steviol and abscisic acid enhance the intracellular levels of ATP in human dopaminergic neuronal cells SH-SY5Y suggesting that the mixture can cause an energy lift, enhancing the energetic potential of cells. The reduction of intracellular ATP is one of the major biological events who lead to cell death. If the noxious conditions persist, a cascade of biological events occurs within cells that lead to irreversible injury and eventually to cell death. There is increasing evidence implicating alterations in the physical state of the biological membrane as a major factor in the evolution of irreversible injury during intracellular ATP depletion.

[00128] Figure 21. Results show that the mixture of steviol and abscisic acid prevents an increase in ATP secretion induced by TRPV1 agonist capsaicin observed upon stimulation with steviol or abscisic acid alone. For more than two decades it has been known that exogenous ATP is lytic to many cell types. Indeed, extracellular ATP is known to be a mediator of cytotoxic cell-dependent lysis. Since massive extracellular release of ATP often occurs after metabolic stress, brain ischemia and trauma, purinergic mechanisms are also correlated to and involved in the etiopathology of many neurodegenerative conditions. Figure 22 is a schematic representation summarizing the effects of SVO+ABS in neuronal and immune cells described in this application.

[00129] Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

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